Publications

Endoscopic ultrasonography (EUS) is a safe, real-time diagnostic and therapeutic tool. Speckle noise, inherent to ultrasonography, degrades the diagnostic precision of EUS. Elevational angular compounding (EAC) can provide real-time speckle noise reduction; however, EAC has never been applied to EUS because current implementations require costly and bulky arrays and are incompatible with the tight spatial constraints of hollow organs. Here we develop a radial implementation of a refraction-based elevational angular compounding technique (REACT) for EUS and demonstrate for the first time spatial compounding in a radial endoscopy. The proposed implementation was investigated in cylindrical phantoms and demonstrated superior suppression of ultrasound speckle noise and up to a two-fold improvement in signal- and contrast- ratios, compared to standard image processing techniques and averaging. The effect of elevational angular deflection on image fidelity was further investigated in a phantom with lymph node-like structures to determine the optimum elevational angular width for high speckle reduction efficiency while maintaining image fidelity. This study introduces REACT as a potential compact and low-cost solution to impart current radial echo-endoscopes with spatial compounding, which could enable accurate identification and precise sizing of lymph nodes in staging of gastrointestinal tract cancers.

Image performance in optoacoustic endoscopy depends markedly on the design of the transducer employed. Ideally, high-resolution performance is required over an expanded depth of focus. Current optoacoustic focused transducers achieve lateral resolutions in the range of tens of microns in the mesoscopic regime, but their depth of focus is limited to hundreds of microns by the nature of their spherical geometry. We designed an ultra-broadband axicon detector with a 2 mm central aperture and investigated whether the imaging characteristics exceeded those of a spherical detector of similar size. We show a previously undocumented ability to achieve a broadband elongated pencil-beam optoacoustic sensitivity with an axicon detection geometry, providing approximately 40 μm-lateral resolution maintained over a depth of focus of 950 μm-3.8 times that of the reference spherical detector. This performance could potentially lead to optoacoustic endoscopes that can visualize optical absorption deeper and with higher resolution than any other optical endoscope today.

Background: Left ventricular ejection fraction (LVEF) is the gold standard for evaluating heart failure (HF) in coronary artery disease (CAD) patients. It is an essential metric in categorizing HF patients as preserved (HFpEF), mid-range (HFmEF), and reduced (HFrEF) ejection fraction but differs, depending on whether the ASE/EACVI or ESC guidelines are used to classify HF. Objectives: We sought to investigate the effectiveness of using deep learning as an automated tool to predict LVEF from patient clinical profiles using regression and classification trained models. We further investigate the effect of utilizing other LVEF-based thresholds to examine the discrimination ability of deep learning between HF categories grouped with narrower ranges. Methods: Data from 303 CAD patients were obtained from American and Greek patient databases and categorized based on the American Society of Echocardiography and the European Association of Cardiovascular Imaging (ASE/EACVI) guidelines into HFpEF (EF > 55%), HFmEF (50% ≤ EF ≤ 55%), and HFrEF (EF < 50%). Clinical profiles included 13 demographical and clinical markers grouped as cardiovascular risk factors, medication, and history. The most significant and important markers were determined using linear regression fitting and Chi-squared test combined with a novel dimensionality reduction algorithm based on arc radial visualization (ArcViz). Two deep learning-based models were then developed and trained using convolutional neural networks (CNN) to estimate LVEF levels from the clinical information and for classification into one of three LVEF-based HF categories. Results: A total of seven clinical markers were found important for discriminating between the three HF categories. Using statistical analysis, diabetes, diuretics medication, and prior myocardial infarction were found statistically significant (p < 0.001). Furthermore, age, body mass index (BMI), anti-arrhythmics medication, and previous ventricular tachycardia were found important after projections on the ArcViz convex hull with an average nearest centroid (NC) accuracy of 94%. The regression model estimated LVEF levels successfully with an overall accuracy of 90%, average root mean square error (RMSE) of 4.13, and correlation coefficient of 0.85. A significant improvement was then obtained with the classification model, which predicted HF categories with an accuracy ≥93%, sensitivity ≥89%, 1-specificity <5%, and average area under the receiver operating characteristics curve (AUROC) of 0.98. Conclusions: Our study suggests the potential of implementing deep learning-based models clinically to ensure faster, yet accurate, automatic prediction of HF based on the ASE/EACVI LVEF guidelines with only clinical profiles and corresponding information as input to the models. Invasive, expensive, and time-consuming clinical testing could thus be avoided, enabling reduced stress in patients and simpler triage for further intervention.

Abstract: Astrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical, metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms. Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21 in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1 cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescence-associated β-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes. Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic strategies based on miRNA manipulation could prevent senescence and may improve the physiological outcome.

The physical properties of each transducer element play a vital role in the quality of images generated in optoacoustic (photoacoustic) tomography using transducer arrays. Thorough experimental characterization of such systems is often laborious and impractical. A shortcoming of the existing impulse response correction methods, however, is the assumption that all transducers in the array are identical and therefore share one electrical impulse response (EIR). In practice, the EIRs of the transducer elements in the array vary, and the effect of this element-to-element variability on image quality has not been investigated so far, to the best of our knowledge. We hereby propose a robust EIR derivation for individual transducer elements in an array using sparse measurements of the total impulse response (TIR) and by solving the linear system for temporal convolution. Thereafter, we combine a simulated spatial impulse response with the derived individual EIRs to obtain a full characterization of the TIR, which we call individual synthetic TIR. Correcting for individual transducer responses, we demonstrate significant improvement in isotropic resolution, which further enhances the clinical potential of array-based. handheld transducers.

Barrett's esophagus (BE) is the main known precursor condition of esophageal adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is probably mediated by chronic esophageal inflammation, secondary to gastroesophageal reflux disease in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high-fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, whereas both NSAIDs were effective chemoprevention agents in the accelerated HFD-fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.

The inability to directly visualize large-scale neural dynamics across the entire mammalian brain in the millisecond temporal resolution regime is among the main limitations of existing neuroimaging methods. Recent advances in optoacoustic imaging systems have led to the establishment of this technology as an alternative method for real-time deep-tissue observations. Particularly, functional optoacoustic neurotomography (FONT) has recently been suggested for three-dimensional imaging of both direct calcium activity and cerebral hemodynamic parameters in rodents. However, the lack of suitable calcium indicators featuring optical absorption peaks within the so-called near-infrared window has hampered the applicability of FONT for imaging neuronal activity deep within the mammalian brain. To surmount this challenge, we developed and validated an intracardially perfused murine brain model labelled with genetically encoded calcium indicator GCaMP6f that closely simulates in vivo conditions. Penetration of light through skull and skin is greatly facilitated after blood is substituted by artificial cerebrospinal fluid (ACSF). The new preparation enabled here the observation of stimulus-evoked calcium dynamics within the mouse brain at penetration depths and spatio-temporal resolution scales not attainable with other neuroimaging techniques.

Image contrast in multispectral optoacoustic tomography can be reduced by electrical noise. We present a deep learning method to remove electrical noise from optoacoustic signals and thereby significantly enhance morphological and spectral contrast.

Image contrast in multispectral optoacoustic tomography can be reduced by electrical noise. We present a deep learning method to remove electrical noise from optoacoustic signals and thereby significantly enhance morphological and spectral contrast.

Fullerenes are candidates for theranostic applications because of their high photodynamic activity and intrinsic multimodal imaging contrast. However, fullerenes suffer from low solubility in aqueous media, poor biocompatibility, cell toxicity, and a tendency to aggregate. C70@lysozyme is introduced herein as a novel bioconjugate that is harmless to a cellular environment, yet is also photoactive and has excellent optical and optoacoustic contrast for tracking cellular uptake and intracellular localization. The formation, water-solubility, photoactivity, and unperturbed structure of C70@lysozyme are confirmed using UV-visible and 2D 1H, 15N NMR spectroscopy. The excellent imaging contrast of C70@lysozyme in optoacoustic and third harmonic generation microscopy is exploited to monitor its uptake in HeLa cells and lysosomal trafficking. Last, the photoactivity of C70@lysozyme and its ability to initiate cell death by means of singlet oxygen (1O2) production upon exposure to low levels of white light irradiation is demonstrated. This study introduces C70@lysozyme and other fullerene-protein conjugates as potential candidates for theranostic applications.

PURPOSE: The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. METHODS: Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. RESULTS: Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized  by human BE dysplastic patient-derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. CONCLUSION: In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

OBJECTIVE: Postprandial lipid profiling (PLP), a risk indicator ofcardiometabolic disease, is based on frequent blood sampling over several hours after a meal, an approach that is invasive and inconvenient. Non-invasive PLP may offer an alternative for disseminated human monitoring. Herein, we investigate the use of clinical Multispectral Optoacoustic Tomography (MSOT) for the non-invasive, label-free PLP via direct lipid-sensing in human vasculature and soft tissues. METHODS: Four (n = 4) subjects (3 females and 1 male, age: 28 ± 7 years) were enrolled in the current pilot study. We longitudinally measured the lipid signals in arteries, veins, skeletal muscles and adipose tissues of all participants at 30 min-intervalsfor 6 hours after the oral consumption of a high-fat meal. RESULTS: Optoacoustic lipid-signal analysis showed on average a 63.4% intra-arterial increase at ∼4 hours postprandially, a 83.9% intra-venous increase at ∼3 hours, a 120.8% intra-muscular increase at ∼3 hours and a 32.8% subcutaneous fat increase at ∼4 hours. CONCLUSION: MSOT provides the potential to study lipid metabolism that could lead to novel diagnostics and prevention strategies by label-free, non-invasive detection of tissue biomarkers implicated in cardiometabolic diseases.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor and executive function. Central to BG function are striatal cells expressing D1 (D1R) and D2 (D2R) dopamine receptors. D1R and D2R cells are considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. However, whether they maintain a uniform function across the striatum and what influence they exert outside the BG is unclear. Here, we address these questions by combining optogenetic activation of D1R and D2R cells in the mouse ventrolateral caudoputamen with fMRI. Striatal D1R/D2R stimulation evokes distinct activity within the BG-thalamocortical network and differentially engages cerebellar and prefrontal regions. Computational modeling of effective connectivity confirms that changes in D1R/D2R output drive functional relationships between these regions. Our results suggest a complex functional organization of striatal D1R/D2R cells and hint toward an interconnected fronto-BG-cerebellar network modulated by striatal D1R and D2R cells.

Genetically engineered bacterial outer membrane vesicles (OMVs) offer promising applications for gene therapy, immunotherapy, and vaccine delivery. Importantly, OMVs are biocompatible, biodegradable, and easy to engineer and produce on a large scale. In this chapter, we discuss the development and application of bioengineered OMVs for optoacoustics-guided phototherapy applications (theranostics). We provide detailed protocols for OMVs preparation, characterization, and in vitro and in vivo validation. The engineered OMVs carry the biopolymer melanin, which generates a strong optoacoustic (OA) signal and intense heat upon absorption of near-infrared (NIR) light, enabling optoacoustics-guided cancer diagnosis and photothermal therapy in vivo.

One of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery3-7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor8, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors-as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors-our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 μm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.

The total impulse response of a clinical optoacoustic system is characterized by combining experimentally acquired signals with a numerical model of the spatial impulse response, resulting in high-resolution images in clinical applications.

Visualizing whole-body dynamics across entire living organisms is crucial for understanding complex biology, disease progression as well as evaluating efficacy of new drugs and therapies. Existing small animal functional and molecular imaging modalities either suffer from low spatial and temporal resolution, limited penetration depth or poor contrast. In this work, we present flash scanning volumetric optoacoustic tomography (fSVOT) imaging system that enables the acquisition speeds required for visualizing fast kinetics and biodistribution of optical contrast agents across whole mice. fSVOT can render images of intricate vascular and organ anatomy with rich contrast by capitalizing on the large angular coverage of a spherical matrix array transducer rapidly scanned around the mouse. Volumetric (three-dimensional) images with 200 μm resolution can be acquired within 45 seconds, which corresponds to an imaging speed gain of an order of magnitude with respect to existing state-of-the-art modalities offering comparable resolution performance. We demonstrate volumetric tracking and quantification of gold nanorod kinetics and their differential uptake across the spleen, liver and kidneys. Overall, fSVOT offers unprecedented capabilities for multi-scale imaging of pharmacokinetics and bio-distribution of agents with high contrast, resolution and image acquisition speed.

Imaging is an essential tool in research, diagnostics and the management of endocrine disorders. Ultrasonography, nuclear medicine techniques, MRI, CT and optical methods are already used for applications in endocrinology. Optoacoustic imaging, also termed photoacoustic imaging, is emerging as a method for visualizing endocrine physiology and disease at different scales of detail: microscopic, mesoscopic and macroscopic. Optoacoustic contrast arises from endogenous light absorbers, such as oxygenated and deoxygenated haemoglobin, lipids and water, or exogenous contrast agents, and reveals tissue vasculature, perfusion, oxygenation, metabolic activity and inflammation. The development of high-performance optoacoustic scanners for use in humans has given rise to a variety of clinical investigations, which complement the use of the technology in preclinical research. Here, we review key progress with optoacoustic imaging technology as it relates to applications in endocrinology; for example, to visualize thyroid morphology and function, and the microvasculature in diabetes mellitus or adipose tissue metabolism, with particular focus on multispectral optoacoustic tomography and raster-scan optoacoustic mesoscopy. We explain the merits of optoacoustic microscopy and focus on mid-infrared optoacoustic microscopy, which enables label-free imaging of metabolites in cells and tissues. We showcase current optoacoustic applications within endocrinology and discuss the potential of these technologies to advance research and clinical practice.

Background: Current imaging assessment of peripheral artery disease (PAD) relies on anatomical cross-sectional visualizations of the affected arteries. Multispectral optoacoustic tomography (MSOT) is a novel molecular imaging technique that provides direct and label-free visualizations of soft tissue perfusion and oxygenation. Methods: MSOT was prospectively assessed in a pilot trial in healthy volunteers (group n1=4, mean age 31, 50% male and group n3=4, mean age 37.3, 75% male) and patients with intermittent claudication (group n2=4, mean age 72, 75% male, PAD stage IIb). We conducted cuff-induced ischemia (group n1) and resting state measurements (groups n2 and n3) over the calf region. Spatially resolved mapping of oxygenated (HbO2), deoxygenated (Hb) and total (THb) hemoglobin, as well as oxygen saturation (SO2), were measured via hand-held hybrid MSOT-Ultrasound based purely on hemoglobin contrast. Results: Calf measurements in healthy volunteers revealed distinct dynamics for HbO2, Hb, THb and SO2 under cuff-induced ischemia. HbO2, THb and SO2 levels were significantly impaired in PAD patients compared to healthy volunteers (P<0.05 for all parameters). Revascularization led to significant improvements in HbO2 of the affected limb. Conclusions: Clinical MSOT allows for non-invasive, label-free and real-time imaging of muscle oxygenation in health and disease with implications for diagnostics and therapy assessment in PAD.

In the original version of this article, in Fig. 1c, the icons for oxygenated haemoglobin and deoxygenated haemoglobin were incorrectly shown outside of the artery and vein. This error has now been corrected in the HTML and PDF versions of the article.

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

Modern imaging technologies, including optoacoustic endoscopy, are based on the optoacoustic effect. Much promise is offered by the all-optical fiber-based approach, because fiber has a miniature cross section, is highly sensitive, and can be used in a variety of imaging and therapeutic techniques. We developed a probe based on a hollow-core microstructured optical waveguide (HC-MOW) with a hybrid nanostructured membrane. The membrane consisted of a free-standing single-walled carbon nanotube film and a Bragg reflector, which can be used as a source and a detector of ultrasound. Membrane vibrations were excited with an IR laser pulse and were read out by recording the intensity of the reflected visible CW laser light. We explained the nature of the intensity modulation of the reflected light and supported our explanation with numerical simulations of the membrane's vibration eigenfrequencies and thermal distribution. The membrane vibrations were also observed with raster-scanning optoacoustic mesoscopy. The transmittance of the HC-MOW between 400 nm and 6.5 μm and that of the hybrid nanostructured membrane in the NIR range enable potential optoacoustic sensing in the IR fingerprint region of biomolecules. This permits the optoacoustic probe to be used for medical endoscopic purposes.

Coronary artery disease (CAD) remains a leading cause of mortality and warrants new imaging approaches to better guide clinical care. We report on a miniaturized, hybrid intravascular catheter and imaging system for comprehensive coronary artery imaging in vivo. Our catheter exhibits a total diameter of 1.0 mm (3.0 French), equivalent to standalone clinical intravascular ultrasound (IVUS) catheters but enables simultaneous near-infrared fluorescence (NIRF) and IVUS molecular-structural imaging. We demonstrate NIRF-IVUS imaging in vitro in coronary stents using NIR fluorophores, and compare NIRF signal strengths for prism and ball lens sensor designs in both low and high scattering media. Next, in vivo intravascular imaging in pig coronary arteries demonstrates simultaneous, co-registered molecular-structural imaging of experimental CAD inflammation on IVUS and distance-corrected NIRF images. The obtained results suggest substantial potential for the NIRF-IVUS catheter to advance standalone IVUS, and enable comprehensive phenotyping of vascular disease to better assess and treat patients with CAD.

Ultrasound (US) and optoacoustic (OA) imaging provide complementary information for quantitative analysis of the tumor microenvironment. Herein, we demonstrate the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS) for characterizing breast cancer in tumor-bearing mice. For this, 4 different mice featuring orthotopic tumor of different sizes were scanned with a full-ring ultrasound transducer array to simultaneously render pulse-echo US images, speed of sound (SoS) maps and OA images. The tumor size, vascular density and its elastic parameters were further quantified in the images. Our results pave the way toward clinical translation of the hybrid TROPUS imaging for tumor detection and characterization.

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the αvβ3 integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics.

CR760, a croconaine dye with excellent optical properties, was synthesized in a single step and subsequently nano-formulated for optoacoustic imaging and photothermal therapy of cancer.

The optoacoustic imaging (OAI) methods are rapidly evolving for resolving optical contrast in medical imaging applications. In practice, measurement strategies are commonly implemented under limited-view conditions due to oversized image objectives or system design limitations. Data acquired by limited-view detection may impart artifacts and distortions in reconstructed optoacoustic (OA) images. We propose a hybrid data-driven deep learning approach based on generative adversarial network (GAN), termed as LV-GAN, to efficiently recover high quality images from limited-view OA images. Trained on both simulation and experiment data, LV-GAN is found capable of achieving high recovery accuracy even under limited detection angles less than 60 degrees. The feasibility of LV-GAN for artifact removal in biological applications was validated by ex vivo experiments based on two different OAI systems, suggesting high potential of a ubiquitous use of LV-GAN to optimize image quality or system design for different scanners and application scenarios.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.Calcium and other analytes can be imaged at super-resolution and in vivo with photo-switchable sensors.

BACKGROUND: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). METHODS: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. RESULTS: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. CONCLUSION: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia.

Magneto-plasmonic nanocomposites can possess properties inherent to both individual components (iron oxide and gold nanoparticles) and are reported to demonstrate high potential in targeted drug delivery and therapy. Herein, we report on Fe O /Au magneto-plasmonic nanocomposites (MPNC) synthesized with the use of amino acid tryptophan via chemical and photochemical reduction of Au ions in the presence of nanosized magnetite. The magnetic field (MF) induced aggregation was accompanied by an increase in the absorption in the near-infrared (NIR) spectral region, which was demonstrated to provide an enhanced photothermal (PT) effect under NIR laser irradiation (at 808 nm). A possibility for therapeutic application of the MPNC was illustrated using cancer cells in vitro. Cultured HeLa cells were treated by MPNC in the presence of MF and without it, following laser irradiation and imaging using confocal laser scanning microscopy. After scanning laser irradiation of the MPNC/MF treated cells, a formation and rise of photothermally-induced microbubbles on the cell surfaces was observed, leading to a damage of the cell membrane and cell destruction. We conclude that the synthesized magneto-plasmonic Fe O /Au nanosystems exhibit magnetic field-induced reversible aggregation accompanied by an increase in NIR absorption, allowing for an opportunity to magnetophoretically control and locally enhance a NIR light-induced thermal effect, which holds high promise for the application in photothermal therapy. 3 4 3 4

Optoacoustic signals are typically reconstructed into images using inversion algorithms applied in the time-domain. However, time-domain reconstructions can be computationally intensive and therefore slow when large amounts of raw data are collected from an optoacoustic scan. Here we consider a fast weighted ω-κ (FWOK) algorithm operating in the frequency domain to accelerate the inversion in raster-scan optoacoustic mesoscopy (RSOM), while seamlessly incorporating impulse response correction with minimum computational burden. We investigate the FWOK performance with RSOM measurements from phantoms and mice in vivo and obtained 360-fold speed improvement over inversions based on the back-projection algorithm in the time-domain. This previously unexplored inversion of in vivo optoacoustic data with impulse response correction in frequency domain reconstructions points to a promising strategy of accelerating optoacoustic imaging computations, toward video-rate tomography.

Die optoakustische Bildgebung (OAB) hat sich in den letzten Jahren stetigweiterentwickelt. Mittels teils gepulsten Lichts in verschiedenen Wellenlängen werdenunterschiedliche Farbträger (Chromophore) zur Bildung von Schallwellen angeregt.Diese werden von den neu entwickelten Systemen detektiert und mittels verschiedenerAlgorithmen in dreidimensionale Bilder umgewandelt. Die Technik zeichnet sich durchein gutes Verhältnis von Kontrast und Eindringtiefe aus und kann aufgrund ihrerSkalierbarkeit makro-, meso- und mikroskopische Bilder erzeugen. Die optoakustischeMakroskopie bestrahlt das zu untersuchende Areal breit mit Laserlicht. Hierdurchkönnen Abbildungen mit hoher Eindringtiefe erzeugt werden, jedoch lediglich miteiner mittleren Auflösung. Klinisch interessante Anwendungsfelder sind z.B. dieErgebnisse von ex-vivo untersuchten Sentinellymphknoten mittels makroskopischerOptoakustik. Aufgrund der Fähigkeit der OAB Melanin darzustellen zeigte sicheine bisherigen bildgebenden Methoden, jedoch nicht der Histologie überlegeneDetektionsrate für Metastasen. Die Fähigkeit, mit einer guten Auflösung dermale undepidermale Strukturen, besonders Gefäße, darzustellen, macht sich die optoakustischeMesoskopie bei der Untersuchung entzündlicher Hauterkrankungen zunutze undkönnte künftig zur Überprüfung des Therapieerfolges, z.B. durch Biologika bei Psoriasisvulgaris oder dem atopischen Ekzem, beitragen. Die bisher v. a. auf präklinische In-vivo-Forschung beschränkte optoakustische Mikroskopie könnte künftig zur Detektion nochfeinerer Gefäßstrukturen und deren Veränderungen verwendet werden. Die klinischenMöglichkeiten der OAB scheinen bisher sehr großen Nutzen bieten zu können und sindein aktuell stark untersuchtes Forschungsfeld.

Laser ablation (LA) is gaining acceptance for the treatment of tumors as a viable alternative to surgical resection. In parallel, optoacoustic tomography (OAT) has enabled defining new regimes for diagnosis and characterization of malignant neoplastic lesions with high sensitivity and specificity. Even though pulsed nanosecond lasers are commonly used for both imaging and therapeutic purposes, real-time thermal treatment monitoring with a single laser source has not been previously attempted. Herein, we demonstrate the feasibility of combined OAT and LA by percutaneous irradiation of subcutaneous tumors with a 100 mJ short-pulsed (∼5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. The OAT images rendered with a spherical ultrasound transducer array enabled real-time monitoring of the LA lesion progression, which is essential for determining the optimal treatment end-point. Local changes in the optoacoustic signal intensity associated with the induced temperature changes as well as structural alterations in the tumor vasculature could clearly be observed. The optoacoustic volumetric projections further correlated with crosssections extracted from the excised tumors. This newly enabled capability anticipates new theranostic approaches in cancer research and treatment with potential applicability in a clinical setting.

We propose a new opto-mechanical ultrasound sensor (OMUS) enabled by an innovative silicon photonics waveguide. We present experimental results up to 30 MHz, a 10-sensor array proof-of-concept and our latest findings.

Tracking of biodynamics across entire living organisms is essential for understanding complex biology and disease progression. The presently available small-animal functional and molecular imaging modalities remain constrained by factors including long image acquisition times, low spatial resolution, limited penetration or poor contrast. Here flash scanning volumetric optoacoustic tomography (fSVOT), a new approach for high-speed imaging of fast kinetics and biodistribution of optical contrast agents in whole mice that simultaneously provides reference images of vascular and organ anatomy with unrivaled fidelity and contrast, is presented. The imaging protocol employs continuous overfly scanning of a spherical matrix array transducer, accomplishing a 200 µm resolution 3D scan of the whole mouse body within 45 s without relying on signal averaging. This corresponds to an imaging speed gain of more than an order of magnitude compared with existing state-of-the-art implementations of comparable resolution performance. Volumetric tracking and quantification of gold nanoagent and near infrared (NIR)-II dye kinetics and their differential uptake in various organs are demonstrated. fSVOT thus offers unprecedented capabilities for multiscale imaging of pharmacokinetics and biodistribution with high contrast, resolution, and speed.

Morphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell’s size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system’s precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.

The non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

Vertebral labelling and segmentation are two fundamental tasks in an automated spine processing pipeline. Reliable and accurate processing of spine images is expected to benefit clinical decision support systems for diagnosis, surgery planning, and population-based analysis of spine and bone health. However, designing automated algorithms for spine processing is challenging predominantly due to considerable variations in anatomy and acquisition protocols and due to a severe shortage of publicly available data. Addressing these limitations, the Large Scale Vertebrae Segmentation Challenge (VERSE) was organised in conjunction with the International Conference on Medical Image Computing and Computer Assisted Intervention (MICCAI) in 2019 and 2020, with a call for algorithms tackling the labelling and segmentation of vertebrae. Two datasets containing a total of 374 multi-detector CT scans from 355 patients were prepared and 4505 vertebrae have individually been annotated at voxel level by a human-machine hybrid algorithm (https://osf.io/nqjyw/, https://osf.io/t98fz/). A total of 25 algorithms were benchmarked on these datasets. In this work, we present the results of this evaluation and further investigate the performance variation at the vertebra level, scan level, and different fields of view. We also evaluate the generalisability of the approaches to an implicit domain shift in data by evaluating the top-performing algorithms of one challenge iteration on data from the other iteration. The principal takeaway from VERSE: the performance of an algorithm in labelling and segmenting a spine scan hinges on its ability to correctly identify vertebrae in cases of rare anatomical variations. The VERSE content and code can be accessed at: https://github.com/anjany/verse.

The recent development of ultrasound sensing using the silicon-photonics platform has enabled super-resolution optoacoustic imaging not possible by piezoelectric technology or polymeric optical microresonators. The silicon waveguide etalon detector (SWED) design exploits the sub-micrometer light confinement in the cross-section of a silicon strip waveguide to achieve a sensor aperture which is 13-fold to 30-fold smaller than the cutoff wavelength of the sensor. While its performance in near-field scanning optoacoustic imaging has been previously studied, the operational characteristics of this sensor as it relates to conventional optoacoustic imaging applications are not known. Here, for the first time, the application of the SWED in optoacoustic mesoscopy is investigated, the interaction of the sensor with ultrasound in the far-field is characterized, the acoustic point spread function up to a depth of 10 mm is measured, and 3D vasculature-mimicking phantoms are imaged. The measured point spread function of the sensor shows that surface acoustic waves can degrade the lateral resolution. Nevertheless, superior resolution is demonstrated over any state-of-the-art ultrasound sensor, over the whole range of imaging depths that are of interest to optoacoustic mesoscopy. Silicon photonics is proposed as a powerful and promising new platform for ultrasonics and optoacoustics.

Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing—depending on the used photoswitching agent and sample—routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal.

Attention to Black Carbon (BC) has been rising due to its effects on human health as well its contribution to climate change. Measurements of BC are challenging, as currently used devices are either expensive or impractical for continuous monitoring. Here, we propose an optoacoustic sensor to address this problem. The sensor utilizes a novel ellipsoidal design for refocusing the optoacoustic signal with minimal acoustic energy losses. To reduce the cost of the system, without sacrificing accuracy, an overdriven laser diode and a Quartz Tuning Fork are used as the light source and the sound detector, respectively. The prototype was able to detect BC particles and to accurately monitor changes in concentration in real time and with very good agreement with a reference instrument. The response of the sensor was linearly dependent on the BC particles concentration with a normalized noise equivalent absorption coefficient (NNEA) for soot equal to 7.39 × 10−9 W cm−1 Hz−1/2. Finally, the prototype was able to perform NO2 measurements, demonstrating its ability to accurately monitor both particulate and gaseous pollutants. The proposed sensor has the potential to offer a significant economic impact for BC environmental measurements and source appointment technologies.

We developed high-density spherical matrix array based on polyvinylidene difluoride films. Ultrawide bandwidth (0.3-38 MHz) and sub-millimeter sized elements enabled non-invasive cerebrovascular imaging of adult mouse with ~60 µm resolution.

Optical fiber sensors can offer robust and miniaturized detection of wideband ultrasound, yielding high sensitivity and immunity to electromagnetic interference. However, the lack of cost-effective manufacturing methods prevents the disseminated use of these sensors in biomedical applications. In this study, we developed and optimized a simple method to create optical cavities with high-quality mirrors for acoustic sensing based on micro-manipulation of UV-curable optical adhesives and electroless chemical silver deposition. This approach enables the manufacturing of ultrasound sensors based on Fabry-Pérot Interferometers (FPI) on optical fiber tips with minimal production costs. Characterization and high-resolution optoacoustic imaging experiments show that the manufacturing process yielded a fiber sensor with a small NEP (11 mPa/ Hz ) over a broad detection bandwidth (25 MHz), generally outperforming conventional piezoelectric based transducers. We discuss how the new manufacturing process leads to a high-performance acoustic detector that, due to low cost, can be used as a disposable sensor.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab’)2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenylisothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab’)2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab’)2 and 89Zr-DFO-Gal3-F(ab’)2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab’)2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab’)2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

Ultrasonography1 and photoacoustic2,3 (optoacoustic) tomography have recently seen great advances in hardware and algorithms. However, current high-end systems still use a matrix of piezoelectric sensor elements, and new applications require sensors with high sensitivity, broadband detection, small size and scalability to a fine-pitch matrix. This work demonstrates an ultrasound sensor in silicon photonic technology with extreme sensitivity owing to an innovative optomechanical waveguide. This waveguide has a tiny 15 nm air gap between two movable parts, which we fabricated using new CMOS-compatible processing. The 20 μm small sensor has a noise equivalent pressure below 1.3 mPa Hz−1/2 in the measured range of 3–30 MHz, dominated by acoustomechanical noise. This is two orders of magnitude better than for piezoelectric elements of an identical size4. The demonstrated sensor matrix with on-chip photonic multiplexing5–7 offers the prospect of miniaturized catheters that have sensor matrices interrogated using just a few optical fibres, unlike piezoelectric sensors that typically use an electrical connection for each element.

Accurate knowledge of the exact stopping location of ions inside the patient would allow full exploitation of their ballistic properties for patient treatment. The localized energy deposition of a pulsed particle beam induces a rapid temperature increase of the irradiated volume and leads to the emission of ionoacoustic (IA) waves. Detecting the time-of-flight (ToF) of the IA wave allows inferring information on the Bragg peak location and can henceforth be used for in-vivo range verification. A challenge for IA is the poor signal-to-noise ratio at clinically relevant doses and viable machines. We present a frequency-based measurement technique, labeled as ionoacoustic tandem phase detection (iTPD) utilizing lock-in amplifiers. The phase shift of the IA signal to a reference signal is measured to derive the ToF. Experimental IA measurements with a 3.5 MHz lead zirconate titanate (PZT) transducer and lock-in amplifiers were performed in water using 22 MeV proton bursts. A digital iTPD was performed in-silico at clinical dose levels on experimental data obtained from a clinical facility and secondly, on simulations emulating a heterogeneous geometry. For the experimental setup using 22 MeV protons, a localization accuracy and precision obtained through iTPD deviates from a time-based reference analysis by less than 15 mu m. Several methodological aspects were investigated experimentally in systematic manner. Lastly, iTPD was evaluated in-silico for clinical beam energies indicating that iTPD is in reach of sub-mm accuracy for fractionated doses < 5 Gy. iTPD can be used to accurately measure the ToF of IA signals online via its phase shift in frequency domain. An application of iTPD to the clinical scenario using a single pulsed beam is feasible but requires further development to reach <1 Gy detection capabilities.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in highrisk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 μm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 μm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 μs in water, corresponding to a frame rate of μ30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

Ultrasound imaging is affected by coherent noise or speckle, which reduces contrast and overall image quality and degrades the diagnostic precision of the collected images. Elevational angular compounding (EAC) is an attractive means of addressing this limitation, since it reduces speckle noise while operating in real-time. However, current EAC implementations rely on mechanically rotating a one-dimensional (1D) transducer array or electronically beam steering of two-dimensional (2D) arrays to provide different elevational imaging angles, which increases the size and cost of the systems. Here we present a novel EAC implementation based on a 1D array, which does not necessitate mechanically rotating the transducer. The proposed refraction-based elevational angular compounding technique (REACT) instead utilizes a translating cylindrical acoustic lens that steers the ultrasound beam along the elevational direction. Applying REACT to investigate phantoms and excised tissue samples demonstrated superior suppression of ultrasound speckle noise compared to previous EAC methods, with up to a two-fold improvement in signal- and contrast-to-noise ratios. The effects of elevational angular width on speckle reduction was further investigated to determine the appropriate conditions for applying EAC. This study introduces acoustic refractive elements as potential low cost solutions to noise reduction, which could be integrated into current medical ultrasound devices.

The miR-221 expression is dependent on the oncogenic RAS-RAF-MEK pathway activation and influences epithelial-to-mesenchymal transition (EMT). The Cancer Genome Atlas (TCGA) database analysis showed high gene significance for ZEB1 with EMT module analysis and miR-221 overexpression within the triple-negative breast cancer (TNBC) and HER2+ subgroups when compared to luminal A/B subgroups. EMT marker expression analysis after MEK1 (TAK-733) inhibitor treatment and irradiation was combined with miR-221 and ZEB1 expression analysis. The interaction of miR-221 overexpression with irradiation and its influence on migration, proliferation, colony formation and subsequent EMT target activation were investigated. The results revealed that MEK1 inhibitor treatment combined with irradiation could decrease the migratory potential of breast cancer cells including reduction of miR-221 and corresponding downstream ZEB1 (EMT) marker expression. The clonogenic survival assays revealed that miR-221 overexpressing SKBR3 cells were more radioresistant when compared to the control. Remarkably, the effect of miR-221 overexpression on migration in highly proliferative and highly HER2-positive SKBR3 cells remained constant even upon 8 Gy irradiation. Further, in naturally miR-221-overexpressing MDA-MB-231 cells, the proliferation and migration significantly decrease after miR-221 knockdown. This leads to the assumption that radiation alone is not reducing migration capacity of miR-221-overexpressing cells and that additional factors play an important role in this context. The miR-221/ZEB1 activity is efficiently targeted upon MEK1 inhibitor (TAK-733) treatment and when combined with irradiation treatment, significant reduction in migration of breast cancer cells was shown.

Scanning optical microscopy techniques are commonly restricted to a sub-millimeter field-of-view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large-field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam-splitting grating and an acousto-optic deflector synchronized with a high-speed camera to attain real-time fluorescence microscopy over a centimeter-scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide-field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide-field microscopes to mitigate image blur due to tissue scattering and attain optimal trade-off between spatial resolution and FOV. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large-scale fluorescent-based biodynamics.

Multispectral Optoacoustic Tomography (MSOT) resolves oxy- (HbO2) and deoxy-hemoglobin (Hb) to perform vascular imaging. MSOT suffers from gradual signal attenuation with depth due to light-tissue interactions: an effect that hinders the precise manual segmentation of vessels. Furthermore, vascular assessment requires functional tests, which last several minutes and result in recording thousands of images. Here, we introduce a deep learning approach with a sparse-UNET (S-UNET) for automatic vascular segmentation in MSOT images to avoid the rigorous and time-consuming manual segmentation. We evaluated the S-UNET on a test-set of 33 images, achieving a median DICE score of 0.88. Apart from high segmentation performance, our method based its decision on two wavelengths with physical meaning for the task-at-hand: 850 nm (peak absorption of oxy-hemoglobin) and 810 nm (isosbestic point of oxy-and deoxy-hemoglobin). Thus, our approach achieves precise data-driven vascular segmentation for automated vascular assessment and may boost MSOT further towards its clinical translation.

The impulse response of optoacoustic (photoacoustic) tomographic imaging system depends on several system components, the characteristics of which can influence the quality of reconstructed images. The effect of these system components on reconstruction quality have not been considered in detail so far. Here we combine sparse measurements of the total impulse response (TIR) with a geometric acoustic model to obtain a full characterization of the TIR of a handheld optoacoustic tomography system with concave limited-view acquisition geometry. We then use this synthetic TIR to reconstruct data from phantoms and healthy human volunteers, demonstrating improvements in image resolution and fidelity. The higher accuracy of optoacoustic tomographic reconstruction with TIR correction further improves the diagnostic capability of handheld optoacoustic tomographic systems.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

Particles with sizes in the order of a few micrometers can significantly enhance the capabilities of optoacoustic imaging systems by improving visualization of arbitrarily oriented vascular structures and achieving resolution beyond the acoustic diffraction barrier. Particle tracking may also be used for mapping the blood flow in two and three dimensions. However, a trade-off exists between the particle absorption properties and size, whereas large sized microparticles also tend to arrest in the capillary network. We analyzed the flow of microparticles in an intracardiac perfusion mouse model in which blood is effectively substituted by artificial cerebrospinal fluid (ACSF). This enables mitigating the strong blood absorption background in the optoacoustic images thus facilitating the visualization of microparticles. A sequence of three-dimensional optoacoustic images of the mouse brain is then acquired at a high frame rate of 100 Hz after injection of the particles in the left heart ventricle. By visualizing the flow of particles of different sizes in microvascular structures it is possible to establish optimal trade-offs between the particle size, their optoacoustic signal and perfusion properties.

Widespread metastasis is the major cause of death from melanoma and other types of cancer. At present, the dynamic aspects of the metastatic cascade remain enigmatic. The feasibility to track circulating melanoma cells deep within living intact organisms can greatly impact our knowledge on tumor metastasis, but existing imaging approaches lack the sensitivity, spatio-temporal resolution or penetration depth to capture flowing tumor cells over large fields of view within optically-opaque biological tissues. Vast progress with the development of optoacoustic tomography technologies has recently enabled two- and three-dimensional imaging at unprecedented frame rates in the order of hundreds of Hertz, effectively mapping up to a million image voxels within a single volumetric snapshot. Herein, we employ volumetric optoacoustic tomography for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain. Detection of individual circulating melanoma cells was facilitated by substituting blood with an artificial cerebrospinal fluid that removes the strong absorption background in the optoacoustic images. The approach can provide new opportunities for studying trafficking and accumulation of metastatic melanoma cells in different organs.

Iterative model-based algorithms are known to enable more accurate and quantitative optoacoustic (photoacoustic) tomographic reconstructions than standard back-projection methods. However, three-dimensional (3D) model-based inversion is often hampered by high computational complexity and memory overhead. Parallel implementations on a graphics processing unit (GPU) have been shown to efficiently reduce the memory requirements by on-the-fly calculation of the actions of the optoacoustic model matrix, but the high complexity still makes these approaches impractical for large 3D optoacoustic datasets. Herein, we show that the computational complexity of 3D model-based iterative inversion can be significantly reduced by splitting the model matrix into two parts: one maximally sparse matrix containing only one entry per voxel-transducer pair and a second matrix corresponding to cyclic convolution. We further suggest reconstructing the images by multiplying the transpose of the model matrix calculated in this manner with the acquired signals, which is equivalent to using a very large regularization parameter in the iterative inversion method. The performance of these two approaches is compared to that of standard back-projection and a recently introduced GPU-based model-based method using datasets from in vivo experiments. The reconstruction time was accelerated by approximately an order of magnitude with the new iterative method, while multiplication with the transpose of the matrix is shown to be as fast as standard back-projection.

In the field of reproductive biology, there is a strong need for a suitable tool capable of non-destructive evaluation of oocyte viability and function. We studied the application of brilliant cresyl blue (BCB) as an intra-vital exogenous contrast agent using multispectral optoacoustic tomography (MSOT) for visualization of porcine ovarian follicles. The technique provided excellent molecular sensitivity, enabling the selection of competent oocytes without disrupting the follicles. We further conducted in vitro embryo culture, molecular analysis (real-time and reverse transcriptase polymerase chain reaction) and DNA fragmentation analysis to comprehensively establish the safety of BCB-enhanced MSOT imaging in monitoring oocyte viability. Overall, the experimental results suggest that the method offers a significant advance in the use of contrast agents and molecular imaging for reproductive studies. Our technique improves the accurate prediction of ovarian reserve significantly and, once standardized for in vivo imaging, could provide an effective tool for clinical infertility management.

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

Bone microvasculature plays a paramount role in bone marrow maintenance, development, and hematopoiesis. Studies of calvarian vascular patterns within living mammalian skull with the available intravital microscopy techniques are limited to small scale observations. We developed an optical-resolution optoacoustic microscopy method combined with ultrasound biomicroscopy in order to reveal and discern the intricate networks of calvarian and cerebral vasculature over large fields of view covering majority of the murine calvaria. The vasculature segmentation method is based on an angle-corrected homogeneous model of the rodent skull, generated using simultaneously acquired three-dimensional pulse-echo ultrasound images. The hybrid microscopy design along with the appropriate skull segmentation method enable high throughput studies of a living bone while facilitating correct anatomical interpretation of the vasculature images acquired with optical resolution optoacoustic microscopy.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

Recently, a new class of prokaryotic compartments, collectively called encapsulins or protein nanocompartments, has been discovered. The shell proteins of these structures self-organize to form icosahedral compartments with a diameter of 25-42 nm, while one or more cargo proteins with various functions can be encapsulated in the nanocompartment. Non-native cargo proteins can be loaded into nanocompartments and the surface of the shells can be further functionalized, which allows for developing targeted drug delivery systems or using encapsulins as contrast agents for magnetic resonance imaging. Since the genes encoding encapsulins can be integrated into the cell genome, encapsulins are attractive for investigation in various scientific fields, including biomedicine and nanotechnology.

Raster-scan optoacoustic mesoscopy (RSOM) is a powerful, non-invasive optical imaging technique for functional, anatomical, and molecular skin and tissue analysis. However, both the manual and the automated analysis of such images are challenging, because the RSOM images have very low contrast, poor signal to noise ratio, and systematic overlaps between the absorption spectra of melanin and hemoglobin. Nonetheless, the segmentation of the epidermis layer is a crucial step for many downstream medical and diagnostic tasks, such as vessel segmentation or monitoring of cancer progression. We propose a novel, shape-specific loss function that overcomes discontinuous segmentations and achieves smooth segmentation surfaces while preserving the same volumetric Dice and IoU. Further, we validate our epidermis segmentation through the sensitivity of vessel segmentation. We found a 20% improvement in Dice for vessel segmentation tasks when the epidermis mask is provided as additional information to the vessel segmentation network.

Scattering phenomena affect light propagation through any kind of medium from free space to biological tissues. Finding appropriate strategies to increase the robustness to scattering is the common requirement in developing both communication protocols and imaging systems. Recently, structured light has attracted attention due to its seeming scattering resistance in terms of transmissivity and spatial behavior. Moreover, correlation between optical polarization and orbital angular momentum (OAM), which characterizes the so-called vector vortex beams (VVBs) states, seems to allow for the preservation of the polarization pattern. We extend the analysis by investigating both the spatial features and the polarization structure of vectorial optical vortexes propagating in scattering media with different concentrations. Among the observed features, we find a sudden swift decrease in contrast ratio for Gaussian, OAM, and VVB modes for concentrations of the adopted scattering media exceeding 0.09%. Our analysis provides a more general and complete study on the propagation of structured light in dispersive and scattering media.

Objective: Fluorescence molecular imaging (FMI) has emerged as a promising tool for surgical guidance in oncology, with one of the few remaining challenges being the ability to offer quality control and data referencing. This paper investigates the use of a novel composite phantom to correct and benchmark FMI systems. Methods: This paper extends on previous work by describing a phantom design that can provide a more complete assessment of FMI systems through quantification of dynamic range and determination of spatial illumination patterns for both reflectance and fluorescence imaging. Various performance metrics are combined into a robust and descriptive "system benchmarking score," enabling not only the comprehensive comparison of different systems, but also for the first time, correction of the acquired data. Results: We show that systems developed for targeted fluorescence imaging can achieve benchmarking scores of up to 70 & x0025;, while clinically available systems optimized for indocyanine green are limited to 50 & x0025;, mostly due to greater leakage of ambient and excitation illumination and lower resolution. The image uniformity can also be approximated and employed for image flat-fielding, an important milestone toward data referencing. In addition, we demonstrate composite phantom use in assessing the performance of a surgical microscope and of a raster-scan imaging system. Conclusion: Our results suggest that the new phantom has the potential to support high-fidelity FMI through benchmarking and image correction. Significance: Standardization of the FMI is a necessary process for establishing good imaging practices in clinical environments and for enabling high-fidelity imaging across patients and multi-center imaging studies.

The Author(s), under exclusive licence to Springer Nature Limited. In the HTML version of the Article originally published, Shy Shoham was mistakenly not denoted as a corresponding author; this has now been corrected. The PDF version was unaffected.

Background Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. Results As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. Conclusions Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy.

Background Differentiation between irritant and allergic skin reactions in epicutaneous patch testing is based largely on subjective clinical criteria, with the risk of high intraobserver and interobserver variability. Novel dermatological imaging using optoacoustic mesoscopy allows quantitative three-dimensional assessment of microvascular biomarkers.Objectives We investigated the potential of optoacoustic imaging to improve the precision of patch test evaluation.Methods Sixty-nine test reactions and 48 healthy skin sections in 52 patients with suspected type IV allergy were examined using raster-scan optoacoustic mesoscopy.Results We identified biomarkers from the optoacoustic images. Allergic reactions were associated with higher fragmentation of skin vasculature than irritant reactions (19.5 +/- 9.7 vs 14.3 +/- 3.7 fragments/100 pixels(2); P < .05), as well as lower ratio of low- to high-frequency acoustic signals (1.6 +/- 0.5 vs 2.0 +/- 0.6, P < .05). Allergic reactions graded "++" showed higher vessel fragmentation than reactions graded "+" (25.4 +/- 13.2 vs 17.1 +/- 6.5 fragments/100 pixels(2); P < .05). A linear model combining the biomarkers fragmentation and frequency ratio could differentiate allergic from irritant test reactions with an area under the receiving operator characteristic curve of 0.80 (95% confidence interval 0.64-0.91), reaching a sensitivity of 81% and specificity of 63%.Conclusions Optoacoustic mesoscopy shows potential to help in differentiating between allergic and irritant test reactions based on novel biomarkers that may reflect vasodilation, vessel tortuosity, and edema.

Die optoakustische Bildgebung ist eine neuartige Bildgebungsmodalität, welche auf der Aufzeichnung und Verarbeitung von Ultraschallsignalen basiert, die im Gewebe durch Absorption von Laserlicht entstehen. Die Methode vereint guten, auf Lichtabsorption beruhenden Kontrast mit hoher Eindringtiefe und ermöglicht sowohl morphologische als auch molekulare und funktionelle Bildgebung. Sie kann mit oder ohne Kontrastmittel eingesetzt werden und ist frei von ionisierender Strahlung. Darüber hinaus ist die Technik skalierbar und kann somit zur makroskopischen, mesoskopischen und mikroskopischen Bildgebung verwendet werden. In den letzten Jahren wurden eine Reihe verschiedener Systeme zur optoakustischen Bildgebung entwickelt und in vielfältigen Bereichen der präklinischen und klinischen Forschung eingesetzt. In der Dermatologie zeigten sich vielversprechende Anwendungsgebiete der Optoakustik insbesondere in der Untersuchung von Melanomen, Wächterlymphknoten und nicht-melanozytärem Hautkrebs sowie in der Charakterisierung von entzündlichen Hauterkrankungen. In diesem Review sollen die technischen Grundlagen der optoakustischen Bildgebung erläutert sowie der derzeitige Stand der Forschung hinsichtlich Anwendungsbereiche makroskopischer, mesoskopischer und mikroskopischer optoakustischer Systeme beschrieben und diskutiert werden.

Phototesting is used to assess individual sensitivity to ultraviolet (UV) radiation in order to determine adequate UV dosage for phototherapy1 . In the standard procedure, small skin areas are exposed to increasing doses of UV radiation. The lowest UV dose that induces a delineated erythema at 24±2 h after UV exposure defines the minimal erythema dose (MED)2 . Visual assessment is the gold standard for MED determination; however, it is prone to observer variability3 . Optical methods have been considered to quantify the magnitude of erythema response. However, they are limited by light scattering therefore high-resolution is restricted to depths of <200 μm resulting in unreliable measurements4,5 .

Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near-infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label-free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand-held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3-minute occlusion challenges. MSOT-recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT-extracted Hb-values showed an increase, while HbO(2)- and total blood volume (TBV)-values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO(2), Hb and TBV within the segmented muscle area. MSOT was found to be capable of label-free non-invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyOmethyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

Optoacoustic tomography systems have attained unprecedented volumetric imaging speeds, thus enabling insights into rapid biological dynamics and marking a milestone in the clinical translation of this modality. Fast imaging performance often comes at the cost of limited field-of-view, which may hinder potential applications looking at larger tissue volumes. The imaged field-of-view can potentially be expanded via scanning and using additional hardware to track the position of the imaging probe. However, this approach turns impractical for high-resolution volumetric scans performed in a freehand mode along arbitrary trajectories. We have developed an accurate framework for spatial compounding of time-lapse optoacoustic data. The method exploits the frequency-domain properties of vascular networks in optoacoustic images and estimates the relative motion and orientation of the imaging probe. This allows rapidly combining sequential volumetric frames into large area scans without additional tracking hardware. The approach is universally applicable for compounding volumetric data acquired with calibrated scanning systems but also in a freehand mode with up to six degrees of freedom. Robust performance is demonstrated for whole-body mouse imaging with spiral volumetric optoacoustic tomography and for freehand visualization of vascular networks in humans using volumetric imaging probes. The newly introduced capability for angiographic observations at multiple spatial and temporal scales is expected to greatly facilitate the use of optoacoustic imaging technology in pre-clinical research and clinical diagnostics. The technique can equally benefit other biomedical imaging modalities, such as scanning fluorescence microscopy, optical coherence tomography or ultrasonography, thus optimizing their trade-offs between fast imaging performance and field-of-view.

Development of imaging methods capable of furnishing tumor-specific morphological, functional, and molecular information is paramount for early diagnosis, staging, and treatment of breast cancer. Ultrasound (US) and optoacoustic (OA) imaging methods exhibit excellent traits for tumor imaging in terms of fast imaging speed, ease of use, excellent contrast, and lack of ionizing radiation. Here, we demonstrate simultaneous tomographic whole body imaging of optical absorption, US reflectivity, and speed of sound (SoS) in living mice. In vivo studies of 4T1 breast cancer xenografts models revealed synergistic and complementary value of the hybrid imaging approach for characterizing mammary tumors. While neovasculature surrounding the tumor areas were observed based on the vascular anatomy contrast provided by the OA data, the tumor boundaries could be discerned by segmenting hypoechoic structures in pulse-echo US images. Tumor delineation was further facilitated by enhancing the contrast and spatial resolution of the SoS maps with a full-wave inversion method. The malignant lesions could thus be distinguished from other hypoechoic regions based on the average SoS values. The reported findings corroborate the strong potential of the hybrid imaging approach for advancing cancer research in small animal models and fostering development of new clinical diagnostic approaches.

Optoacoustic imaging: Revealing the details of tumour patterns A technique that can image the entire tumour volume with high resolution may help oncologists optimize specific treatments for breast cancer. Jiao Li (Tianjin University, China), Vasilis Ntziachristos (Technical University of Munich, Germany), and colleagues have designed a multispectral optoacoustic mesoscope (MSOM) that illuminates millimetre-sized tumours with laser light of various wavelengths, and detects the ultrasound waves generated by internal absorbers, such as haemoglobin, or external nanoparticle probes. By reconstructing the ultrasound signals over multiple frequencies, the team produced 3D images of features that included the vascular network of a tumour with micrometre-scale detail. Experiments with live mice demonstrated that specific tumours could be identified through differences in spatial patterns, such as altered oxygen levels between tumour cores and peripheries. The study highlights the power of MSOM as a tool for preclinical cancer studies.The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 mu m throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacousticmethod to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO(2)) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. Aninitial sharp drop in tumor sO(2) andTHb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today.Significance: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.

The Hessian-based Frangi vesselness filter is commonly used to enhance vasculature in optoacoustic (photoacoustic) images, but its accuracy and limitations have never been rigorously assessed. Here we validate the ability of the filter to enhance vessel-like structures in phantoms, and we introduce an experimental approach that uses measurements before and after the administration of gold nanorods (AuNRs) to examine filter performance in vivo. We evaluate the influence of contrast, filter scales, angular tomographic coverage, out-of-plane signals and light fluence on image quality, and gain insight into the performance of the filter. We observe the generation of artifactual structures that can be misinterpreted as vessels and provide recommendations to ensure appropriate use of Frangi and other vesselness filters and avoid misinterpretation of post-processed optoacoustic images.

The fidelity and quality of reconstructed images in optoacoustic mesoscopy (OPAM) can be significantly improved by considering the spatial impulse response (SIR) of the employed focused transducer into reconstruction. However, the traditional method fully taking the SIR into account can hardly meet the data-intensive requirements of high resolution OPAM because of excessive memory and time consumption. Herein, a modified back-projection method using a space-variant filter for full-frequency correction of the SIR is presented, and applied to the OPAM system with a sphere-focused transducer. The proposed method can readily manage the large datasets of the OPAM and effectively reduce the extra time consumption. The performance of the proposed method is showcased by simulations and experiments of phantoms and biological tissue. The results demonstrate that the modified back-projection method exhibits better image fidelity, resolution and contrast compared to the common and weighted back-projection methods that are not or not fully accounting for the SIR.

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson's correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Optoacoustic (photoacoustic) mesoscopy offers unique capabilities in skin imaging and resolves skin features associated with detection, diagnosis, and management of disease. A critical first step in the quantitative analysis of clinical optoacoustic images is to identify the skin surface in a rapid, reliable, and automated manner. Nevertheless, most common edge- and surface-detection algorithms cannot reliably detect the skin surface on 3D raster-scan optoacoustic mesoscopy (RSOM) images, due to discontinuities and diffuse interfaces in the image. We present herein a novel dynamic programming approach that extracts the skin boundary as a 2D surface in one single step, as opposed to consecutive extraction of several independent 1D contours. A domain-specific energy function is introduced, taking into account the properties of volumetric optoacoustic mesoscopy images. The accuracy of the proposed method is validated on scans of the volar forearm of 19 volunteers with different skin complexions, for which the skin surface has been traced manually to provide a reference. In addition, the robustness and the limitations of the method are demonstrated on data where the skin boundaries are low-contrast or ill-defined. The automatic skin surface detection method can improve the speed and accuracy in the analysis of quantitative features seen on the RSOM images and accelerate the clinical translation of the technique. Our method can likely be extended to identify other types of surfaces in the RSOM and other imaging modalities.

The autoimmune disease systemic sclerosis (SSc) causes microvascular changes that can be easily observed cutaneously at the finger nailfold. Optoacoustic imaging (OAI), a combination of optical and ultrasound imaging, specifically raster-scanning optoacoustic mesoscopy (RSOM), offers a noninvasive high-resolution 3D visualization of capillaries allowing for a better view of microvascular changes and an extraction of volumetric measures. In this study, nailfold capillaries of patients with SSc and healthy controls are imaged and compared with each other for the first time using OAI. The nailfolds of 23 patients with SSc and 19 controls were imaged using RSOM. The acquired images were qualitatively compared to images from state-of-the-art imaging tools for SSc, dermoscopy and high magnification capillaroscopy. The vascular volume in the nailfold capillaries were computed from the RSOM images. The vascular volumes differ significantly between both cohorts (0.216 +/- 0.085 mm(3) and 0.337 +/- 0.110 mm(3); p < 0.0005). In addition, an artificial neural network was trained to automatically differentiate nailfold images from both cohorts to further assess whether OAI is sensitive enough to visualize anatomical differences in the capillaries between the two cohorts. Using transfer learning, the model classifies images with an area under the ROC curve of 0.897, and a sensitivity of 0.783 and specificity of 0.895. In conclusion, this study demonstrates the capabilities of RSOM as an imaging tool for SSc and establishes it as a modality that facilitates more in-depth studies into the disease mechanisms and progression.

Label free imaging of oxygenation distribution in tissues is highly desired in numerous biomedical applications, but is still elusive, in particular in sub-epidermal measurements. Eigenspectra multispectral optoacoustic tomography (eMSOT) and its Bayesian-based implementation have been introduced to offer accurate label-free blood oxygen saturation (sO(2)) maps in tissues. The method uses the eigenspectra model of light fluence in tissue to account for the spectral changes due to the wavelength dependent attenuation of light with tissue depth. eMSOT relies on the solution of an inverse problem bounded by a number of ad hoc hand-engineered constraints. Despite the quantitative advantage offered by eMSOT, both the non-convex nature of the optimization problem and the possible sub-optimality of the constraints may lead to reduced accuracy. We present herein a neural network architecture that is able to learn how to solve the inverse problem of eMSOT by directly regressing from a set of input spectra to the desired fluence values. The architecture is composed of a combination of recurrent and convolutional layers and uses both spectral and spatial features for inference. We train an ensemble of such networks using solely simulated data and demonstrate how this approach can improve the accuracy of sO(2) computation over the original eMSOT, not only in simulations but also in experimental datasets obtained from blood phantoms and small animals (mice) in vivo. The use of a deep-learning approach in optoacoustic sO(2) imaging is confirmed herein for the first time on ground truth sO(2) values experimentally obtained in vivo and ex vivo.

Researchers around the world join forces to reconstruct the molecular processes of the virus-host interactions aiming to combat the cause of the ongoing pandemic.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

To date, the vast majority of intra-vital neuroimaging systems applied in clinic and diagnostics is stationary with a rigid scanning element, requires specialized facilities and costly infrastructure. Here, we describe a simple yet radical approach for optoacoustic (photoacoustic) brain imaging in vivo using a light-weight handheld probe. It enables multispectral video-rate visualization of hemoglobin gradient changes in the cortex of adult rats induced by whisker and forelimb sensory inputs, as well as by optogenetic stimulation of intra-cortical connections. With superb penetration and molecular specificity, described here in method holds major promises for future applications in research, routine ambulatory neuroimaging, and diagnostics.

The recently developed optoacoustic tomography systems have attained volumetric frame rates exceeding 100 Hz, thus opening up new venues for studying previously invisible biological dynamics. Further gains in temporal resolution can potentially be achieved via partial data acquisition, though a priori knowledge on the acquired data is essential for rendering accurate reconstructions using compressed sensing approaches. In this work, we suggest a machine learning method based on principal component analysis for high-frame-rate volumetric cardiac imaging using only a few tomographic optoacoustic projections. The method is particularly effective for discerning periodic motion, as demonstrated herein by non-invasive imaging of a beating mouse heart. A training phase enables efficiently compressing the heart motion information, which is subsequently used as prior information for image reconstruction from sparse sampling at a higher frame rate. It is shown that image quality is preserved with a 64-fold reduction in the data flow. We demonstrate that, under certain conditions, the volumetric motion could effectively be captured by relying on time-resolved data from a single optoacoustic detector. Feasibility of capturing transient (non-periodic) events not registered in the training phase is further demonstrated by visualizing perfusion of a contrast agent in vivo. The suggested approach can be used to significantly boost the temporal resolution of optoacoustic imaging and facilitate development of more affordable and data efficient systems.

Understanding the mechanisms of cardiac disorders largely depends on availability of multi-dimensional and multiparametric imaging methods capable of quantitative assessment of cardiac morphology and function. The imaging modalities commonly employed in cardiac research, such as ultrasonography and magnetic resonance imaging, are lacking sufficient contrast and/or spatio-temporal resolution in 3D in order to reveal the multi-scale nature of rapid electromechanical activity in a beating heart. Our recently developed volumetric optoacoustic tomography (VOT) platform offers versatile observations of the heart function with rich optical contrast at otherwise unattainable temporal and spatial resolutions. Herein, we further advance the imaging performance by developing compressed acquisition scheme to boost the temporal resolution of VOT into the kilohertz range, thus enabling 3D mapping of electromechanical wave propagation in the heart. Experiments in isolated mouse hearts were performed by exciting the entire imaged tissue volume with nanosecond-duration laser pulses at 1 kHz repetition rate pulse operating at 532 nm and sparse tomographic signal sampling using a custom-made 512-element spherical matrix ultrasound array. By analyzing the strain maps obtained from the rapid VOT image sequence, it was possible to quantify the phase velocity of the electromechanical cardiac waves, in good agreement with previously reported values.

Laser ablation (LA) is a promising approach for minimally invasive cancer treatments. Its in vivo applicability is often impeded by the lack of efficient monitoring tools that can help to minimize collateral tissue damage and aid in determining the optimal treatment end-points. We have devised a new, to the best of our knowledge, hybrid LA approach combining simultaneous volumetric optoacoustic (OA) imaging to monitor the lesion progression accurately in real time and 3D. Time-lapse imaging of laser ablation of solid tumors was performed in a murine breast cancer model in vivo by irradiation of subcutaneous tumors with a 100 mJ short-pulsed (similar to 5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. Local changes in the OA signal intensity ascribed to structural alterations in the tumor vasculature were clearly observed, while the OA volumetric projections recorded in vivo appeared to correlate with cross sections of the excised tumors.

We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.Mid-infrared optoacoustic microscopy enables label-free, bond-selective imaging in living cells

Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 degrees C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 degrees C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.

SIGNIFICANCE: Expanded use of fluorescence-guided surgery with devices approved for use with indocyanine green (ICG) has led to a range of commercial systems available. There is a compelling need to be able to independently characterize system performance and allow for cross-system comparisons. AIM: The goal of this work is to expand on previous proposed fluorescence imaging standard designs to develop a long-term stable phantom that spectrally matches ICG characteristics and utilizes 3D printing technology for incorporating tissue-equivalent materials. APPROACH: A batch of test targets was created to assess ICG concentration sensitivity in the 0.3- to 1000-nM range, tissue-equivalent depth sensitivity down to 6 mm, and spatial resolution with a USAF test chart. Comparisons were completed with a range of systems that have significantly different imaging capabilities and applications, including the Li-Cor® Odyssey, Li-Cor® Pearl, PerkinElmer® Solaris, and Stryker® Spy Elite. RESULTS: Imaging of the ICG-matching phantoms with all four commercially available systems showed the ability to benchmark system performance and allow for cross-system comparisons. The fluorescence tests were able to assess differences in the detectable concentrations of ICG with sensitivity differences >10× for preclinical and clinical systems. Furthermore, the tests successfully assessed system differences in the depth-signal decay rate, as well as resolution performance and image artifacts. The manufacturing variations, photostability, and mechanical design of the tests showed promise in providing long-term stable standards for fluorescence imaging. CONCLUSIONS: The presented ICG-matching phantom provides a major step toward standardizing performance characterization and cross-system comparisons for devices approved for use with ICG. The developed hybrid manufacturing platform can incorporate long-term stable fluorescing agents with 3D printed tissue-equivalent material. Further, long-term testing of the phantom and refinements to the manufacturing process are necessary for future implementation as a widely adopted fluorescence imaging standard.

Optical microscopy improves in resolution and signal-to-noise ratio by correcting for the system's point spread function; a measure of how a point source is resolved, typically determined by imaging nanospheres. Optical-resolution optoacoustic (photoacoustic) microscopy could be similarly corrected, especially to account for the spatially-dependent signal distortions induced by the acoustic detection and the time-resolved and bi-polar nature of optoacoustic signals. Correction algorithms must therefore include the spatial dependence of signals' origins and profiles in time, i.e. the four-dimensional total impulse response (TIR). However, such corrections have been so far impeded by a lack of efficient TIR-characterization methods. We introduce high-quality TIR determination based on spatially-distributed optoacoustic point sources (SOAPs), produced by scanning an optical focus on an axially-translatable 250nm gold layer. Using a spatially-dependent TIR-correction improves the signal-to-noise ratio by >10dB and the axial resolution by similar to 30%. This accomplishment displays a new performance paradigm for optoacoustic microscopy.

Loss mechanisms in fluid heating of cobalt ferrite (CFO) nanoparticles and CFO-Pd heterodimer colloidal suspensions are investigated as a function of particle size, fluid concentration and magnetic field amplitude. The specific absorption rate (SAR) is found to vary with increasing particle size due to a change in dominant heating mechanism from susceptibility to hysteresis and frictional loss. The maximum SAR is obtained for particle diameters of 11-15 nm as a result of synergistic contributions of susceptibility loss, including Néel and Brownian relaxation and especially hysteresis loss, thereby validating the applicability of linear response theory to superparamagnetic CFO nanoparticles. Our results show that the ferrofluid concentration and magnetic field amplitude alter interparticle interactions and associated heating efficiency. The SAR of the CFO nanoparticles could be maximized by adjusting the synthesis parameters. Despite the paramagnetic properties of individual palladium nanoparticles, CFO-Pd heterodimer suspensions were observed to have surprisingly improved magnetization as well as SAR values, when compared with CFO ferrofluids. This difference is attributed to interfacial interactions between the magnetic moments of paramagnetic Pd and superparamagnetic/ferrimagnetic CFO. SAR values measured from CFO-Pd heterodimer suspensions were found to be 47-52 W gFerrite-1, which is up to a factor of two higher than the SAR values of commercially available ferrofluids, demonstrating their potential as efficient heat mediators. Our results provide insight into the utilization of CFO-Pd heterodimer suspensions as potential nanoplatforms for diagnostic and therapeutic biomedical applications, e.g., in cancer hyperthermia, cryopreserved tissue warming, thermoablative therapy, drug delivery and bioimaging.

The widely available silicon-on-insulator technology is used to develop a miniaturized ultrasound detector, which is 200 times smaller than the wavelengths of sound that it can detect.Ultrasound detectors use high-frequency sound waves to image objects and measure distances, but the resolution of these readings is limited by the physical dimensions of the detecting element. Point-like broadband ultrasound detection can greatly increase the resolution of ultrasonography and optoacoustic (photoacoustic) imaging(1,2), but current ultrasound detectors, such as those used for medical imaging, cannot be miniaturized sufficiently. Piezoelectric transducers lose sensitivity quadratically with size reduction(3), and optical microring resonators(4)and Fabry-Perot etalons(5)cannot adequately confine light to dimensions smaller than about 50 micrometres. Micromachining methods have been used to generate arrays of capacitive(6)and piezoelectric(7)transducers, but with bandwidths of only a few megahertz and dimensions exceeding 70 micrometres. Here we use the widely available silicon-on-insulator technology to develop a miniaturized ultrasound detector, with a sensing area of only 220 nanometres by 500 nanometres. The silicon-on-insulator-based optical resonator design provides per-area sensitivity that is 1,000 times higher than that of microring resonators and 100,000,000 times better than that of piezoelectric detectors. Our design also enables an ultrawide detection bandwidth, reaching 230 megahertz at -6 decibels. In addition to making the detectors suitable for manufacture in very dense arrays, we show that the submicrometre sensing area enables super-resolution detection and imaging performance. We demonstrate imaging of features 50 times smaller than the wavelength of ultrasound detected. Our detector enables ultra-miniaturization of ultrasound readings, enabling ultrasound imaging at a resolution comparable to that achieved with optical microscopy, and potentially enabling the development of very dense ultrasound arrays on a silicon chip.

Optical sensors developed for the assessment of oxygen in tissue microvasculature, such as those based on near-infrared spectroscopy, are limited in application by light scattering. Optoacoustic methods are insensitive to light scattering, and therefore, they can provide higher specificity and accuracy when quantifying local vascular oxygenation. However, currently, to the best of our knowledge, there is no low-cost, single point, optoacoustic sensor for the dedicated measurement of oxygen saturation in tissue microvasculature. This work introduces a spectroscopic optoacoustic sensor (SPOAS) for the non-invasive measurement of local vascular oxygenation in real time. SPOAS employs continuous wave laser diodes and measures at a single point, which makes it low-cost and portable. The SPOAS performance was benchmarked using blood phantoms, and it showed excellent linear correlation (R2 = 0.98) with a blood gas analyzer. Subsequent measurements of local vascular oxygenation in living mice during an oxygen stress test correlated well with simultaneous readings from a reference instrument.

Cerebrovascular imaging of rodents is one of the trending applications of optoacoustics aimed at studying brain activity and pathology. Imaging of deep brain structures is often hindered by sub-optimal arrangement of the light delivery and acoustic detection systems. In our work we revisit the physics behind opto-acoustic signal generation for theoretical evaluation of optimal laser wavelengths to perform cerebrovascular optoacoustic angiography of rodents beyond the penetration barriers imposed by light diffusion in highly scattering and absorbing brain tissues. A comprehensive model based on diffusion approximation was developed to simulate optoacoustic signal generation using optical and acoustic parameters closely mimicking a typical murine brain. The model revealed three characteristic wavelength ranges in the visible and near-infrared spectra optimally suited for imaging cerebral vasculature of different size and depth. The theoretical conclusions are confirmed by numerical simulations while in vivo imaging experiments further validated the ability to accurately resolve brain vasculature at depths ranging between 0.7 and 7 mm.

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.

Photoacoustic (PA) imaging is a hybrid imaging technique that can provide both structural and functional information of biological tissues. Due to limited permissible laser energy deposited on tissues, highly sensitive PA imaging is required. Here, we developed a 20 MHz lead zirconium titanate (PZT) transducer (1.5 mm x 3 mm) with front-end amplifier circuits for local signal processing to achieve sensitivity enhanced PA imaging. The electrical and acoustic performance was characterized. Experiments on phantoms and chicken breast tissue were conducted to validate the imaging performance. The fabricated prototype shows a bandwidth of 63% and achieves a noise equivalent pressure (NEP) of 0.24 mPa/root Hz and a receiving sensitivity of 62.1 mu V/Pa at 20 MHz without degradation of the bandwidth. PA imaging of wire phantoms demonstrates that the prototype is capable of improving the detection sensitivity by 10 dB compared with the traditional transducer without integrated amplifier. In addition, in vitro experiments on chicken breast tissue show that structures could be imaged with enhanced contrast using the prototype and the imaging depth range was improved by 1 mm. These results demonstrate that the transducer with an integrated front-end amplifier enables highly sensitive PA imaging with improved penetration depth. The proposed method holds the potential for visualization of deep tissue structures and enhanced detection of weak physiological changes.

Using the same ultrasound detector, hybrid optoacoustic-ultrasound (OPUS) imaging provides concurrent scans of tissue slices or volumes and visualizes complementary sound- and light-based contrast at similar resolutions. In addition to the benefit of hybrid contrast, spatial co-registration enables images from one modality to be employed as prior information for improving an aspect of the performance of the other modality. We consider herein a handheld OPUS system and utilize structural information from ultrasound images to guide regional Laplacian regularization-based reconstruction of optoacoustic images. Using phantoms and data from OPUS scans of human radial and carotid arteries, we show that ultrasound-driven optoacoustic inversion reduces limited-view artefacts and improves image contrast. In phantoms, prior-integrated reconstruction leads to a 50 % higher contrast-to-noise ratio (CNR) of the image than standard reconstruction, and a 17 % higher structural similarity (SSIM) index. In clinical data, prior-integrated reconstruction detects deep-seated radial arteries with higher CNR than the standard method at three different depths. In this way, the prior-integrated method offers unique insights into atherosclerotic carotid plaques in humans (with p < 0.01 between patients and healthy volunteers), potentially paving the way for new abilities in vascular imaging and more generally in optoacoustic imaging.

Raster-scan optoacoustic mesoscopy (RSOM) offers high-resolution non-invasive insights into skin pathophysiology, which holds promise for disease diagnosis and monitoring in dermatology and other fields. However, RSOM is quite vulnerable to verticalmotion of the skin, which can depend on the part of the body being imaged. Motion correction algorithms have already been proposed, but they are not fully automated, they depend on anatomical segmentation pre-processing steps that might not be performed successfully, and they are not site-specific. Here, we determined for the first time the magnitude of themicrometric vertical skin displacements at different sites on the body that affect RSOM. The quantification of motion allowed us to develop a site-specific correction algorithm. The algorithm is fully automated and does not need prior anatomical information. We found that the magnitude of the vertical motion depends strongly on the site of imaging and is caused by breathing, heart beating, and arterial pulsation. The developed algorithm resulted in more than 2-fold improvement in the signal-to-noise ratio of the reconstructed images at every site tested. Proposing an effective automatedmotion correction algorithm paves the way for realizing the full clinical potential of RSOM.

This publisher’s note contains corrections to Opt. Lett. 39, 6297 (2014).

Fluorescence imaging opens new possibilities for intraoperative guidance and early cancer detection, in particular when using agents that target specific disease features. Nevertheless, photon scattering in tissue degrades image quality and leads to ambiguity in fluorescence image interpretation and challenges clinical translation. We introduce the concept of capturing the spatially-dependent impulse response of an image and investigate Spatially Adaptive Impulse Response Correction (SAIRC), a method that is proposed for improving the accuracy and sensitivity achieved. Unlike classical methods that presume a homogeneous spatial distribution of optical properties in tissue, SAIRC explicitly measures the optical heterogeneity in tissues. This information allows, for the first time, the application of spatially-dependent deconvolution to correct the fluorescence images captured in relation to their modification by photon scatter. Using experimental measurements from phantoms and animals, we investigate the improvement in resolution and quantification over non-corrected images. We discuss how the proposed method is essential for maximizing the performance of fluorescence molecular imaging in the clinic.

Photoacoustic imaging (or optoacoustic imaging) is an upcoming biomedical imaging modality availing the benefits of optical resolution and acoustic depth of penetration. With its capacity to offer structural, functional, molecular and kinetic information making use of either endogenous contrast agents like hemoglobin, lipid, melanin and water or a variety of exogenous contrast agents or both, PAI has demonstrated promising potential in a wide range of preclinical and clinical applications. This review provides an overview of the rapidly expanding clinical applications of photoacoustic imaging including breast imaging, dermatologic imaging, vascular imaging, carotid artery imaging, musculoskeletal imaging, gastrointestinal imaging and adipose tissue imaging and the future directives utilizing different configurations of photoacoustic imaging. Particular emphasis is placed on investigations performed on human or human specimens.

Despite the importance of placental function in embryonic development, it remains poorly understood and challenging to characterize, primarily due to the lack of non-invasive imaging tools capable of monitoring placental and foetal oxygenation and perfusion parameters during pregnancy. We developed an optoacoustic tomography approach for real-time imaging through entire ~4 cm cross-sections of pregnant mice. Functional changes in both maternal and embryo regions were studied at different gestation days when subjected to an oxygen breathing challenge and perfusion with indocyanine green. Structural phenotyping of the cross-sectional scans highlighted different internal organs, whereas multi-wavelength acquisitions enabled non-invasive label-free spectroscopic assessment of blood-oxygenation parameters in foeto-placental regions, rendering a strong correlation with the amount of oxygen administered. Likewise, the placental function in protecting the embryo from extrinsically administered agents was substantiated. The proposed methodology may potentially further serve as a probing mechanism to appraise embryo development during pregnancy in the clinical setting.

Detailed assessment of skin conditions or the efficacy of skin treatments could greatly benefit from noninvasively assessing the distribution of cutaneous and subcutaneous structures and biomolecules. We considered ultrawideband raster scan optoacoustic mesoscopy with an extended wavelength range from visible to short-wave infrared and observed previously unseen high-resolution images of lipids colocalized with water, melanin, and hemoglobin distribution in human skin. Based on this contrast, the technique resolves subcutaneous fat, the pilosebaceous unit with complete hair strand and bulb, dermal microvasculature, and epidermal structures. We further visualize melanoidins that form via the Maillard reaction in the ultrathin stratum corneum layer, analyze their absorption spectrum, and separate them from the melanin layer. The suggested method may allow novel interrogation of skin conditions, possibly impacting diagnostics and medical and cosmetic treatments.

Optoacoustic imaging is a highly scalable and versatile method that can be used for optical resolution (OR) microscopy applications at superficial depth yet can be adapted for tomographic imaging with ultrasonic resolution at centimeter penetration scales. However, imaging speed of the commonly employed scanning-based microscopy methods is slow as far as concerned with acquisition of volumetric data. Herein, we propose a new approach using multifocal structured illumination in conjunction with a spherical matrix ultrasonic array detection to achieve fast volumetric optoacoustic imaging in both optical and acoustic resolution modes. In our approach, the laser beam is raster scanned by an acousto-optic deflector running at hundred hertz scanning rate with the beam then split into hundreds of mini-beams by a beamsplitting grating, which are subsequently focused by a condensing lens to generate multifocal structured illumination. Phantom experimental results show that 10 x 10 x 5 cm 3 volumetric imaging can be accomplished with spatial resolution around 29 μm. We believe by further speeding up the data acquisition in the further, the system will be operated in full power, making it possible to study functional, kinetic and metabolic processes across multiple penetration scales.

Intravenously administered liposomes and other nano-sized particles are known to passively accumulate in solid tumors via the enhanced permeability and retention (EPR) effect, which is extensively explored toward the improvement of diagnosis and drug delivery in oncology. Agent extravasation into tumors is often hampered by the mononuclear phagocytic and renal systems, which sequester and/or eliminate most of the nanoparticles from the body. Dynamic imaging of the tumor microcirculation and bolus perfusion can thus facilitate optimization of the nanoparticle delivery. When it comes to non-invasive visualization of rapid biological dynamics in whole tumors, the currently available preclinical imaging modalities are commonly limited by shallow penetration, lack of suitable contrast or otherwise insufficient spatial or temporal resolution. Herein, we demonstrate the unique capabilities of a combined epi-fluorescence and optoacoustic tomography (FLOT) system for characterizing contrast agent dynamics in orthotopic breast tumors in mice. A liposomal indocyanine green (Lipo-ICG) preparation was administered intravenously with the time-lapse data continuously acquired during and after the injection procedure. In addition to the highly sensitive detection of the fluorescence agent by the epi-fluorescence modality, the volumetric multi-spectral optoacoustic tomography readings further enabled resolving deep-seated vascular structures with high spatial resolution and hence provided accurate readings of the dynamic bio-distribution of nanoparticles in the entire tumor in 3D. The synergetic combination of the two modalities can become a powerful tool in cancer research and potentially aid the diagnosis, staging and treatment guidance of certain types of cancer in the clinical setting.

Biomedical optoacoustic (photoacoustic) imaging is generally performed with short laser pulses with durations in the order of a few nanoseconds. This enables maximizing the conversion efficiency of optical energy into acoustic (ultrasound) energy when light is absorbed in biological tissues. The generated ultrasound waves are generally very broadband, with typical frequency spectra ranging from tens of kHz to tens of MHz. Most ultrasound transducers used for the detection of optoacoustic signals have a finite detection bandwidth, in a way that they are not optimal for the acquisition of optoacoustic signals generated with a single pulse. In this work, we analyze a narrowband excitation approach based on a tone-burst consisting of multiple equally-delayed short pulses. We compare the power spectral density of the signals generated with a tone-burst with those generated with a single pulse having the same energy under safety exposure limits. We further analyze the performance of tone-burst excitation when non-linear effects take place. Specifically, we consider non-linearities associated to temperature increase and to optical absorption saturation.

A myriad of optoacoustic imaging systems based on scanning focused ultrasound transducers or on tomographic acquisition of pressure signals are available. In all cases, image formation is based on the assumption that ultrasound waves undergo no distortion and propagate with constant velocity across the sample and coupling medium (typically water). Thereby, ultrasound time-of-flight readings from multiple time-resolved signals are required to form an image. Acoustic scattering is known to cause distortion in the signals and is generally to be avoided. In this work, we exploit acoustic scattering to physically encode the position of optical absorbers in the acquired time-resolved signals and hence reduce the amount of data required to reconstruct an image. This new approach was experimentally tested with an array of cylindrically-focused transducers, where a cluster of acoustic scatterers was introduced in the ultrasound propagating path between the sample and the array elements. Ultrasound transmission was calibrated by raster scanning a lightabsorbing particle across the effective field of view. The acquired calibrating signals were then used for the development of a regularized model-based iterative algorithm that enabled reconstructing an image from a relatively low number of optoacoustic signals. A relatively short acquisition time window was needed to capture the entire optoacoustic field, which demonstrates the high signal compression efficiency. The feasibility to form an image with a relatively low number of signals is expected to play a major role in the development of a new generation of optoacoustic imaging systems.

Optoacoustic image formation is conventionally based upon ultrasound time-of-flight readings from multiple detection positions. Herein, we exploit acoustic scattering to physically encode the position of optical absorbers in the acquired signals, thus reducing the amount of data required to reconstruct an image from a single waveform. This concept is experimentally tested by including a random distribution of scatterers between the sample and an ultrasound detector array. Ultrasound transmission through a randomized scattering medium was calibrated by raster scanning a light-absorbing microparticle across a Cartesian grid. Image reconstruction from a single time-resolved signal was then enabled with a regularized model-based iterative algorithm relying on the calibration signals. The signal compression efficiency is facilitated by the relatively short acquisition time window needed to capture the entire scattered wave field. The demonstrated feasibility to form an image using a single recorded optoacoustic waveform paves a way to the development of faster and affordable optoacoustic imaging systems.

One main limitation of established neuroimaging methods is the inability to directly visualize large-scale neural dynamics in whole mammalian brains at subsecond speeds. Optoacoustic imaging has advanced in recent years to provide unique advantages for real-time deep-tissue observations, which have been exploited for three-dimensional imaging of both cerebral hemodynamic parameters and direct calcium activity in rodents. Due to a lack of suitable calcium indicators excitable in the near-infrared window, optoacoustic imaging of neuronal activity at deep-seated areas of the mammalian brain has been impeded by the strong absorption of blood in the visible range of the light spectrum. To overcome this, we have developed and validated an intracardially perfused mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f that closely resembles in vivo conditions. By overcoming the limitations of hemoglobin-based light absorption, this new technique was used to observe stimulus-evoked calcium dynamics in the brain at penetration depths and spatio-temporal resolution scales not attainable with existing neuroimaging techniques.

Model-based reconstruction techniques have been successfully implemented in optoacoustic tomography and acoustic-resolution microscopy to retrieve improved image quality over delay-and-sum methods. In scanning optical resolution optoacoustic microscopy (OR-OAM), no reconstruction methods are employed while post- processing is usually limited to basic frequency filtering and envelope extraction with the Hilbert transform. This results in considerable deterioration of the acoustically-determined resolution in the axial (depth) direction. In addition, when OR-OAM is used for transcranial mouse brain imaging, the skull strongly attenuates high ultrasonic frequencies and induces reverberations, which need to be accounted for during the reconstruction process to avoid image distortions and further deterioration of the axial resolution. Here we show a basic implementation of a model-based reconstruction to increase the axial resolution in OR-OAM. The model matrix is calculated using Field II for free field conditions, taking into account the shape and bandwidth of the spherically focused transducer. Assuming the confinement of the optoacoustic sources within the limits of the optical focus, one may calculate the model matrix by assuming a line source of small absorbing spheres equal in size to the optical beam. In addition, a plate model used in the recently reported virtual-craniotomy deconvolution algorithm is incorporated into the model matrix to tackle the transcranial acoustic transmission problem. The free-field model-based results are compared against the plate model for transcranial brain data obtained in-vivo.

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.

Fluorescence-guided intervention is increasingly considered for real-time intra-operative oncological applications. Herein we propose a novel composite phantom for standardization and quality control, which could serve as a framework toward good clinical practices.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with existing neuroimaging methods having limited capacity when it comes to imaging large tissue volumes at high speeds. Optoacoustic imaging has been shown to be capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep within the mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as a lack of activity labels excitable in the near-infrared window. We have developed and validated an isolated whole mouse brain preparation labeled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions. An optoacoustic imaging system coupled to a superfusion system was further designed and used for rapid volumetric monitoring of stimulus-evoked calcium dynamics in the brain. These new imaging setup and isolated preparation's protocols and characteristics are described here in detail. Our new technique captures calcium fluxes as true three-dimensional information across the entire brain with temporal resolution of 10 ms and spatial resolution of 150 μm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with any existing neuroimaging techniques.

Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMVMel) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMVMel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMVMel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMVMel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS-fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS-fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS-fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration.

Detection and monitoring of esophageal cancer severity require an imaging technique sensitive enough to detect early pathological changes in the esophagus and capable of analyzing the esophagus over 360 degrees in a non-invasive manner. Optoacoustic endoscopy (COE) has been shown to resolve superficial vascular structure of the esophageal lumen in rats and rabbits using catheter-type probes. Although these systems can work well in small animals, they are unsuitable for larger lumens with thicker walls as required for human esophageal screening, due to their lack of position stability along the full organ circumference, sub-optimal acoustic coupling and limited signal-to-noise ratio (SNR). In this work, we introduce a novel capsule COE system that provides high-quality 360 degrees images of the entire lumen, specifically designed for typical dimensions of human esophagus. The pill-shaped encapsulated probe consists of a novel and highly sensitive ultrasound transducer fitted with an integrated miniature pre-amplifier, which increases SNR of 10 dB by minimizing artifacts during signal transmission compared to the configuration without the preamplifier. The scanner rotates helically around the central axis of the probe to capture three-dimensional images with uniform quality. We demonstrate for the first time ex vivo volumetric vascular network images to a depth of 2 mm in swine esophageal lining using COE. Vascular information can be resolved within the mucosa and submucosa layers as confirmed by histology of samples stained with hematoxylin and eosin and with antibody against vascular marker CD31. COE creates new opportunities for optoacoustic screening of esophageal cancer in humans.

In recent years, several non-invasive imaging methods have been introduced to facilitate diagnostics and therapy monitoring in dermatology. The microscopic imaging methods are restricted in their penetration depth, while the mesoscopic methods probe deeper but provide only morphological, not functional, information. 'Raster-scan optoacoustic mesoscopy' (RSOM), an emerging new imaging technique, combines deep penetration with contrast based on light absorption, which provides morphological, molecular and functional information. Here, we compare the capabilities and limitations of currently available dermatological imaging methods and highlight the principles and unique abilities of RSOM. We illustrate the clinical potential of RSOM, in particular for non-invasive diagnosis and monitoring of inflammatory and oncological skin diseases.

Background: Multispectral optical imaging has the capability of resoling hemoglobin, lipid,and water. Volumetric multispectral optoacoustic tomography (MSOT) is a hybrid imaging technique that provides a unique combination of functional and molecular contrast with real-time handheld imaging.Purpose: To investigate whether volumetric MSOT can provide real-time assessment of the anatomic and functional status of the human carotid artery bifurcation nonoinvasively.Materials and Methods: Imaging of healthy volunteers (n = 16) was performed with a custom-designed handheld volumetric MSOT scanner capable. of high-spatial-resolution (approximately 200 mu m) and real-time (10 volumes/sec) three-dimensional imaging, while further providing spectrosccopic capacity through fast tuning of the excitation light wavelength. For comparison and anatomic cross-validation, volunteers were also scanned with clinical B-mode US.Results: Volumetric MSOT achieved real-time imaging and characterization of the entire carotid bifurcation area across three dimensions simultaneously captured in a single volumetric image frame. Analysis of the acquired data further showed that a higher contrast-to-noise ratio can be achieved for wavelengths corresponding to a high optical absorption of oxygenated hemoglobin.Conclusion: The human carotid artery was visualized by using handheld volumetric multispectral optoacoustic tomography. This imaging approach is less prone to motion artifacts than are the conventional clinical imaging methods, holding promise for providing additional image-based biomarkers for noninvasive label-free assesment of carotid artery disease. (C) RSNA, 2019

Chronic hypoxia in pulmonary diseases is known to have a severe negative impact on heart function, including right heart hypertrophy, increased workload on the heart and arrhythmia. Yet, the direct effect of the chronic hypoxic environment on the cardiovascular system is still not fully understood. Usual pre-clinical analytic methods analysing this effect are limited to ex vivo histology or highly invasive approaches such as right heart catheterisation, which inevitably interfere with cardiac tissue. In this work, we propose volumetric optoacoustic tomography as a method for assessing heart function in response to chronic hypoxia non-invasively. Hypoxic and normoxic murine hearts were imaged in vivo at high temporal (100 Hz) and spatial resolution (200 μm). Analysis of the murine models on a beat-to-beat scale enabled identifying and characterizing arrhythmic events in hypoxic models. In addition, blood flow was tracked using indocyanide green (ICG) contrast agent, which revealed a clear difference in the pulmonary transit time (PTT) between the hypoxic and normoxic models. Validation for presence of hypoxia in the lungs was carried out by α-smooth muscle actin staining for muscularization of the pulmonary vasculature. We expect that the novel capabilities offered by volumetric optoacoustic tomography for analysing impaired heart function under hypoxic conditions in pre-clinical models will provide important insights into early diagnosis and treatment methods for pulmonary diseases.

Exposure to chronic hypoxia results in pulmonary hypertension characterized by increased vascular resistance and pulmonary vascular remodeling, changes in functional parameters of the pulmonary vasculature, and right ventricular hypertrophy, which can eventually lead to right heart failure. The underlying mechanisms of hypoxia-induced pulmonary hypertension have still not been fully elucidated while no curative treatment is currently available. Commonly employed pre-clinical analytic methods are largely limited to invasive studies interfering with cardiac tissue or otherwise ex vivo functional studies and histopathology. In this work, we suggest volumetric optoacoustic tomography (VOT) for non-invasive assessment of heart function in response to chronic hypoxia. Mice exposed for 3 consecutive weeks to normoxia or chronic hypoxia were imaged in vivo with heart perfusion tracked by VOT using indocyanide green contrast agent at high temporal (100 Hz) and spatial (200 µm) resolutions in 3D. Unequivocal difference in the pulmonary transit time was revealed between the hypoxic and normoxic conditions concomitant with the presence of pulmonary vascular remodeling within hypoxic models. Furthermore, a beat-to-beat analysis of the volumetric image data enabled identifying and characterizing arrhythmic events in mice exposed to chronic hypoxia. The newly introduced non-invasive methodology for analysis of impaired pulmonary vasculature and heart function under chronic hypoxic exposure provides important inputs into development of early diagnosis and treatment strategies in pulmonary hypertension.

Imaging has become an indispensable tool in the research and clinical management of cardiovascular disease (CVD). An array of imaging technologies is considered for CVD diagnostics and therapeutic assessment, ranging from ultrasonography, X-ray computed tomography and magnetic resonance imaging to nuclear and optical imaging methods. Each method has different operational characteristics and assesses different aspects of CVD pathophysiology; nevertheless, more information is desirable for achieving a comprehensive view of the disease. Optoacoustic (photoacoustic) imaging is an emerging modality promising to offer novel information on CVD parameters by allowing high-resolution imaging of optical contrast several centimeters deep inside tissue. Implemented with illumination at several wavelengths, multi-spectral optoacoustic tomography (MSOT) in particular, is sensitive to oxygenated and deoxygenated hemoglobin, water and lipids allowing imaging of the vasculature, tissue oxygen saturation and metabolic or inflammatory parameters. Progress with fast-tuning lasers, parallel detection and advanced image reconstruction and data-processing algorithms have recently transformed optoacoustics from a laboratory tool to a promising modality for small animal and clinical imaging. We review progress with optoacoustic CVD imaging, highlight the research and diagnostic potential and current applications and discuss the advantages, limitations and possibilities for integration into clinical routine.

Doxorubicin (DOX) is a widely used chemotherapeutic anticancer drug. Its intrinsic fluorescence properties enable investigation of tumor response, drug distribution and metabolism. First phantom studies in vitro showed optoacoustic property of DOX. We therefore aimed to further investigate the optoacoustic properties of DOX in biological tissue in order to explore its potential as theranostic agent. We analysed doxorubicin hydrochloride (Dox center dot HCl) and liposomal encapsulated doxorubicin hydrochloride (Dox center dot Lipo), two common drugs for anticancer treatment in clinical medicine. Optoacoustic measurements revealed a strong signal of both doxorubicin substrates at 488 nm excitation wavelength. Post mortem analysis of intra-tumoral injections of DOX revealed a detectable optoacoustic signal even at three days after the injection. We thereby demonstrate the general feasibility of doxorubicin detection in biological tissue by means of optoacoustic tomography, which could be applied for high resolution imaging at mesoscopic depths dictated by effective penetration of visible light into the biological tissues.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

Raster Scanning Optoacoustic Mesoscopy (RSOM) is a novel optoacoustic imaging modality that offers non-invasive, label-free, high resolution (~7 mu m axial, ~30 mu m lateral) imaging up to 1 to 2 mm below the skin, providing novel quantitative insights into skin pathophysiology. As the RSOM image contrast mechanism is based on light absorption, it is expected that the amount of melanin present in the skin will affect RSOM images. However, the effect of skin tone in the performance of RSOM has not been addressed so far. Herein, we present the efficiency of RSOM for in vivo skin imaging of human subjects with Fitzpatrick (FP) skin types between II to V. RSOM images acquired from the volar forearms of the subjects were used to derive metrics used in RSOM studies, such as total blood volume, vessel diameter and melanin signal intensity. Our study shows that the melanin signal intensity derived from the RSOM images exhibited an excellent correlation with that obtained from a clinical colorimeter for the subjects of varying FP skin types. We could successfully estimate the vessel diameter at different depths of the dermis. Furthermore, our study shows that there is a need to compensate for total blood volume calculated for subjects with higher FP skin types due to the lower signal-to-noise ratio in dermis, owing to strong absorption of light by melanin. This study sheds light into how RSOM can be used for studying various skin conditions in populations with different skin phenotypes.

Nanoparticles with effective tumor accumulation and efficient renal clearance have attracted significant interests for clinical applications. We prepared 2.5 nm tyrosine based carbon dots (TCDs) with phenolic hydroxyl groups on the surface for directly I-125 labeling. The I-125 labeled polyethylene glycol (PEG) functionalized TCDs (I-125-TCDPEGs) showed excellent radiochemical stability both in vitro and in vivo. Due to the enhanced permeability and retention effect, these I-125-TCDPEGs demonstrated a tumor accumulation around 4%-5% of the injected dose per gram (ID/g) for U87MG, 4T1, HepG2 and MCF7 tumor-bearing mice at 1 h post-injection. Meanwhile, the I-125-TCDPEGs also could be fast renally excreted, with less than 0.6% ID/g left in the liver and spleen within 24 h. These radioactive carbon dots not only can be used for cellular fluorescence imaging due to their intrinsic optical property, but are also effective single photon emission computed tomography (SPECT) imaging agents for tumor. Together with their excellent biocompatibility and stability, we anticipate these I-125-TCDPEGs of great potential for early tumor diagnosis in clinic. What's more, our TCDPEGs are also proved to be feasible carriers for other iodine isotopes such as 127I and 131I for different biomedical application.

Accurate image reconstruction in volumetric optoacoustic tomography implies the efficient generation and collection of ultrasound signals around the imaged object. Non-uniform delivery of the excitation light is a common problem in optoacoustic imaging often leading to a diminished field of view, limited dynamic range and penetration, as well as impaired quantification abilities. Presented here is an optimized illumination concept for volumetric tomography that utilizes additive manufacturing via 3D printing in combination with custom-made optical fiber illumination. The custom-designed sample chamber ensures convenient access to the imaged object along with accurate positioning of the sample and a matrix array ultrasound transducer used for collection of the volumetric image data. Ray tracing is employed to optimize the positioning of the individual fibers in the chamber. Homogeneity of the generated light excitation field was confirmed in tissue-mimicking agar spheres. Applicability of the system to image entire mouse organs ex vivo has been showcased. The new approach showed a clear advantage over conventional, single-sided illumination strategies by eliminating the need to correct for illumination variances and resulting in enhancement of the effective field of view, greater penetration depth and significant improvements in the overall image quality.

Rapid progress in the development of multispectral optoacoustic tomography techniques has enabled unprecedented insights into biological dynamics and molecular processes in vivo and noninvasively at penetration and spatiotemporal scales not covered by modern optical microscopy methods. Ultrasound imaging provides highly complementary information on elastic and functional tissue properties and further aids in enhancing optoacoustic image quality. We devised the first hybrid transmission-reflection optoacoustic ultrasound (TROPUS) small animal imaging platform that combines optoacoustic tomography with both reflection- and transmission-mode ultrasound computed tomography. The system features full-view cross-sectional tomographic imaging geometry for concomitant noninvasive mapping of the absorbed optical energy, acoustic reflectivity, speed of sound, and acoustic attenuation in whole live mice with submillimeter resolution and unrivaled image quality. Graphics-processing unit (GPU)-based algorithms employing spatial compounding and bent-ray-tracing iterative reconstruction were further developed to attain real-time rendering of ultrasound tomography images in the full-ring acquisition geometry. In vivo mouse imaging experiments revealed fine details on the organ parenchyma, vascularization, tissue reflectivity, density, and stiffness. We further used the speed of sound maps retrieved by the transmission ultrasound tomography to improve optoacoustic reconstructions via two-compartment modeling. The newly developed synergistic multimodal combination offers unmatched capabilities for imaging multiple tissue properties and biomarkers with high resolution, penetration, and contrast.

Photocontrollable proteins revolutionized life-science imaging due to their contribution to subdiffraction-resolution optical microscopy. They might have yet another lasting impact on photo- or optoacoustic imaging (OA). OA combines optical contrast with ultrasound detection enabling high-resolution real-time in vivo imaging well-beyond the typical penetration depth of optical methods. While OA already showed numerous applications relying on endogenous contrast from blood hemoglobin or lipids, its application in the life-science was limited by a lack of labels overcoming the strong signal from the aforementioned endogenous absorbers. Here, a number of recent studies showed that photocontrollable proteins provide the means to overcome this barrier eventually enabling OA to image small cell numbers in a complete organism in vivo. In this Feature article, we introduce the key photocontrollable proteins, explain the basic concepts, and highlight achievements that have been already made.

A new type of bimodal contrast agent was made that is based on the self-quenching of indocyanine green (ICG) encapsulated in a biocompatible and biodegradable polymer shell. The increasing of a local ICG concentration that is necessary for the obtaining of self-quenching effect was achieved by freezing-induced loading and layer-by-layer assembly. As a result, an efficient photoacoustic(optoacoustic)/fluorescent contrast agent based on composite indocyanine green/polymer particles was successfully prepared and was characterized by fluorescence and photoacoustic(optoacoustic) tomography in vitro. This type of contrast agent holds good promise for clinical application owing to its high efficiency and biosafety.

Background and aims Minirhizotrons are commonly used to study root turnover which is essential for understanding ecosystem carbon and nutrient cycling. Yet, extracting data from minirhizotron images requires extensive annotation effort. Existing annotation tools often lack flexibility and provide only a subset of the required functionality. To facilitate efficient root annotation in minirhizotrons, we present the user-friendly open source tool rhizoTrak. Methods and results rhizoTrak builds on TrakEM2 and is publicly available as Fiji plugin. It uses treelines to represent branching structures in roots and assigns customizable status labels per root segment. rhizoTrak offers configuration options for visualization and various functions for root annotation mostly accessible via keyboard shortcuts. rhizoTrak allows time-series data import and particularly supports easy handling and annotation of time-series images. This is facilitated via explicit temporal links (connectors) between roots which are automatically generated when copying annotations from one image to the next. rhizoTrak includes automatic consistency checks and guided procedures for resolving inconsistencies. It facilitates easy data exchange with other software by supporting open data formats. Conclusions rhizoTrak covers the full range of functions required for user-friendly and efficient annotation of time-series images. Its flexibility and open source nature will foster efficient data acquisition procedures in root studies using minirhizotrons.

Purpose Identification of morphological characteristics of skin lesions is of vital importance in diagnosing diseases with dermatological manifestations. This task is often performed manually or in an automated way based on intensity level. Recently, ultra-broadband raster-scan optoacoustic mesoscopy (UWB-RSOM) was developed to offer unique cross-sectional optical imaging of the skin. A machine learning (ML) approach is proposed here to enable, for the first time, automated identification of skin layers in UWB-RSOM data. Materials and methods The proposed method, termed SkinSeg, was applied to coronal UWB-RSOM images obtained from 12 human participants. SkinSeg is a multi-step methodology that integrates data processing and transformation, feature extraction, feature selection, and classification. Various image features and learning models were tested for their suitability at discriminating skin layers including traditional machine learning along with more advanced deep learning algorithms. An support vector machines-based postprocessing approach was finally applied to further improve the classification outputs. Results Random forest proved to be the most effective technique, achieving mean classification accuracy of 86.89% evaluated based on a repeated leave-one-out strategy. Insights about the features extracted and their effect on classification accuracy are provided. The highest accuracy was achieved using a small group of four features and remained at the same level or was even slightly decreased when more features were included. Convolutional neural networks provided also promising results at a level of approximately 85%. The application of the proposed postprocessing technique was proved to be effective in terms of both testing accuracy and three-dimensional visualization of classification maps. Conclusions SkinSeg demonstrated unique potential in identifying skin layers. The proposed method may facilitate clinical evaluation, monitoring, and diagnosis of diseases linked to skin inflammation, diabetes, and skin cancer.

Due to the implication of altered metabolism in a large spectrum of tissue function and disease, assessment of metabolic processes becomes essential in managing health. In this regard, imaging can play a critical role in allowing observation of biochemical and physiological processes. Nuclear imaging methods, in particular positron emission tomography, have been widely employed for imaging metabolism but are mainly limited by the use of ionizing radiation and the sensing of only one parameter at each scanning session. Observations in healthy individuals or longitudinal studies of disease could markedly benefit from non-ionizing, multi-parameter imaging methods. We therefore focus this review on progress with the non-ionizing radiation methods of MRI, hyperpolarized magnetic resonance and magnetic resonance spectroscopy, chemical exchange saturation transfer, and emerging optoacoustic (photoacoustic) imaging. We also briefly discuss the role of nuclear and optical imaging methods for research and clinical protocols.

In traditional optical imaging, limited light penetration constrains high-resolution interrogation to tissue surfaces. Optoacoustic imaging combines the superb contrast of optical imaging with deep penetration of ultrasound, enabling a range of new applications. We used multispectral optoacoustic tomography (MSOT) for functional and structural neuroimaging in mice at resolution, depth, and specificity unattainable by other neuroimaging modalities. Based on multispectral readouts, we computed hemoglobin gradient and oxygen saturation changes related to processing of somatosensory signals in different structures along the entire subcortical-cortical axis. Using temporal correlation analysis and seed-based maps, we reveal the connectivity between cortical, thalamic, and sub-thalamic formations. With the same modality, high-resolution structural tomography of intact mouse brain was achieved based on endogenous contrasts, demonstrating near-perfect matches with anatomical features revealed by histology. These results extend the limits of noninvasive observations beyond the reach of standard high-resolution neuroimaging, verifying the suitability of MSOT for small-animal studies.

Fuelled by innovation, optical microscopy plays a critical role in the life sciences and medicine, from basic discovery to clinical diagnostics. However, optical microscopy is limited by typical penetration depths of a few hundred micrometres for in vivo interrogations in the visible spectrum. Optoacoustic microscopy complements optical microscopy by imaging the absorption of light, but it is similarly limited by penetration depth. In this Review, we summarize progress in the development and applicability of optoacoustic mesoscopy (OPAM); that is, optoacoustic imaging with acoustic resolution and wide-bandwidth ultrasound detection. OPAM extends the capabilities of optical imaging beyond the depths accessible to optical and optoacoustic microscopy, and thus enables new applications. We explain the operational principles of OPAM, its placement as a bridge between optoacoustic microscopy and optoacoustic macroscopy, and its performance in the label-free visualization of tissue pathophysiology, such as inflammation, oxygenation, vascularization and angiogenesis. We also review emerging applications of OPAM in clinical and biological imaging.

Synaptic transmission is a fundamental neurobiological process enabling exchange of signals between neurons as well as neurons and their non-neuronal effectors. The complex molecular machinery of the synaptic vesicle cycle and transmitter release has emerged and developed in the course of the evolutionary race, to ensure adaptive gain and survival of the fittest. In parallel, a generous arsenal of biomolecules and neuroactive peptides have co-evolved, which selectively target the transmitter release machinery, with the aim of subduing natural rivals or neutralizing prey. With advances in neuropharmacology and quantitative biology, neurotoxins targeting pre synaptic mechanisms have attracted major interest, revealing considerable potential as carriers of molecular cargo and probes for meddling synaptic transmission mechanisms for research and medical benefit. In this review, we investigate and discuss key facets employed by the most prominent bacterial and animal toxins targeting the presynaptic secretory machinery. We explore the cellular basis and molecular grounds for their tremendous potency and selectivity, with effects on a wide range of neural functions. Finally, we consider the emerging preclinical and clinical data advocating the use of active ingredients of neurotoxins for the advancement of molecular medicine and development of restorative therapies. (C) 2018 Elsevier Inc. All rights reserved.

The bulk of brain energy expenditure is allocated for maintenance of perpetual intrinsic activity of neurons and neural circuits. Long-term electrophysiological and neuroimaging studies in anesthetized and behaving animals show, however, that the great majority of nerve cells in the intact brain do not fire action potentials, i.e., are permanently silent. Herein, I review emerging data suggesting massive redundancy of nerve cells in mammalian nervous system, maintained in inhibited state at high energetic costs. Acquired in the course of evolution, these collections of dormant neurons and circuits evade routine functional undertakings, and hence, keep out of the reach of natural selection. Under penetrating stress and disease, however, they occasionally switch in active state and drive a variety of neuro-psychiatric symptoms and behavioral abnormalities. The increasing evidence for widespread occurrence of silent neurons warrants careful revision of functional models of the brain and entails unforeseen reserves for rehabilitation and plasticity.

Unlike traditional optical methods, optoacoustic imaging is less sensitive to scattering of ballistic photons, so it is capable of high-resolution interrogation at a greater depth. By integrating video-rate visualization with multiplexing and sensing a range of endogenous and exogenous chromophores, optoacoustic imaging has matured into a versatile noninvasive investigation modality with rapidly expanding use in biomedical research. We review the principal features of the technology and discuss recent advances it has enabled in structural, functional, and molecular neuroimaging in small-animal models. In extending the boundaries of noninvasive observation beyond the reach of customary photonic methods, the latest developments in optoacoustics have substantially advanced neuroimaging inquiry, with promising implications for basic and translational studies.

Efficient monitoring of radiofrequency ablation procedures is essential to optimize the lesions induced to treat cancer, cardiac arrhythmias and other conditions. Recently, optoacoustic imaging and sensing methods have been suggested as a promising approach to address this challenge, offering unique advantages such as high sensitivity to temperature changes and chemical transformations in coagulated tissues, real-time operation and use of non- ionizing radiation. However, assessing how the ablation lesion boundary progresses is still challenged by changes in optical properties induced during the interventions. Herein, we suggest a new approach for dimensional characterization of the induced lesion based on detecting sharp positive variations in the time derivative of optoacoustic signals. Experiments in porcine tissue samples indicate that such variations are uniquely associated to the onset of ablation and that the method can robustly visualize the evolution of the lesion in three dimensions.

Laser ablation (LA) represents a minimally invasive intervention that is gaining acceptance for the treatment of different types of cancer, leading to important advantages such as less pain and shorter recovery time. Accurate monitoring of ablation progression is crucial to prevent damage of non-cancerous tissues and optimize the outcome of the intervention. To this end, imaging techniques such as ultrasound, computed tomography or magnetic resonance imaging have been used for monitoring LA. However, these techniques feature important drawbacks such as the need of contrast agents, poor spatio-temporal resolution or high cost. Optoacoustics (OA, photoacoustic) has recently been shown to provide unique properties to monitor thermal treatments. Herein, we demonstrate the feasibility of optoacoustic laser-ablation (OLA) monitoring in a murine breast tumor model using a single short-pulsed 1064 nm laser source. The effect of irradiation was volumetrically tracked with the OA images acquired with a 256-element spherical array. Structural damage of the tissue was clearly seen during the LA procedure.

The morphology, physiology and immunology, of solid tumors exhibit spatial heterogeneity which complicates our understanding of cancer progression and therapy response. Understanding spatial heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information. We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). We show that endogenous bacteriochlorophyll a in Rhodobacter gives rise to strong optoacoustic signals >800 nm away from interfering endogenous absorbers. Importantly, our results suggest that changes in the spectral signature of Rhodobacter which depend on macrophage activity inside the tumor can be used to reveal heterogeneity of the tumor microenvironment. Employing non-invasive high resolution MSOT in longitudinal studies we show spatiotemporal changes of Rhodobacter spectral profiles in mice bearing 4T1 and CT26.WT tumor models. Accessibility of Rhodobacter to genetic modification and thus to sensory and therapeutic functions suggests potential for a theranostic platform organism.

Objective: Optoacoustic (photoacoustic) tomography is aimed at reconstructing maps of the initial pressure rise induced by the absorption of light pulses in tissue. In practice, due to inaccurate assumptions in the forward model, noise, and other experimental factors, the images are often afflicted by artifacts, occasionally manifested as negative values. The aim of this work is to develop an inversion method which reduces the occurrence of negative values and improves the quantitative performance of optoacoustic imaging. Methods: We present a novel method for optoacoustic tomography based on an entropy maximization algorithm, which uses logarithmic regularization for attaining non-negative reconstructions. The reconstruction image quality is further improved using structural prior-based fluence correction. Results: We report the performance achieved by the entropy maximization scheme on numerical simulation, experimental phantoms, and in-vivo samples. Conclusion: The proposed algorithm demonstrates superior reconstruction performance by delivering non-negative pixel values with no visible distortion of anatomical structures. Significance: Our method can enable quantitative optoacoustic imaging, and has the potential to improve preclinical and translational imaging applications.

Breast cancer and Glioblastoma brain cancer are aggressive malignancies with poor prognosis. In this study primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. A custom multi-angle Light Sheet Fluorescence Microscope is employed for high resolution imaging of both cancer cell spheroids. Such a technique is successful in realizing pre-clinical drug screening, while enables the discrimination among physiologic tumor parameters. LSFM technique, parameters and method followed are also presented.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.

Early and comprehensive endoscopic detection of colonic dysplasia-the most clinically significant precursor lesion to colorectal adenocarcinoma-provides an opportunity for timely, minimally invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis is described. FSNs are biodegradable (t(1/2) of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm(2) with high tumor-to-background ratios. Furthermore, in the human scale, APC(1311/+) porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demon-strated. Since nanoparticles of similar size (e.g., 100-150 nm) or composition (i.e., silica and silica/gold hybrid) have already been successfully translated to the clinic, and clinical fluorescent/white-light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

This study aimed at evaluating hybrid multispectral optoacoustic tomography/ultrasound for imaging of thyroid disorders, including Graves' disease and thyroid nodules. Methods: The functional biomarkers and tissue parameters deoxygenated hemoglobin, oxygenated hemoglobin, total hemoglobin, saturation of hemoglobin, fat content, and water content were analyzed in thyroid lobes affected by Graves' disease (n = 6), thyroid lobes with healthy tissue (n = 8), benign thyroid nodules (n = 13), and malignant thyroid nodules (n = 3). Results: In Graves' disease, significantly higher deoxygenated hemoglobin (3.18 +/- 0.52 vs. 2.13 +/- 0.62; P = 0.0055) and total hemoglobin (8.34 +/- 0.88 vs. 6.59 +/- 1.16; P = 0.0084) and significantly lower fat content (0.64 +/- 0.37 vs. 1.69 +/- 1.25; P = 0.0293) were found than in healthy controls. Malignant thyroid nodules showed significantly lower saturation of hemoglobin (55.4% +/- 2.6% vs. 60.8% +/- 7.2%; P = 0.0393) and lower fat content (0.62 +/- 0.19 vs. 1.46 +/- 0.87; P = 0.1295) than benign nodules. Conclusion: This pilot study showed the applicability and the potential of hybrid multispectral optoacoustic tomography/ultrasound to semiquantitatively provide tissue characterization and functional parameters in thyroid disorders for improved noninvasive diagnostics of thyroid diseases.

Mapping tumor heterogeneity and hypoxia within a living intact organism is essential for understanding the processes involved in cancer progression and assessing long-term responses to therapies. Efficient investigations into tumor hypoxia mechanisms have been hindered by the lack of intravital imaging tools capable of multiparametric probing of entire solid tumors with high spatial and temporal resolution. Here, we exploit volumetric multispectral optoacoustic tomography (vMSOT) for accurate, label-free delineation of tumor heterogeneity and dynamic oxygenation behavior. Mice bearing orthotopic MDA-MB-231 breast cancer xenografts were imaged noninvasively during rest and oxygen stress challenge, attaining time-lapse three-dimensional oxygenation maps across entire tumors with 100 mm spatial resolution. Volumetric quantification of the hypoxic fraction rendered values of 3.9% to 21.2%, whereas the oxygen saturation (sO(2)) rate declined at 1.7% to 2.3% permmin all tumors when approaching their core. Three distinct functional areas (the rim, hypoxic, and normoxic cores) were clearly discernible based on spatial sO(2) profiles and responses to oxygen challenge. Notably, although sO(2) readings were responsive to the challenge, deoxyhemoglobin (HbR) trends exhibited little to no variations in all mice. Dynamic analysis further revealed the presence of cyclic hypoxia patterns with a 21% average discrepancy between cyclic fractions assessed via sO(2) (42.2% +/- 17.3%) and HbR fluctuations (63% +/- 14.1%) within the hypoxic core. These findings corroborate the strong potential of vMSOT for advancing preclinical imaging of cancer and informing clinical decisions on therapeutic interventions.Significance: vMSOT provides quantitative measures of volumetric hypoxic fraction and cyclic hypoxia in a label-free and noninvasive manner, providing new readouts to aid tumor staging and treatment decision making.

Respiratory motion in living organisms is known to result in image blurring and loss of resolution, chiefly due to the lengthy acquisition times of the corresponding image acquisition methods. Optoacoustic tomography can effectively eliminate in vivo motion artifacts due to its inherent capacity for collecting image data from the entire imaged region following a single nanoseconds-duration laser pulse. However, multi-frame image analysis is often essential in applications relying on spectroscopic data acquisition or for scanning-based systems. Thereby, efficient methods to correct for image distortions due to motion are imperative. Herein, we demonstrate that efficient motion rejection in optoacoustic tomography can readily be accomplished by frame clustering during image acquisition, thus averting excessive data acquisition and post-processing. The algorithm’s efficiency for two- and three-dimensional imaging was validated with experimental whole-body mouse data acquired by spiral volumetric optoacoustic tomography (SVOT) and full-ring cross-sectional imaging scanners.

We present the investigation of in vivo small model organisms, which are well established in biological and biomedical research, using a hybrid multiphoton and optoacoustic microscope (HyMPOM). The unique capabilities of HyMPOM for multimodal and potentially label-free signal acquisition, high resolution, as well as deep and fast imaging allow extraction of detailed information across large areas of living tissue on the microscale. Applying HyMPOM to living zebrafish-like fish larvae allowed exploration of the structural composition of the entire brain, including the brain vasculature and the neuronal network. Applying HyMPOM to the ears of living mice enabled accurate imaging of vasculature, connective tissue, keratinocytes, and sebaceous glands. The hybrid microscope proposed here constitutes a novel approach to explore small model organisms in vivo in great detail by revealing the spatial distribution and interplay of various tissue compartments on the microscale.

In this work we developed a novel near-infrared two-path optoacoustic spectrometer (NiR-TAOS) that could sense OA intensity changes due to metabolite concentration changes in-vivo. The main aim of dividing the optical path in two is 1) perform real time correction of the laser emission profile of the laser source at different wavelengths and, 2) perform pulse to pulse correction to remove laser beam fluctuation and instability to increase signal to noise ratio. Signal to noise ratio improvement was significant not only at spectral peaks, but also at all other wavelengths. The system can be used for broad applications in biomedical measurements such as various metabolites in the SWIR.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of similar to 43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

The sampling patterns of the light field microscope (LFM) are highly depth-dependent, which implies non-uniform recoverable lateral resolution across depth. Moreover, reconstructions using state-of-the-art approaches suffer from strong artifacts at axial ranges, where the LFM samples the light field at a coarse rate. In this work, we analyze the sampling patterns of the LFM, and introduce a flexible light field point spread function model (LFPSF) to cope with arbitrary LFM designs. We then propose a novel aliasing-aware deconvolution scheme to address the sampling artifacts. We demonstrate the high potential of the proposed method on real experimental data.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 x 10(3) haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 x 10(4) cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

Objectives: Brown adipose tissue (BAT) dissipates nutritional energy as heat through uncoupling protein 1 (UCP1). The discovery of functional BAT in healthy adult humans has promoted the search for pharmacological interventions to recruit and activate brown fat as a treatment of obesity and diabetes type II. These efforts require in vivo models to compare the efficacy of novel compounds in a relevant physiological context.Methods: We generated a knock-in mouse line expressing firefly luciferase and near-infrared red florescent protein (iRFP713) driven by the regulatory elements of the endogenous Ucp1 gene.Results: Our detailed characterization revealed that firefly luciferase activity faithfully reports endogenous Ucp1 gene expression in response to physiological and pharmacological stimuli. The iRFP713 fluorescence signal was detected in the interscapular BAT region of cold-exposed reporter mice in an allele-dosage dependent manner. Using this reporter mouse model, we detected a higher browning capacity in female peri-ovarian white adipose tissue compared to male epididymal WAT, which we further corroborated by molecular and morphological features. In situ imaging detected a strong luciferase activity signal in a previously unappreciated adipose tissue depot adjunct to the femoral muscle, now adopted as femoral brown adipose tissue. In addition, screening cultured adipocytes by bioluminescence imaging identified the selective Salt-Inducible Kinase inhibitor, HG-9-91-01, to increase Ucp1 gene expression and mitochondrial respiration in brown and brite adipocytes.Conclusions: In our mouse model, firefly luciferase activity serves as a bona fide reporter for dynamic regulation of Ucp1. In addition, by means of iRFP713 we are able to monitor Ucp1 expression in a non-invasive fashion. (C) 2018 Published by Elsevier GmbH.

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.

The concentrations of contrast agents for optoacoustic imaging of small animals must usually be optimized through extensive pilot experiments on a case-by-case basis. The present work describes a streamlined approach for determining the minimum detectable concentration (MDC) of a contrast agent given experimental conditions and imaging system parameters. The developed Synthetic Data Framework (SDF) allows estimation of MDCs of various contrast agents under different tissue conditions without extensive animal experiments. The SDF combines simulated optoacoustic signals from exogenously administered contrast agents with in vivo experimental signals from background tissue to generate realistic synthetic multispectral optoacoustic images. In this paper, the SDF is validated with in vivo measurements and demonstrates close agreement between SDF synthetic data and experimental data in terms of both image intensity and MDCs. Use of the SDF to estimate MDCs for fluorescent dyes and nanoparticles at different tissue depths and for imaging lesions of different sizes is illustrated.

Even though the speed of sound (SoS) is non-homogeneous in biological tissue, most reconstruction algorithms for optoacoustic imaging neglect its variation. In addition, when heavy water is used as coupling medium to enable imaging of certain biological chromophores such as lipids and proteins, the SoS also differs significantly between couplant and tissue. While the assumption of uniform SoS is known to introduce visible deformations of features in single-wavelength optoacoustic images, the spectral error introduced by the assumption of uniform SoS is not fully understood. In this work, we provide an in-depth spectral analysis of multi-spectral optoacoustic imaging artifacts that result from the assumption of uniform SoS in situations where SoS changes substantially. We propose a dual-SoS model to incorporate the SoS variation between the couplant and the sample. Tissue-mimicking phantom experiments and in vivo measurements show that uniform SoS reconstruction causes spectral smearing, which dual-SoS modeling can largely eliminate. Due to this increased spectral accuracy, the method has the potential to improve clinical studies that rely on quantitative optoacoustic imaging of biomolecules like hemoglobin or lipids.

Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex viva murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 +/- 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 +/- 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines.

Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.

Nailfold capillaroscopy, based on bright-field microscopy, is widely used to diagnose systemic sclerosis (SSc). However it cannot reveal information about venules and arterioles lying deep under the nailfold, nor can it provide detailed data about surface microvasculature when the skin around the nail is thick. These limitations reflect the fact that capillaroscopy is based on microscopy methods whose penetration depth is restricted to about 200 μm. We investigated whether ultra-wideband raster-scan optoacoustic mesoscopy (UWB-RSOM) can resolve small capillaries of the nailfold in healthy volunteers and compared the optoacoustic data to conventional capillaroscopy examinations. We quantified UWB-RSOM-resolved capillary density and capillary diameter as features that relate to SSc biomarkers, and we obtained the first three-dimensional, images of the deeper arterioles and venules. These results establish the potential of UWB-RSOM for analyzing SSc-relevant markers.

Intraoperative imaging (IOI) is performed to guide delineation and localization of regions of surgical interest. While oncological surgical planning predominantly utilizes x-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US), intraoperative guidance mainly remains on surgeon interpretation and pathology for confirmation. Over the past decades however, intraoperative guidance has evolved significantly with the emergence of several novel imaging technologies, including fluorescence-, Raman, photoacoustic-, and radio-guided approaches. These modalities have demonstrated the potential to further optimize precision in surgical resection and improve clinical outcomes for patients. Not only can these technologies enhance our understanding of the disease, they can also yield large imaging datasets intraoperatively that can be analyzed by deep learning approaches for more rapid and accurate pathological diagnosis. Unfortunately, many of these novel technologies are still under preclinical or early clinical evaluation. Organizations like the Intra-Operative Imaging Study Group of the European Society for Molecular Imaging (ESMI) support interdisciplinary interactions with the aim to improve technical capabilities in the field, an approach that can succeed only if scientists, engineers, and physicians work closely together with industry and regulatory bodies to resolve roadblocks to clinical translation. In this review, we provide an overview of a variety of novel IOI technologies, discuss their challenges, and present future perspectives on the enormous potential of IOI for oncological surgical navigation.

PURPOSE: Here we demonstrate the potential of multispectral optoacoustic tomography (MSOT), a new non-invasive structural and functional imaging modality, to track the growth and changes in blood oxygen saturation (sO) in orthotopic glioblastoma (GBMs) and the surrounding brain tissues upon administration of a vascular disruptive agent (VDA). METHODS: Nude mice injected with U87MG tumor cells were longitudinally monitored for the development of orthotopic GBMs up to 15 days and observed for changes in sO upon administration of combretastatin A4 phosphate (CA4P, 30 mg/kg), an FDA approved VDA for treating solid tumors. We employed a newly-developed non-negative constrained approach for combined MSOT image reconstruction and unmixing in order to quantitatively map sO in whole mouse brains. RESULTS: Upon longitudinal monitoring, tumors could be detected in mouse brains using single-wavelength data as early as 6 days post tumor cell inoculation. Fifteen days post-inoculation, tumors had higher sO of 63 ± 11% (n = 5, P < .05) against 48 ± 7% in the corresponding contralateral brain, indicating their hyperoxic status. In a different set of animals, 42 days post-inoculation, tumors had lower sO of 42 ± 5% against 49 ± 4% (n = 3, P < .05) in the contralateral side, indicating their hypoxic status. Upon CA4P administration, sO in 15 days post-inoculation tumors dropped from 61 ± 9% to 36 ± 1% (n = 4, P < .01) within one hour, then reverted to pre CA4P treatment values (63 ± 6%) and remained constant until the last observation time point of 6 hours. CONCLUSION: With the help of advanced post processing algorithms, MSOT was capable of monitoring the tumor growth and assessing hemodynamic changes upon administration of VDAs in orthotopic GBMs.

We have reported two methods to analyze glucose in the interstitial fluid of skin based on mid-infrared excitation with a tunable quantum cascade laser and photoacoustic or photothermal detection. These methods were evaluated for optimum skin locations to obtain reproducible glucose information. The lower part of the arm, the hypothenar, the tips of the index finger and the thumb were tested. The thumb appears to be the optimal skin location, followed by the index finger. Basic requirements for an optimum site are good capillary blood perfusion, low Stratum corneum thickness and the absence of fat layers. To obtain a correlation on such a site, spectra were recorded on volunteers continuously after blood glucose manipulation. However, continuous measurements on an in vivo sample such as the skin have to cope with physiological alterations such as the formation of sweat. We have used both detection schemes to investigate the acid mantle reformation after washing during time scales similar to continuous measurements for calibration spectra. We found that reconstitution of the acid mantle of skin may be seen in less than one hour. Precleaning of the measurement site may thus be useful for intermittent, but not for long term continuous measurements. [GRAPHICS] .

The aim of this study was to explore the unique imaging abilities of optoacoustic mesoscopy to visualize skin structures and microvasculature with the view of establishing a robust approach for monitoring heat-induced hyperemia in human skin in vivo. Using raster-scan optoacoustic mesoscopy (RSOM), we investigated whether optoacoustic (photoacoustic) mesoscopy can identify changes in skin response to local heating at microvasculature resolution in a cross-sectional fashion through skin in the human forearm. We visualized the heat-induced hyperemia for the first time with single-vessel resolution throughout the whole skin depth. We quantified changes in total blood volume in the skin and their correlation with local heating. In response to local heating, total blood volume increased 1.83- and 1.76-fold, respectively, in the volar and dorsal aspects of forearm skin. We demonstrate RSOM imaging of the dilation of individual vessels in the skin microvasculature, consistent with hyperemic response to heating at the skin surface. Our results demonstrate great potential of RSOM for elucidating the morphology, functional state and reactivity of dermal microvasculature, with implications for diagnostics and disease monitoring. Image: Cross-sectional view of skin microvasculature dilated in response to hyperthermia.

Hybrid intravascular fluorescence-ultrasound imaging is emerging for reading anatomical and biological information in vivo. By operating through blood, intravascular near-infrared fluorescence (NIRF) detection is affected by hemoglobin attenuation. Improved quantification has been demonstrated with methods that correct for the attenuation of the optical signal as it propagates through blood. These methods assume an attenuation coefficient for blood and measure the distance between detector and the vessel wall by observing the intravascular ultrasound images. Assumptions behind the attenuation employed in correction models may reduce the accuracy of these methods. Herein, we explore a novel approach to dynamically estimate optical absorption by using optoacoustic (photoacoustic) measurements. Adaptive correction is based on a trimodal intravascular catheter that integrates fluorescence, ultrasound and optoacoustic measurements. Using the novel catheter, we show how optoacoustic measurements can determine variations of blood absorption, leading to accurate quantification of the detected NIRF signals at different hematocrit values.

Fluorescent contrast agents are widely employed in biomedical research. While many studies have reported deep tissue imaging of fluorescent moieties using either fluorescence-based or absorption-based (optoacoustic) imaging systems, no systematic comparison has been performed regarding the actual performance of these imaging modalities in detecting deep-seated fluorescent agents. Herein, an integrated imager combining epifluorescence and volumetric optoacoustic imaging capabilities has been employed in order to evaluate image degradation with depth for several commonly-used near-infrared dyes in both modes. We performed controlled experiments in tissue-mimicking phantoms containing deeply embedded targets filled with different concentrations of Alexa Fluor 700, Alexa Fluor 750, indocyanine green (ICG) and IRDye 800CW. The results are further corroborated by multi-modal imaging of ICG through mouse tissues in vivo. It is shown that optoacoustics consistently provides better sensitivity in differentiating fluorescent targets located at depths beyond 2 mm in turbid tissues, as quantified by evaluating image contrast, signal to noise ratio and spatial resolution performance.

Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

Fluorescence imaging is widely employed in biological discovery due to its excellent molecular sensitivity and contrast. However, due to light scattering wide-field fluorescence images are blurred resulting in very low spatial resolution and low image contrast. The existing scanning optical microscopy techniques are commonly restricted to sub-millimeter field-of-view or otherwise slow imaging speeds, limiting their applicability for imaging of fast biological dynamics occurring on larger spatial scales. Herein, we developed a rapid scanning wide-field multifocal structured illumination microscopy method based on a beam-splitting grating and an acousto-optic deflector synchronized with a high speed camera. The multi-beam pattern is focused by a condensing lens and a macroscopic objective to generate multifocal structured illumination profile on the imaged sample that is rapidly scanned at kHz rates. Experimental results show that the proposed method can achieve real-time fluorescence microscopy over a centimeter-scale field of view. Owing to the low numerical aperture of the diffracted beams, the illumination has a large depth of focus and hence is generally not affected by the sample's curvature, which allowed here imaging of perfusion in the entire mouse cerebral cortex noninvasively. The new approach can be readily incorporated into traditional wide-field microscopes to attain optimal tradeoff between spatial resolution and field of view. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus anticipated to find broad applicability in a variety of applications looking at large-scale fluorescent-based biodynamics.

Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3β4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3β4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.

In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.

Signal propagation in cardiac cell networks can be modulated by heat stimulation. Here, the response of a connected HL-1 cardiomyocyte cell network to the application of confined heat stimuli using Ca 2+ imaging is investigated. Localized temperature gradients are generated by resistive heating via microwire arrays on a chip surface, which serves as a substrate for growing a confluent cell network. It is demonstrated that upon heat stimulation, the velocity of the propagating Ca 2+ wave in the network is locally increased, leading to a deformation of the wavefront. Furthermore, evidence of a change in the signal propagation direction caused by a relocation of the pacemaker cell is shown. This effect might be used in future applications, where heat is employed as an alternative modality for cell stimulation protocols.

Localization-based imaging has revolutionized fluorescence optical microscopy and has also enabled unprecedented ultrasound images of microvascular structures in deep tissues. Herein, we introduce a new concept of localization optoacoustic tomography (LOT) that employs rapid sequential acquisition of three-dimensional optoacoustic images from flowing absorbing particles. We show that the new method enables breaking through the spatial resolution barrier of acoustic diffraction while further enhancing the visibility of structures under limited-view tomographic conditions. Given the intrinsic sensitivity of optoacoustics to multiple hemodynamic and oxygenation parameters, LOT may enable a new level of performance in studying functional and anatomical alterations of microcirculation.

Downloading of the abstract is permitted for personal use only. Optical microscopy remains a major workhorse in biological discovery despite the fact that light scattering limits its applicability to depths of ∼ 1 mm in scattering tissues. Optoacoustic imaging has been shown to overcome this barrier by resolving optical absorption with microscopic resolution in significantly deeper regions. Yet, the time domain is paramount for the observation of biological dynamics in living systems that exhibit fast motion. Commonly, acquisition of microscopy data involves raster scanning across the imaged volume, which significantly limits temporal resolution in 3D. To overcome these limitations, we have devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth around 25 MHz. We performed experiments by imaging tissue-mimicking phantoms and zebrafish larvae, demonstrating that OMT can provide nearly cellular resolution and imaging speed of 100 volumetric frames per second. As opposed to other optical microscopy techniques, OMT is a hybrid method that resolves optical absorption contrast acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Diffraction causes blurring of high-resolution features in images and has been traditionally associated to the resolution limit in light microscopy and other imaging modalities. The resolution of an imaging system can be generally assessed via its point spread function, corresponding to the image acquired from a point source. However, the precision in determining the position of an isolated source can greatly exceed the diffraction limit. By combining the estimated positions of multiple sources, localization-based imaging has resulted in groundbreaking methods such as super-resolution fluorescence optical microscopy and has also enabled ultrasound imaging of microvascular structures with unprecedented spatial resolution in deep tissues. Herein, we introduce localization optoacoustic tomography (LOT) and discuss on the prospects of using localization imaging principles in optoacoustic imaging. LOT was experimentally implemented by real-time imaging of flowing particles in 3D with a recently-developed volumetric optoacoustic tomography system. Provided the particles were separated by a distance larger than the diffraction-limited resolution, their individual locations could be accurately determined in each frame of the acquired image sequence and the localization image was formed by superimposing a set of points corresponding to the localized positions of the absorbers. The presented results demonstrate that LOT can significantly enhance the well-established advantages of optoacoustic imaging by breaking the acoustic diffraction barrier in deep tissues and mitigating artifacts due to limited-view tomographic acquisitions.

© 2018 The Author(s). Ultrasound-mediated transcranial images of the brain often suffer from acoustic distortions produced by the skull bone. In high-resolution optoacoustic microscopy, the skull-induced acoustic aberrations are known to impair image resolution and contrast, further skewing the location and intensity of the different absorbing structures. We present a virtual craniotomy deconvolution algorithm based on an ultrasound wave propagation model that corrects for the skull-induced distortions in optically-resolved optoacoustic transcranial microscopy data. The method takes advantage of the geometrical and spectral information of a pulse-echo ultrasound image of the skull simultaneously acquired by our multimodal imaging system. Transcranial mouse brain imaging experiments confirmed the ability to accurately account for the signal amplitude decay, temporal delay and pulse broadening introduced by the rodent's skull. Our study is the first to demonstrate skull-corrected transcranial optoacoustic imaging in vivo.

Human skull poses a significant barrier for the propagation of ultrasound waves. Development of methods enabling more efficient ultrasound transmission into and from the brain is therefore critical for the advancement of ultrasound-mediated transcranial imaging or actuation techniques. We report on the first observation of guided acoustic waves in the near field of an ex vivo human skull specimen in the frequency range between 0.2 and 1.5 MHz. In contrast to what was previously observed for guided wave propagation in thin rodent skulls, the guided wave observed in a higher-frequency regime corresponds to a quasi-Rayleigh wave, confined mostly to the cortical bone layer. The newly discovered near-field properties of the human skull are expected to facilitate the development of more efficient diagnostic and therapeutic techniques based on transcranial ultrasound. (C) 2018 World Federation for Ultrasound in Medicine & Biology. All rights reserved.

Current knowledge on the ultrasound wave propagation in the cranial bone is restricted to far-field observations. In order to extend our understanding on how ultrasound waves propagate in the skull, we use short laser pulses to excite ultrasound waves in water-immersed ex vivo mouse and human skulls and explored their near-field. The laser pulses (10 ns duration) of 532 nm are absorbed by a small layer of black burnish deposited on the skull's inner surface and generate ultrasound waves due to the thermoelastic effect. The acoustic near-field is mapped using a needle hydrophone close to the skull surface, following a three-dimensional scanning path derived from a previous pulse-echo scan of the skull with a spherically focused ultrasound transducer. The results for mouse and human skulls show different wave propagation regimes according to their differences in size, thickness, and internal structure. Leaky and non-leaky waves have been observed for both skull samples. Zero order Lamb modes were observed in the mouse skull, whereas Rayleigh-Lamb higher order modes can be observed in the human skull sample, presumably propagating in the outer cortical bone layer. Good agreement is found between the experiments and the multilayered flat plate model.

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.

Glucose sensing is pursued extensively in biomedical research and clinical practice for assessment of the carbohydrate and fat metabolism as well as in the context of an array of disorders, including diabetes, morbid obesity, and cancer. Currently used methods for real-time glucose measurements are invasive and require access to body fluids, with novel tools and methods for non-invasive sensing of the glucose levels highly desired. In this study, we introduce a near-infrared (NIR) optoacoustic spectrometer for sensing physiological concentrations of glucose within aqueous media and describe the glucose spectra within 850-1,900 nm and various concentration ranges. We apply the ratiometric and dictionary learning methods with a training set of data and validate their utility for glucose concentration measurements with optoacoustics in the probe dataset. We demonstrate the superior signal-to-noise ratio (factor of ~3.9) achieved with dictionary learning over the ratiometric approach across the wide glucose concentration range. Our data show a linear relationship between the optoacoustic signal intensity and physiological glucose concentration, in line with the results of optical spectroscopy. Thus, the feasibility of detecting physiological glucose concentrations using NIR optoacoustic spectroscopy is demonstrated, enabling the sensing glucose with ±10 mg/dl precision.

Background. Lichen planus (LP) is a T-cell mediated autoimmune disorder of unknown aetiology that affects the skin, nails, oral and genital mucous membranes. Conventionally, oral LP (OLP) is diagnosed through clinical assessment and histopathological confirmation by oral biopsy.Aim. To explore the use of time-resolved fluorescence spectroscopy (TRFS) to detect fluorescence lifetime changes between lesional OLP and perilesional normal mucosa.Methods. In this pilot study, measurements of lesional and perilesional buccal and mouth floor mucosa were conducted in vivo with a TRFS system. Histopathological findings were consistent with OLP in 8 out of 10 patients biopsied. Two patients with histopathological diagnoses of frictional hyperkeratosis and oral candidiasis, respectively, were excluded from the study.Results. Our preliminary data show that lifetime values in the 360-560 nm spectral range indicate a significant differentiation between normal and diseased tissue. In contrast to the standard oral biopsy procedure, this technique is noninvasive, painless, time-efficient and safe.Conclusions. Future studies are needed to better elucidate the diagnostic capability of TRFS and to further explore the sources of fluorescence contrast. This pilot study suggests that, based on fluorescence lifetime parameters, TRFS is a very promising technology for the development of a novel OLP diagnostic technique.

A critical issue associated with the clinical translation of fluorescence molecular imaging relates to the reproducibility of the collected measurements. In particular, images acquired from the same target using different fluorescence cameras may vary considerably when the employed systems have markedly different specifications. Methods that standardize fluorescence imaging are therefore becoming necessary for assessing the performance of fluorescence systems and agents and for providing a reference to the data collected. In the work presented herein we propose a composite phantom for integrating multiple targets within the field of view of a fluorescence camera. Each quadrant of this phantom resolves different fluorescence features: (1) sensitivity as a function of the optical properties; (2) sensitivity as a function of the depth from the top surface; (3) resolution of the fluorescence and optical imaging; and (4) cross-talk from the excitation light. In addition, there exist structures in the phantom for assessing homogeneity of the incident illumination. In order to validate our main hypothesis that standardization of fluorescence imaging systems is feasible through imaging such a phantom, we employed two systems of different specifications and quantified all relevant performance metrics. The derived results showcase the feasibility of fluorescence cameras calibration. Additionally, we demonstrate a methodology of comparing fluorescence cameras by means of benchmarking scoring. We expect that such approaches will boost the clinical translation of fluorescence molecular imaging and will allow for the investigation of novel fluorescence agents.

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas.Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME.VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas (<3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL.signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas.These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.

IEEE Optoacoustic (photoacoustic) endoscopy has shown potential to reveal complementary contrast to optical endoscopy methods, indicating clinical relevance. However operational parameters for accurate optoacoustic endoscopy must be specified for optimal performance. Recent support from the EU Horizon 2020 program ESOTRAC to develop a next-generation optoacoustic esophageal endoscope directs the interrogation of the optimal frequency required for accurate implementation. We simulated the frequency response of the esophagus wall and then validated the simulation results with experimental measurements of pig esophagus. Phantoms and fresh pig esophagus samples were measured using two detectors with central frequencies of 15 or 50 MHz, and the imaging performance of both detectors was compared. We analyzed the frequency bandwidth of optoacoustic signals in relation to morphological layer structures of the esophagus and found the 50 MHz detector to differentiate layer structures better than the 15 MHz detector. Furthermore, we identify the necessary detection bandwidth for visualizing esophagus morphology and selecting ultrasound transducers for future optoacoustic endoscopy of the esophagus.

In this work, two dedicated endoscopy systems are developed, and the imaging performance is characterized on phantoms and biological samples. Besides, optoacoustic endoscopy probes based on different scanning configurations, illumination settings and ultrasound transducers are built and analyzed regarding their influence on the endoscopic imaging performance. In addition to the technical development of the instrumentation, the necessary detection bandwidth for visualizing esophagus morphology and selecting ultrasound transducers for future optoacoustic endoscopy of the esophagus is studied. Finally, excised esophageal samples from pigs and human are measured to test the tissue imaging capability of the developed endoscopy systems, showing great clinical potential of esophagus imaging.

We introduce a contrast mechanism for visualizing blood vessels based on radiofrequency-induced second harmonic acoustic (RISHA) signals sensing blood conductivity. We develop a novel imaging system using commonly available inexpensive components, and demonstrate in vivo RISHA visualization of blood vessels based on low-power quasi-continuous radiofrequency excitation of tissue at frequencies of a few MHz. We show how the novel approach also implicitly enables radiofrequency-induced passive ultrasound imaging and can be readily applied to non-invasive imaging of blood vessels ex vivo and in vivo. We discuss the implications of non-invasive conductivity measurements in the context of biomedical applications.

MSOT has revolutionized biomedical imaging because it allows anatomical, functional, and molecular imaging of deep tissues in vivo in an entirely noninvasive, label-free, and real-time manner. This imaging modality works by pulsing light onto tissue, triggering the production of acoustic waves, which can be collected and reconstructed to provide high-resolution images of features as deep as several centimeters below the body surface. Advances in hardware and software continue to bring MSOT closer to clinical translation. Most recently, a clinical handheld MSOT system has been used to image brown fat tissue (BAT) and its metabolic activity by directly resolving the spectral signatures of hemoglobin and lipids. This opens up new possibilities for studying BAT physiology and its role in metabolic disease without the need to inject animals or humans with contrast agents. In this chapter, we overview how MSOT works and how it has been implemented in preclinical and clinical contexts. We focus on our recent work using MSOT to image BAT in resting and activated states both in mice and humans.

Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.

In vivo optical imaging modalities are mostly limited to cell cultures, superficial tissues, and intravital imaging since they lack either resolution or penetration depth.1 In contrast, optoacoustic (OA) imaging—combining features of optical and ultrasound imaging—has been used to visualize hemoglobin in depths of approximately 3 cm in patients with Crohn’s disease.2,3 Realizing an even higher resolution, raster-scanning OA mesoscopy (RSOM) provides intrinsic optical tissue contrast down to 10-20 μm resolution at still high penetration depths of several millimeters.

The emerging clinical use of targeted fluorescent agents heralds a shift in intraoperative imaging practices that overcome the limitations of human vision. However, in contrast to established radiological methods, no appropriate performance specifications and standards have been established in fluorescence molecular imaging. Moreover, the dependence of fluorescence signals on many experimental parameters and the use of wavelengths ranging from the visible to short-wave infrared (400–1,700 nm) complicate quality control in fluorescence molecular imaging. Here, we discuss the experimental parameters that critically affect fluorescence molecular imaging accuracy, and introduce the concept of high-fidelity fluorescence imaging as a means for ensuring reliable reproduction of fluorescence biodistribution in tissue.

The molecular mediator and functional significance of meal-assosiated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-synmpathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.

The Grüneisen parameter is an essential factor in biomedical photoacoustic (PA) diagnostics. In most PA imaging applications, the variation of the Grüneisen parameter with tissue type is insignificant. This is not the case for PA imaging and characterization of lipids, as they have a very distinct Grüneisen parameter compared with other tissue types. One example of PA applications involving lipids is the imaging and characterization of atherosclerotic plaques. Intravascular photoacoustic (IVPA) imaging is a promising diagnostic tool that can evaluate both plaque severity and composition. The literature for IVPA has mainly focused on using the difference in absorption coefficients between plaque components and healthy arterial tissues. However, the Grüneisen parameters for lipids and their behavior with temperature have not been well established in the literature. In this study we employ frequency-domain photoacoustic measurements to estimate the Grüneisen parameter by virtue of the ability of this modality to independently measure the absorption coefficient and the Grüneisen parameter through the use of the phase channel. The values of the Grüneisen parameters of some lipids are calculated as functions of temperature in the range 25-45 °C.

The Langendorff-perfused heart technique has become the model of choice for multiparametric optical mapping of cardiac function and electrophysiology. However, photon scattering in tissues represents a significant drawback of the optical imaging approach, fundamentally limiting its mapping capacity to the heart surface. This work presents the first implementation of the optoacoustic approach for 4D imaging of the entire beating isolated mouse heart. The method combines optical excitation and acoustic detection to simultaneously render rich optical contrast and high spatio-temporal resolution at centimeter-scale depths. We demonstrate volumetric imaging of deeply located cardiac features, including the interventricular septum, chordae tendineae, and papillary muscles while further tracking the heart beat cycle and the motion of the pulmonary, mitral, and tricuspid valves in real time. The technique possesses a powerful combination between high imaging depth, fast volumetric imaging speed, functional and molecular imaging capacities not available with other imaging modalities currently used in cardiac research.

Photoacoustic mesoscopy (PAMe), offering high-resolution (sub-100-μm) and high optical contrast imaging at the depth of 1-10 mm, generally obtains massive collection data using a high-frequency focused ultrasonic transducer. The spatial impulse response (SIR) of this focused transducer causes the distortion of measured signals in both duration and amplitude. Thus, the reconstruction method considering the SIR needs to be investigated in the computation-economic way for PAMe. Here, we present a modified back-projection algorithm, by introducing a SIR-dependent calibration process using a non-satationary convolution method. The proposed method is performed on numerical simulations and phantom experiments of microspheres with diameter of both 50 μm and 100 μm, and the improvement of image fidelity of this method is proved to be evident by methodology parameters. The results demonstrate that, the images reconstructed when the SIR of transducer is accounted for have higher contrast-to-noise ratio and more reasonable spatial resolution, compared to the common back-projection algorithm.

Optoacoustic (photoacoustic) imaging assumes that the detected signal varies linearly with laser energy. However, nonlinear intensity responses as a function of light fluence have been suggested in optoacoustic microscopy, that is, within the first millimeter of tissue. In this study, we explore the presence of nonlinearity deeper in tissue (similar to 4 mm), as it relates to optoacoustic mesoscopy, and investigate the fluence required to delineate a switch from linear to nonlinear behavior. Optoacoustic signal nonlinearity is studied for different materials, different wavelengths and as a function of changes in the scattering and absorption coefficient of the medium imaged. We observe fluence thresholds in the mJ/cm(2) range and preliminary find that different materials may exhibit different nonlinearity patterns. We discuss the implications of nonlinearity in relation to image accuracy and quantification in optoacoustic tomography.

Nanomaterials are of enormous value for biomedical applications because of their customizable features. However, the material properties of nanomaterials can be altered substantially by interactions with tissue thus making it important to assess them in the specific biological context to understand and tailor their effects. Here, a genetically controlled system is optimized for cellular uptake of superparamagnetic ferritin and subsequent trafficking to lysosomes. High local concentrations of photoabsorbing magnetoferritin give robust contrast in optoacoustic imaging and allow for selective photoablation of cells overexpressing ferritin receptors. Genetically controlled uptake of the biomagnetic nanoparticles also strongly enhances third-harmonic generation due to the change of refractive index caused by the magnetite-protein interface of ferritins entrapped in lysosomes. Selective uptake of magnetoferritin furthermore enables sensitive detection of receptor-expressing cells by magnetic resonance imaging, as well as efficient magnetic cell sorting and manipulation. Surprisingly, a substantial increase in the blocking temperature of lysosomally entrapped magnetoferritin is observed, which allows for specific ablation of genetically defined cell populations by local magnetic hyperthermia. The subcellular confinement of superparamagnetic ferritins thus enhances their physical properties to empower genetically controlled interrogation of cellular processes with deep tissue penetration.

The study aimed to evaluate the clinical feasibility of hybrid ultrasound/multispectral optoacoustic tomography (MSOT) for assessing microvascular dysfunction in systemic sclerosis (SSc). A handheld US/MSOT imaging system was applied for imaging patients diagnosed with SSc (n = 7) and healthy volunteers (n = 8). Semiquantitative MSOT values for deoxygenated (HbR), oxygenated (HbO(2)) and total haemoglobin (HbT) were analysed for subcutaneous finger tissue of both hands (8 fingers per subject, 120 fingers in total) and used to assess disease activity (progressive vs stable). Grouped data were compared by one-way nested analysis of variance, Tukey post-hoc test as well as student's t test were used for statistical analysis.Subcutaneous finger tissue of patients with SSc provided significantly lower MSOT values for HbO(2) (26.16 +/- 0.71 vs 38.2 +/- 1.54, P =.023) and HbT (55.92 +/- 1.62 vs 72.46 +/- 1.90, P =.018) compared to healthy volunteers. Patients with progressive SSc had significantly lower MSOT values compared to patients with stable disease and healthy volunteers.This pilot study shows the feasibility of MSOT imaging to resolve microvascular dysfunction in SSc as a marker of disease activity. By providing biological tissue properties not revealed by other imaging modalities, MSOT might help to grade SSc non-invasively and monitor early therapy response.

IMPORTANCE Differential diagnosis of congenital vascular anomalies is challenging, and misdiagnosis is frequent. Vascular malformations are considered one of the most difficult vascular diseases to treat. A new imaging approach that visualizes anatomical features and quantitatively assesses molecular biomarkers noninvasively would aid diagnosis and monitoring of treatment response of vascular malformations.OBJECTIVE To evaluate multispectral optoacoustic tomography (MSOT) for noninvasive assessment of molecular biomarkers for diagnosis and therapeutic monitoring of vascular malformations.DESIGN, SETTING, AND PARTICIPANTS This pilot study examined 6 patients with arteriovenous malformation (AVM) and 6 patients with venous malformation (VM) diagnosed according to the classification system of the International Society for the Study of Vascular Anomalies. All patients underwent clinical hybrid MSOT/ultrasonographic (US) imaging before and after treatment at an interdisciplinary vascular malformations clinic by trained MSOT and US examiners. Examiners were blinded to the patient history and stage of disease. Data were collected from April 11 to 25, 2017, and analyzed from May 1 to October 31, 2017.INTERVENTIONS Clinical hybrid MSOT/US imaging was performed before or within 1 week after endovascular embolization (for AVM) or percutaneous sclerotherapy (for VM).MAIN OUTCOMES AND MEASURES Region-of-interest analysis of the lesion and contralateral healthy tissue revealed quantitative values for oxygenated (HbO(2)) and deoxygenated (HbR) hemoglobin by spectral unmixing of optoacoustic data acquired at multiple wavelengths. The HbO(2):HbR ratio was calculated for healthy tissue and for AVM and VM before and after treatment.RESULTS Twelve patients (9 female and 3 male; mean [SD] age, 23 [18] years; age range, 6-59 years) with vascular malformations (6 with AVMs and 6 with VMs) were included. Significantly higher HbO(2):HbR ratios for AVMs (mean [SEM], 1.82 [0.08] vs 0.89 [0.03]; P < .001) and for VMs (mean [SEM], 1.12 [0.04] vs 0.89 [0.03]; P = .001) were found on MSOT tissue compared with healthy tissue. Significantly higher HbO(2):HbR ratios for AVMs compared with VMs (mean [SEM], 1.82 [0.08] vs 1.12 [0.04]; P < .001) were also found. Therefore, MSOT provided intrinsic biomarker patterns to distinguish both vascular malformations. After therapy, the HbO(2):HbR ratio dropped in correlation to treatment success validated by magnetic resonance imaging or angiography.CONCLUSIONS AND RELEVANCE This study suggests that different types of vascular malformations are clearly distinguished by MSOT-based, noninvasive assessment of hemoglobin levels in vascular malformations. The therapy effects found in this study could be instantly visualized, and this may offer a new tool for noninvasive diagnosis and monitoring of vascular malformations.

Changes in hemodynamic parameters are directly linked to biological function and physiological activity. Characterization of hemodynamics is commonly performed by Doppler ultrasound, which provides accurate measurements of blood flow velocity. Multi-spectral optoacoustic tomography is rapidly undergoing clinical translation fostered by its unique and complementary capacity for label-free mapping of the blood volume and the distribution of oxy- and deoxy-hemoglobin in blood. Here we report on a hybrid optoacoustic and ultrasound imaging approach that enables multi-modal imaging of blood flow and oxygen state using a multi-segment detector array. We further demonstrate rendering of multi-modal pulse-echo ultrasound, multi-spectral optoacoustic tomography, and color Doppler images from carotid artery of a healthy subject.

Changes in hemodynamic parameters are directly linked to biological function and physiological activity. Characterization of hemodynamics is commonly performed by Doppler ultrasound, which provides accurate measurements of blood flow velocity. Multi-spectral optoacoustic tomography is rapidly undergoing clinical translation fostered by its unique and complementary capacity for label-free mapping of the blood volume and the distribution of oxy- and deoxy-hemoglobin in blood. Here we report on a hybrid optoacoustic and ultrasound imaging approach that enables multi-modal imaging of blood flow and oxygen state using a multi-segment detector array. We further demonstrate rendering of multi-modal pulse-echo ultrasound, multi-spectral optoacoustic tomography, and color Doppler images from carotid artery of a healthy subject.

An impediment to a mechanistic understanding of how some species sense the geomagnetic field ("magnetoreception") is the lack of vertebrate genetic models that exhibit well-characterized magnetoreceptive behavior and are amenable to whole-brain analysis. We investigated the genetic model organisms zebrafish and medaka, whose young stages are transparent and optically accessible. In an unfamiliar environment, adult fish orient according to the directional change of a magnetic field even in darkness. To enable experiments also in juveniles, we applied slowly oscillating magnetic fields, aimed at generating conflicting sensory inputs during exploratory behavior. Medaka (but not zebrafish) increase their locomotor activity in this assay. Complementary brain activity mapping reveals neuronal activation in the lateral hindbrain during magnetic stimulation. These comparative data support magnetoreception in teleosts, provide evidence for a light-independent mechanism, and demonstrate the usefulness of zebrafish and medaka as genetic vertebrate models for studying the biophysical and neuronal mechanisms underlying magnetoreception.

In the original version of this Article, Oryzias latipes was incorrectly spelt Oryzias lapites in the main text and in Fig. 1. These errors have been corrected in both the PDF and HTML versions of the Article.

Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous monitoring of NP delivery. BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-1×10 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed by paw volume measurements with µCT and by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by optical imaging and by fluorescence microscopy. Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. , Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h , where they were preferentially taken up by peribronchial and alveolar M2 macrophages. Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.

Objective: Monitoring emerging vascular-targeted photodynamic therapy (VTP) and understanding the time-dynamics of treatment effects remains challenging. We interrogated whether handheld multispectral optoacoustic tomography (MSOT) could noninvasively monitor the effect of VTP using WST11, a vascular-acting photosensitizer, on tumor tissues over time using a renal cell cancer mouse model. We also investigated whether MSOT illumination can induce VTP, to implement a single-modality theranostic approach. Materials and Methods: Eight BalB/c mice were subcutaneously implanted with murine renal adenocarcinoma cells (RENCA) on the flank. Three weeks later VTP was performed (10 min continuous illumination at 753 nm following intravenous infusion) using WST11 or saline as control. Handheld MSOT images were collected prior to VTP administration and subsequently thereafter over the course of the first hour, at 24 and 48 h. Data collected were unmixed for blood oxygen saturation in tissue (SO 2 ) based on the spectral signatures of deoxy- and oxygenated hemoglobin. Changes in oxygen saturation over time, relative to baseline, were examined by paired t-test for statistical significance (p < 0.05). In-vivo findings were corroborated by histological analyses of the tumor tissue. Results: MSOT is shown to prominently resolve changes in oxygen saturation in tumors within the first 20 min post WST11-VTP treatment. Within the first hour post-treatment, SO 2 decreased by more than 60% over baseline (p < 0.05), whereas it remained unchanged (p > 0.1) in the sham-treated group. Moreover, unlike in the control group, SO 2 in treated tumors further decreased over the course of 24 to 48 h post-treatment, concomitant with the propagation of profound central tumor necrosis present in histological analysis. We further show that pulsed MSOT illumination can activate WST11 as efficiently as the continuous wave irradiation employed for treatment. Conclusion: Handheld MSOT non-invasively monitored WST11-VTP effects based on the SO 2 signal and detected blood saturation changes within the first 20 min post-treatment. MSOT may potentially serve as a means for both VTP induction and real-time VTP monitoring in a theranostic approach.

Ambient inhalable particulate matter (PM) is a serious health concern worldwide, but especially so in China where high PM concentrations affect huge populations. Atmospheric processes and emission sources cause spatial and temporal variations in PM concentration and chemical composition, but their influence on the toxicological characteristics of PM are still inadequately understood.In this study, we report an extensive chemical and toxicological characterization of size-segregated urban air inhalable PM collected in August and October 2013 from Nanjing, and assess the effects of atmospheric processes and likely emission sources. A549 human alveolar epithelial cells were exposed to day- and nighttime PM samples (25, 75, 150, 200, 300 mu g/ml) followed by analyses of cytotoxicity, genotoxicity, cell cycle, and inflammatory response.PM10-2.5 and PM0.2 caused the greatest toxicological responses for different endpoints, illustrating that particles with differing size and chemical composition activate distinct toxicological pathways in A549 cells. PM10-2.5 displayed the greatest oxidative stress and genotoxic responses; both were higher for the August samples compared with October. In contrast, PM0.2 and PM2.5-1.0 samples displayed high cytotoxicity and substantially disrupted cell cycle; August samples were more cytotoxic whereas October samples displayed higher cell cycle disruption. Several components associated with combustion, traffic, and industrial emissions displayed strong correlations with these toxicological responses. The lower responses for PM1.0-0.2 compared to PM0.2 and PM2.5-1.0 indicate diminished toxicological effects likely due to aerosol aging and lower proportion of fresh emission particles rich in highly reactive chemical components in the PM1.0-0.2 fraction.Different emission sources and atmospheric processes caused variations in the chemical composition and toxicological responses between PM fractions, sampling campaigns, and day and night. The results indicate different toxicological pathways for coarse-mode particles compared to the smaller particle fractions with typically higher content of combustion-derived components. The variable responses inside PM fractions demonstrate that differences in chemical composition influence the induced toxicological responses.

Purpose of Review Advances in technology have expanded telemedicine opportunities covering medical practice, research, and education. This is of particular importance in movement disorders (MDs), where the combination of disease progression, mobility limitations, and the sparse distribution of MD specialists increase the difficulty to access. In this review, we discuss the prospects, challenges, and strategies for telemedicine in MDs.Recent Findings Telemedicine for MDs has been mainly evaluated in Parkinson's disease (PD) and compared to in-office care is cost-effective with similar clinical care, despite the barriers to engagement. However, particular groups including pediatric patients, rare MDs, and the use of telemedicine in underserved areas need further research.Summary Interdisciplinary telemedicine and tele-education for MDs are feasible, provide similar care, and reduce travel costs and travel time compared to in-person visits. These benefits have been mainly demonstrated for PD but serve as a model for further validation in other movement disorders.

The quantification of hemoglobin oxygen saturation (sO(2)) with multispectral optoacoustic (OA) (photoacoustic) tomography (MSOT) is a complex spectral unmixing problem, since the OA spectra of hemoglobin are modified with tissue depth due to depth (location) and wavelength dependencies of optical fluence in tissue. In a recent work, a method termed eigenspectra MSOT (eMSOT) was proposed for addressing the dependence of spectra on fluence and quantifying blood sO(2) in deep tissue. While eMSOT offers enhanced sO(2) quantification accuracy over conventional unmixing methods, its performance may be compromised by noise and image reconstruction artifacts. In this paper, we propose a novel Bayesian method to improve eMSOT performance in noisy environments. We introduce a spectral reliability map, i.e., a method that can estimate the level of noise superimposed onto the recorded OA spectra. Using this noise estimate, we formulate eMSOT as a Bayesian inverse problem where the inversion constraints are based on probabilistic graphical models. Results based on numerical simulations indicate that the proposed method offers improved accuracy and robustness under high noise levels due the adaptive nature of the Bayesian method.

Purpose of Review Advances in technology have expanded telemedicine opportunities covering medical practice, research, and education. This is of particular importance in movement disorders (MDs), where the combination of disease progression, mobility limitations, and the sparse distribution of MD specialists increase the difficulty to access. In this review, we discuss the prospects, challenges, and strategies for telemedicine in MDs.Recent Findings Telemedicine for MDs has been mainly evaluated in Parkinson's disease (PD) and compared to in-office care is cost-effective with similar clinical care, despite the barriers to engagement. However, particular groups including pediatric patients, rare MDs, and the use of telemedicine in underserved areas need further research.Summary Interdisciplinary telemedicine and tele-education for MDs are feasible, provide similar care, and reduce travel costs and travel time compared to in-person visits. These benefits have been mainly demonstrated for PD but serve as a model for further validation in other movement disorders.

Deposition of amyloid plaques in limbic and associative cortices is amongst the most recognized histopathologic hallmarks of Alzheimer’s disease. Despite decades of research, there is a lack of consensus over the impact of plaques on neuronal function, with their role in cognitive decline and memory loss undecided. Evidence has emerged suggesting complex and localized axonal pathology around amyloid plaques, with a significant fraction of swellings and dystrophies becoming enriched with putative synaptic vesicles and presynaptic proteins normally colocalized at hotspots of transmitter release. In the absence of hallmark active zone proteins and postsynaptic receptive elements, the axonal swellings surrounding amyloid plaques have been suggested as sites for ectopic release of glutamate, which under reduced clearance can lead to elevated local excitatory drive. Throughout this review, we consider the emerging data suggestive of amyloid plaques as hotspots of compulsive glutamatergic activity. Evidence for local and long-range effects of nonsynaptic glutamate is discussed in the context of circuit dysfunctions and neurodegenerative changes of Alzheimer’s disease.

While the extensive hunt for therapeutics combating Alzheimer's disease (AD) has fallen short of delivering effective treatments, breakthroughs towards understanding the disease mechanisms and identifying areas for future research have nevertheless been enabled. The majority of clinical trials with beta- and gamma-secretase modulators have been suspended from additional studies or terminated due to toxicity issues and health concerns. The lack of progress in developing innovative AD therapies has also prompted a resurgence of interest in more traditional symptomatic treatments with cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists, as well as in the research of immune response modulators. Recently, evidence has emerged showing that inhibitors of arginine metabolism and in particular blockers of arginase, an enzyme that catalyzes the breakdown of L-arginine, could present an effective therapeutic candidate for halting the progression of AD and boosting cognition and memory. In this commentary, we present a brief overview of reports on arginase inhibitors in AD mouse models and discuss emerging advantages and areas for careful consideration on the road to clinical translation.

Downloading of the abstract is permitted for personal use only. Medium intensity focused ultrasound (MIFU) holds promise in important clinical applications. Generally, the aim in MIFU is to stimulate physiological mechanisms that reinforce healing responses, avoiding reaching temperatures that can cause permanent tissue damage. The outcome of interventions is then strongly affected by the temperature distribution in the treated region, and accurate monitoring represents a significant clinical need. In this work, we showcase the capacities of 4D optoacoustic imaging to monitor tissue heating during MIFU. The proposed method allows localizing the ultrasound focus, estimating the peak temperature and measuring the size of the heat-affected volume. Calibration experiments in a tissue-mimicking phantom demonstrate that the optoacoustically-estimated temperature accurately matches thermocouple readings. The good performance of the suggested approach in real tissues is further showcased in experiments with bovine muscle samples.

Downloading of the abstract is permitted for personal use only. Photoablative laser therapy is in common use for selective destruction of malignant masses, vascular and brain abnormalities. Tissue ablation and coagulation are irreversible processes occurring shortly after crossing a certain thermal exposure threshold. As a result, accurate mapping of the temperature field is essential for optimizing the outcome of these clinical interventions. Here we demonstrate four-dimensional optoacoustic temperature mapping of the entire photoablated region. Accuracy of the method is investigated in tissue-mimicking phantom experiments. Deviations of the volumetric optoacoustic temperature readings provided at 40ms intervals remained below 10% for temperature elevations above 3°C, as validated by simultaneous thermocouple measurements. The excellent spatio-temporal resolution of the new temperature monitoring approach aims at improving safety and efficacy of laser-based photothermal procedures.

Medium-intensity focused ultrasound (MIFU) concerns therapeutic ultrasound interventions aimed at stimulating physiological mechanisms to reinforce healing responses without reaching temperatures that can cause permanent tissue damage. The therapeutic outcome is strongly affected by the temperature distribution in the treated region and its accurate monitoring represents an unmet clinical need. In this work, we investigate on the capacities of four-dimensional optoacoustic tomography to monitor tissue heating with MIFU. Calibration experiments in a tissue-mimicking phantom have confirmed that the optoacoustically-estimated temperature variations accurately match the simultaneously acquired thermocouple readings. The performance of the suggested approach in real tissues was further shown with bovine muscle samples. Volumetric temperature maps were rendered in real time, allowing for dynamic monitoring of the ultrasound focal region, estimation of the peak temperature and the size of the heat-affected volume.

State-of-the-art optoacoustic tomographic imaging systems have been shown to attain three-dimensional (3D) frame rates of the order of 100 Hz. While such a high volumetric imaging speed is beyond reach for other bio-imaging modalities, it may still be insufficient to accurately monitor some faster events occurring on a millisecond scale. Increasing the 3D imaging rate is usually hampered by the limited throughput capacity of the data acquisition electronics and memory used to capture vast amounts of the generated optoacoustic (OA) data in real time. Herein, we developed a sparse signal acquisition scheme and a total-variation-based reconstruction approach in a combined space-time domain in order to achieve 3D OA imaging at kilohertz rates. By continuous monitoring of freely swimming zebrafish larvae in a 3D region, we demonstrate that the new approach enables significantly increasing the volumetric imaging rate by using a fraction of the tomographic projections without compromising the reconstructed image quality. The suggested method may benefit studies looking at ultrafast biological phenomena in 3D, such as large-scale neuronal activity, cardiac motion, or freely behaving organisms.

Flatworms of the species Schmidtea mediterranea are immortal-adult animals contain a large pool of pluripotent stem cells that continuously differentiate into all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. By combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from transcriptional changes, and a lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterized gene expression changes during differentiation and discovered cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at high resolution.

Fluorescence-guided surgery (FGS) and other interventions are rapidly evolving as a class of technologically driven interventional approaches in which many surgical specialties visualize fluorescent molecular tracers or biomarkers through associated cameras or oculars to guide clinical decisions on pathological lesion detection and excision/ablation. The technology has been commercialized for some specific applications, but also presents technical challenges unique to optical imaging that could confound the utility of some interventional procedures where real-time decisions must be made. Accordingly, the AAPM has initiated the publication of this Blue Paper of The Emerging Technology Working Group (TETAWG) and the creation of a Task Group from the Therapy Physics Committee within the Treatment Delivery Subcommittee. In describing the relevant issues, this document outlines the key parameters, stakeholders, impacts, and outcomes of clinical FGS technology and its applications. The presentation is not intended to be conclusive, but rather to inform the field of medical physics and stimulate the discussions needed in the field with respect to a seemingly low-risk imaging technology that has high potential for significant therapeutic impact. This AAPM Task Group is working toward consensus around guidelines and standards for advancing the field safely and effectively.

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.

Successful breast conserving cancer surgeries come along with tumor free resection margins and account for cosmetic outcome. Positive margins increase the likelihood of tumor recurrence. Intra-operative fluorescence molecular imaging (IFMI) aims to focus surgery on malignant tissue thus substantially lowering the presence of positive margins as compared with standard techniques of breast conservation (ST). A goal of this paper is to assess the incremental number of surgeries and costs of IFMI vs. ST. METHODS: We developed a decision analytical model and applied it for an early evaluation approach. Given uncertainty we considered that IFMI might reduce the proportion of positive margins found by ST from all to none and this proportion is assumed to be reduced to 10% for the base case. Inputs included data from the literature and a range of effect estimates. For the costs of IFMI, respective cost components were added to those of ST. RESULTS: The base case reduction lowered number of surgeries (mean [95% confidence interval]) by 0.22 [0.15; 0.30] and changed costs (mean [95% confidence interval]) by €-663 [€-1,584; €50]. A tornado diagram identified the Diagnosis Related Group (DRG) costs, the proportion of positive margins of ST, the staff time saving factor and the duration of frozen section analysis (FSA) as important determinants of this cost. CONCLUSIONS: These early results indicate that IFMI may be more effective than ST and through the reduction of positive margins it is possible to save follow-up surgeries-indicating further health risk-and to save costs through this margin reduction and the avoidance of FSA.

Breast cancer and Glioblastoma brain cancer are severe malignancies with poor prognosis. In this study, primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. High resolution imaging of both primary and secondary cancer cell spheroids is possible using a custom multi-angle Light Sheet Fluorescence Microscope. Such a technique is successful in realizing preclinical drug screening, while enables the discrimination among physiologic tumor parameters.

The guest editors introduce a feature issue containing papers based on research presented at the OSA Biophotonics Congress (the former BIOMED) held in Hollywood, FL, 2-6 April, 2018.

Metabolism is a fundamental process of life. However, non-invasive measurement of local tissue metabolism is limited today by a deficiency in adequate tools for in vivo observations. We designed a multi-modular platform that explored the relation between local tissue oxygen consumption, determined by label-free optoacoustic measurements of hemoglobin, and concurrent indirect calorimetry obtained during metabolic activation of brown adipose tissue (BAT). By studying mice and humans, we show how video-rate handheld multi-spectral optoacoustic tomography (MSOT) in the 700-970 nm spectral range enables non-invasive imaging of BAT activation, consistent with positron emission tomography findings. Moreover, we observe BAT composition differences between healthy and diabetic tissues. The study consolidates hemoglobin as a principal label-free biomarker for longitudinal non-invasive imaging of BAT morphology and bioenergetics in situ. We also resolve water and fat components in volunteers, and contrast MSOT readouts with magnetic resonance imaging data.

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade-offs between the spatiotemporal resolution, field-of-view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual-wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large-scale cerebral vascular networks. The system offers 3-dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 x 8 mm(2), thus covering the entire cortical vasculature in mice. The large-scale OA imaging capacity is complemented by simultaneously acquired pulse-echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.

Radio frequency (RF) catheter ablation is commonly used to eliminate dysfunctional cardiac tissue by heating via an alternating current. Clinical outcomes are highly dependent on careful anatomical guidance, electrophysiological mapping, and careful RF power titration during the procedure. Yet, current treatments rely mainly on the expertise of the surgeon to assess lesion formation, causing large variabilities in the success rate. We present an integrated catheter design suitable for simultaneous RF ablation and real-time optoacoustic monitoring of the forming lesion. The catheter design utilizes copper-coated multimode light guides capable of delivering both ablation current and near-infrared pulsed-laser illumination to the target tissue. The generated optoacoustic responses were used to visualize the ablation lesion formation in an ex-vivo bovine heart specimen in 3D. The presented catheter design enables the monitoring of ablation lesions with high spatiotemporal resolution while the overall therapy-monitoring approach remains compatible with commercially available catheter designs.

Electrosurgery, i.e. the application of radiofrequency current for tissue ablation, is a frequently used treatment for many cardiac arrhythmias. Electrophysiological and anatomic mapping, as well as careful radiofrequency power control typically guide the radiofrequency ablation procedure. Despite its widespread application, accurate monitoring of the lesion formation with sufficient spatio-temporal resolution remains challenging with the existing imaging techniques. We present a novel integrated catheter for simultaneous radiofrequency ablation and optoacoustic monitoring of the lesion formation in real time and 3D. The design combines the delivery of both electric current and optoacoustic excitation beam in a single catheter consisting of copper-coated multimode light-guides and its manufacturing is described in detail. The electrical current causes coagulation and desiccation while the excitation light is locally absorbed, generating OA responses from the entire treated volume. The combined ablation-monitoring capabilities were verified using ex-vivo bovine tissue. The formed ablation lesions showed a homogenous coagulation while the ablation was monitored in realtime with a volumetric frame rate of 10 Hz over 150 seconds.

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca2+-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.

PurposeDiabetes is associated with a deterioration of the microvasculature in brown adipose tissue (BAT) and with a decrease in its metabolic activity. Multispectral optoacoustic tomography has been recently proposed as a new tool capable of differentiating healthy and diabetic BAT by observing hemoglobin gradients and microvasculature density in cross-sectional (2D) views. We report on the use of spiral volumetric optoacoustic tomography (SVOT) for an improved characterization of BAT.ProceduresA streptozotocin-induced diabetes model and control mice were scanned with SVOT. Volumetric oxygen saturation (sO(2)) as well as total blood volume (TBV) in the subcutaneous interscapular BAT (iBAT) was quantified. Segmentation further enabled separating feeding and draining vessels from the BAT anatomical structure.ResultsScanning revealed a 46% decrease in TBV and a 25% decrease in sO(2) in the diabetic iBAT with respect to the healthy control.ConclusionsThese results suggest that SVOT may serve as an effective tool for studying the effects of diabetes on BAT. The volumetric optoacoustic imaging probe used for the SVOT scans can be operated in a handheld mode, thus potentially providing a clinical translation route for BAT-related studies with this imaging technology.

Optoacoustic imaging was for a long time concerned with the reconstruction of energy density or optical properties. In this work, we present the full inverse problem with respect to optical absorption and diffusion as well as speed of sound and mass density. The inverse problem is solved by an iterative gradient-based optimization procedure. Since the ill-conditioning increases with the number of sought parameters, we propose two approaches to improve the conditioning. The first approach is based on the reduction of the size of the basis for the parameter spaces, that evolves according to the particular characteristics of the solution, while maintaining the flexibility of element-wise parameter selection. The second approach is a material identification technique that incorporates prior knowledge of expected material types and uses the acoustical gradients to identify materials uniquely. We present numerical studies to illustrate the properties and functional principle of the proposed methods. Significant convergence speed-ups are gained by the two approaches countering ill-conditioning. Additionally, we show results for the reconstruction of a mouse brain from in vivo measurements.

The prostate-specific membrane antigen (PSMA)-targeted radiotracers Ga-68/Lu-177-PSMA-I&T and Tc-99m-PSMA-I&S (for imaging and surgery) are currently successfully used for clinical PET imaging, radionuclide therapy, and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a PSMA-I&T-based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated. Methods: The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T, and their corresponding Ga-nat-/Ga-68- and Lu-nat/Lu-177 counterparts was determined in LNCaP cells via competitive binding assays (IC50) and dual-tracer radioligand and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17 SCID and LNCaP xenograft-bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of 1 tumor-bearing animal as well as PSMA immunohistochemistry and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed. Results: Compared with the parent PSMA-I&T analogs, the PSMA affinities of PSMA-I&F and its Ga-nat-/Lu-nat-complexes remained high and unaffected by dye conjugation (7.9 < IC50 < 10.5 nM for all ligands). The same was observed for the internalization of Ga-68- and Lu-177-PSMA-I&F. In vivo, blood clearance of Ga-68- and Lu-177-PSMA-I&F was only slightly delayed by high plasma protein binding (94%-95%), and very low accumulation in nontarget organs was observed already at 1 h after injection. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5% +/- 1.8 percentage injected dose per gram) and the kidneys (106% +/- 23 percentage injected dose per gram). Tumor-to-background ratios of 2.1, 5.2, 9.6, and 9.6 for blood, liver, intestines, and muscle, respectively, at 1 h after injection led to excellent imaging contrast in Ga-68-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by immunohistochemistry. Conclusion: Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, Ga-68/Lu-177-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T-based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

Downloading of the abstract is permitted for personal use only. Optoacoustic microscopy (OAM) has enabled high-resolution, label-free imaging of tissues at depths not achievable with purely optical microscopy. However, widespread implementation of OAM into existing epi-illumination microscopy setups is often constrained by the performance and size of the commonly used piezoelectric ultrasound detectors. In this work, we introduce a novel acoustic detector based on a π-phase-shifted fiber Bragg grating (π-FBG) interferometer embedded inside an ellipsoidal acoustic cavity. The cavity enables seamless integration of epi-illumination OAM into existing microscopy setups by decoupling the acoustic and optical paths between the microscope objective and the sample. The cavity also acts as an acoustic condenser, boosting the sensitivity of the π-FBG and enabling cost effective CW-laser interrogation technique. We characterize the sensor's sensitivity and bandwidth and demonstrate hybrid OAM and second-harmonic imaging of phantoms and mouse tissue in vivo.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Pulsed laser diodes may offer a smaller, less expensive alternative to conventional optoacoustic laser sources; however they do not provide pulse rates faster than a few tens of kHz and emit at wavelengths only within the near-infrared region. We investigated whether continuous wave (CW) laser diodes, which are available in visible and near-infrared regions, can be good optoacoustic light sources when overdriven with a peak current >40-fold higher than the CW absolute maximum. We found that overdriven CW diodes provided ∼10 ns pulses of ∼200 nJ/pulse and repetition rates higher than 600 kHz without being damaged, outperforming many pulsed laser diodes. Using this system, we obtained images of phantoms and mouse ear and human arm in vivo, confirming their use in optoacoustic imaging and sensing.

Reversibly switchable fluorescent proteins (rsFPs) have had a revolutionizing effect on life science imaging due to their contribution to sub-diffraction-resolution optical microscopy (nanoscopy). Initial studies showed that their use as labels could also be highly beneficial for emerging photo- or optoacoustic imaging. It could be shown that their use in optoacoustics (i) strongly improves the imaging contrast-to-noise ratio due to modulation and locked-in detection, (ii) facilitates fluence calibration, affording precise measurements of physiological parameters, and finally (iii) could boost spatial resolution following similar concepts as used for nanoscopy. However, rsFPs show different photophysical behavior in optoacoustics than in optical microscopy because optoacoustics requires pulsed illumination and depends on signal generation via nonradiative energy decay channels. This implies that rsFPs optimized for fluorescence imaging may not be ideal for optoacoustics. Here, we analyze the photophysical behavior of a broad range of rsFPs with optoacoustics and analyze how the experimental factors central to optoacoustic imaging influence the different types of rsFPs. Finally, we discuss how knowledge of the switching behavior can be exploited for various optoacoustic imaging approaches using sophisticated temporal unmixing schemes.

Atherosclerotic plaques can remain quiescent for years, but become life threatening upon rupture or disruption, initiating clot formation in the vessel lumen and causing acute myocardial infarction and ischemic stroke. Whether and how a plaque ruptures is determined by its macroscopic structure and microscopic composition. Rupture-prone plaques usually consist of a thin fibrous cap with few smooth muscle cells, a large lipid core, a dense infiltrate of inflammatory cells, and neovessels. Such lesions, termed high-risk plaques, can remain asymptomatic until the thrombotic event. Various imaging technologies currently allow visualization of morphological and biological characteristics of high-risk atherosclerotic plaques. Conventional protocols are often complex and lack specificity for high-risk plaque. Conversely, new imaging approaches are emerging which may overcome these limitations. Validation of these novel imaging techniques in preclinical models of atherosclerosis is essential for effective translational to clinical practice. Imaging the vessel wall, as well as its biological milieu in small animal models, is challenging because the vessel wall is a small structure that undergoes continuous movements imposed by the cardiac cycle as it is adjacent to circulating blood. The focus of this paper is to provide a state-of-the-art review on techniques currently available for preclinical imaging of atherosclerosis in small animal models and to discuss the advantages and limitations of each approach.

Originally developed for diagnostic ultrasound imaging, piezoelectric transducers are the most widespread technology employed in optoacoustic (photoacoustic) signal detection. However, the detection requirements of optoacoustic sensing and imaging differ from those of conventional ultrasonography and lead to specifications not sufficiently addressed by piezoelectric detectors. Consequently, interest has shifted to utilizing entirely optical methods for measuring optoacoustic waves. All-optical sound detectors yield a higher signal-to-noise ratio per unit area than piezoelectric detectors and feature wide detection bandwidths that may be more appropriate for optoacoustic applications, enabling several biomedical or industrial applications. Additionally, optical sensing of sound is less sensitive to electromagnetic noise, making it appropriate for a greater spectrum of environments. In this review, we categorize different methods of optical ultrasound detection and discuss key technology trends geared towards the development of all-optical optoacoustic systems. We also review application areas that are enabled by all-optical sound detectors, including interventional imaging, non-contact measurements, magnetoacoustics, and non-destructive testing.

Imaging plays a critical role in the diagnosis and assessment of dermatological conditions. However, optical or optoacoustic microscopy techniques are limited to visualizing superficial skin features owing to strong photon scattering, whereas ultrasound methods, which can probe deeper-seated tissue, lack the contrast to image pathophysiological mechanisms in detail. Here, we demonstrate that raster-scan optoacoustic mesoscopy (RSOM) implemented in ultra-broadband (10–180 MHz) detection mode bridges the depth capabilities of ultrasound and the resolution range and high contrast of optical methods in clinical dermatology. Using tomographic reconstruction and frequency equalization to represent low and high spatial-frequency components, we visualize skin morphology and vascular patterns in the dermis and sub-dermis of psoriasis patients, enabling quantification of inflammation and other biomarkers of psoriasis without the need for contrast agents. Implemented in a handheld device, we showcase how label-free biomarkers detected by RSOM correlate with clinical score. The method can also be extended to assess a larger spectrum of dermatological conditions.

Despite recent advances in fluorescence imaging, standardization of systems remains an unmet need. We developed a new comprehensive phantom that resolves multiple system parameters simultaneously and could be used for system performance comparison.

Currently, imaging technologies that enable dermsurgeons to visualize non-melanoma skin cancers (NMSC) in vivo preoperatively are lacking, resulting in excessive or incomplete removal. Multispectral optoacoustic tomography (MSOT) is a volumetric imaging tool to differentiate tissue chromophores and exogenous contrast agents, based on differences in their spectral signatures and used for high-resolution imaging of functional and molecular contrast at centimeter scale depth. We performed MSOT imaging with two- and three-dimensional handheld scanners on 21 Asian patients with NMSC. The tumors and their oxygenation parameters could be distinguished from normal skin endogenously. The lesion dimensions and depths were extracted from the spectral melanin component with three-dimensional spatial resolution up to 80 μm. The intraclass correlation coefficient correlating tumor dimension measurements between MSOT and ex vivo histology of excised tumors, showed good correlation. Real-time 3D imaging was found to provide information on lesion morphology and its underlying neovasculature, indicators of the tumor's aggressiveness.

Existing clinical intravascular imaging modalities are not capable of accurate detection of critical plaque pathophysiology in the coronary arteries. This study reports the first intravascular catheter combining intravascular ultrasound (IVUS) with multispectral fluorescence lifetime imaging (FLIm) that enables label-free simultaneous assessment of morpho logical and biochemical features of coronary vessels in vivo. A 3.7 Fr catheter with a fiber-optic channel was constructed based on a 40 MHz clinical IVUS catheter. The ability to safely acquire co-registered FLIm-IVUS data in vivo using Dextran40 solution flushing was demonstrated in swine coronary arteries. FLIm parameters from the arterial wall were consistent with the emission of fluorophores present in healthy arterial wall (collagen, elastin). Additionally, structural and biochemical features from atherosclerotic lesions were acquired in ex vivo human coronary samples and corroborated with histological findings. Current results show that FLIm parameters linked to the amount of structural proteins (e.g. collagen, elastin) and lipids (e.g. foam cells, extracellular lipids) in the first 200 μm of the intima provide important biochemical information that can supplement IVUS data for a comprehensive assessment of plaques pathophysiology. The unique FLIm-IVUS system evaluated here has the potential to provide a comprehensive insight into atherosclerotic lesion formation, diagnostics and response to therapy.

OBJECTIVES: To assess labelling efficiency of rabbit mesenchymal stem cells (MSCs) using the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and detection of labelled MSCs for osteochondral defect repair in a rabbit model using fluorescence molecular tomography-X-ray computed tomography (FMT-XCT). METHODS: MSCs were isolated from New Zealand White rabbits and labelled with DiR (1.25-20 μg/mL). Viability and induction of apoptosis were assessed by XTT- and Caspase-3/-7-testing. Chondrogenic potential was evaluated by measurement of glycosaminoglycans. Labelled cells and unlabeled controls (n = 3) underwent FMT-XCT imaging before and after chondrogenic differentiation. Osteochondral defects were created surgically in rabbit knees (n = 6). Unlabeled and labelled MSCs were implanted in fibrin-clots and imaged by FMT-XCT. Statistical analyses were performed using multiple regression models. RESULTS: DiR-labelling of MSCs resulted in a dose-dependent fluorescence signal on planar images in trans-illumination mode. No significant reduction in viability or induction of apoptosis was detected at concentrations below 10 μg DiR/mL (p > .05); the chondrogenic potential of MSCs was not affected (p > .05). FMT-XCT of labelled MSCs in osteochondral defects showed a significant signal of the transplant (p < .05) with additional high-resolution anatomical information about its osteochondral integration. CONCLUSIONS: FMT-XCT allows for detection of stem cell implantation within osteochondral regeneration processes. KEY POINTS: • DiR-labelling of MSCs shows no toxic side effects or impairment of chondrogenesis. • Fluorescence molecular tomography allows for detection of MSCs for osteochondral defect repair. • FMT-XCT helps to improve evaluation of cell implantation and osteochondral regeneration processes.

The distribution of intramyocardially injected rabbit MSCs, labeled with the near-infrared dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbo-cyanine-iodide (DiR) using hybrid Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) and Multispectral Optoacoustic Tomography (MSOT) imaging technologies, was investigated. Viability and induction of apoptosis of DiR labeled MSCs were assessed by XTT- and Caspase-3/-7-testing in vitro. 2 × 106, 2 × 105 and 2 × 104 MSCs labeled with 5 and 10 μg DiR/ml were injected into fresh frozen rabbit hearts. FMT-XCT, MSOT and fluorescence cryosection imaging were performed. Concentrations up to 10 μg DiR/ml did not cause apoptosis in vitro (p > 0.05). FMT and MSOT imaging of labeled MSCs led to a strong signal. The imaging modalities highlighted a difference in cell distribution and concentration correlated to the number of injected cells. Ex-vivo cryosectioning confirmed the molecular fluorescence signal. FMT and MSOT are sensitive imaging techniques offering high-anatomic resolution in terms of detection and distribution of intramyocardially injected stem cells in a rabbit model.

Aims: (i) to evaluate a novel hybrid near-infrared fluorescence - intravascular ultrasound (NIRF-IVUS) system in coronary and peripheral swine arteries in vivo; (ii) to assess simultaneous quantitative biological and morphological aspects of arterial disease. Methods and results: Two 9F/15MHz peripheral and 4.5F/40MHz coronary near-infrared fluorescence (NIRF)-IVUS catheters were engineered to enable accurate co-registrtation of biological and morphological readings simultaneously in vivo. A correction algorithm utilizing IVUS information was developed to account for the distance-related fluorescence attenuation due to through-blood imaging. Corrected NIRF (cNIRF)-IVUS was applied for in vivo imaging of angioplasty-induced vascular injury in swine peripheral arteries and experimental fibrin deposition on coronary artery stents, and of atheroma in a rabbit aorta, revealing feasibility to intravascularly assay plaque structure and inflammation. The addition of ICG-enhanced NIRF assessment improved the detection of angioplasty-induced endothelial damage compared to standalone IVUS. In addition, NIRF detection of coronary stent fibrin by in vivo cNIRF-IVUS imaging illuminated stent pathobiology that was concealed on standalone IVUS. Fluorescence reflectance imaging and microscopy of resected tissues corroborated the in vivo findings. Conclusions: Integrated cNIRF-IVUS enables simultaneous co-registered through-blood imaging of disease related morphological and biological alterations in coronary and peripheral arteries in vivo. Clinical translation of cNIRF-IVUS may significantly enhance knowledge of arterial pathobiology, leading to improvements in clinical diagnosis and prognosis, and helps to guide the development of new therapeutic approaches for arterial diseases.

To extend sensitivity field for effective optoacoustic imaging, a novel concept of a non-mechanical point spread function (PSF) adjustment is proposed. Method was validated on phantoms and showed to be useful for distance-adaptive imaging.

A wide variety of subcutaneous soft-tissue masses may be seen in clinical practice. Clinical examination based on palpation alone is often insufficient to identify the nature and exact origin of the mass, in which case imaging is necessary. We used handheld multispectral optoacoustic imaging technology (MSOT) in a proof-of-principle study to image superficial fatty tumors and compare the images with diagnostic ultrasound. Fatty tumors were clearly visualized by MSOT and exhibited a spectral signature which differed from normal fatty tissue or muscle tissue. Our findings further indicated that MSOT offers highly complementary contrast to sonography. Based on the performance achieved, we foresee a promising role for MSOT in the diagnosis and evaluation of subcutaneous soft-tissue masses. Picture: Pseudo-color representation of a cross-sectional multi-spectral optoacoustic slice through a subcutaneous lipoma. Multi-spectral information is encoded in color. The lipoma can clearly be distinguished from the surrounding tissue based on its color. Scalebar 1 cm.

Inflammation drives atherosclerosis complications and is a promising therapeutic target for plaque stabilization. At present, it is unknown whether local stenting approaches can stabilize plaque inflammation in vivo. Here, we investigate whether everolimus-eluting stents (EES) can locally suppress plaque inflammatory protease activity in vivo using intravascular near-infrared fluorescence (NIRF) molecular imaging. METHODS AND RESULTS: Balloon-injured, hyperlipidaemic rabbits with atherosclerosis received non-overlapping EES and bare metal stents (BMS) placement into the infrarenal aorta (n = 7 EES, n = 7 BMS, 3.5 mm diameter x 12 mm length). Four weeks later, rabbits received an injection of the cysteine protease-activatable NIRF imaging agent Prosense VM110. Twenty-four hours later, co-registered intravascular 2D NIRF, X-ray angiography and intravascular ultrasound imaging were performed. In vivo EES-stented plaques contained substantially reduced NIRF inflammatory protease activity compared with untreated plaques and BMS-stented plaques (P = 0.006). Ex vivo macroscopic NIRF imaging of plaque protease activity corroborated the in vivo results (P = 0.003). Histopathology analyses revealed that EES-treated plaques showed reduced neointimal and medial arterial macrophage and cathepsin B expression compared with unstented and BMS-treated plaques. CONCLUSIONS: EES-stenting stabilizes plaque inflammation as assessed by translational intravascular NIRF molecular imaging in vivo. These data further support that EES may provide a local approach for stabilizing inflamed plaques.

Epifluorescence imaging is widely used in cell and molecular biology due to its excellent sensitivity, contrast, and ease of implementation. Optoacoustic imaging has been shown to deliver a highly complementary and unique set of capabilities for biological discovery, such as high spatial resolution in noninvasive deep tissue observations, fast volumetric imaging capacity, and spectrally enriched contrast. In this Letter, we report on a hybrid system combining planar fluorescence and real-time volumetric four-dimensional optoacoustic imaging by means of a fiberscope integrated within a handheld hemispherical ultrasound detection array. The in vivo imaging performance is demonstrated by noninvasive visualization of fast contrast agent perfusion through the mouse brain. The proposed synergistic combination of fluorescence and optoacoustic imaging can benefit numerous studies looking at multi-scale in vivo dynamics, such as functional neuroimaging, visualization of organ perfusion and contrast agent uptake, cell tracking, and pharmacokinetic and bio-distribution analysis.

Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between 'on' and 'off' states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells for ~100 × 100 μm(2) fields of view. Individual keratin filaments were rendered at a FWHM of ~60-80 nm, with effective resolution for the filaments of ~80-100 nm. We discuss the impact of novel image reconstruction algorithms featuring background elimination by spatial bandpass filtering, as well as strategies that incorporate complete image formation models.

BACKGROUND: Recent advances in technology have enabled the development of various non-invasive skin imaging tools to aid real-time diagnosis of both benign and malignant skin tumours, minimizing the need for invasive skin biopsy. Multispectral optoacoustic tomography (MSOT) is a recently developed non-invasive imaging tool, which offers the unique capacity for high resolution three dimensional (3D) optical mapping of tissue by further delivering highly specific optical contrast from a depth of several millimetres to centimetres in living tissues. MSOT enables volumetric, spectroscopic differentiation of tissue, both in vivo and in real time, with and without the application of biomarker-specific probes, and is further able of providing spatial maps of skin chromophores, as well as underlying blood vasculature. METHODS: Three patients with suspicious skin tumours consented to have their lesions imaged with MSOT prior to excision. The histological findings and measurements were compared. RESULTS: We demonstrated the first in vivo clinical use of MSOT for 3D reconstruction of skin tumours in three patients with good histological correlation. CONCLUSION: Our findings confirm the potential benefit of the new imaging method in guiding surgical intervention to achieve a more precise excision with better clearance and lower relapse rates. It can also potentially help to shorten the duration of Mohs' micrographic surgery. Further large-scale studies are necessary to ensure correlation between MSOT and histology.

Imaging dynamics at different temporal and spatial scales is essential for understanding the biological complexity of living organisms, disease state and progression. Optoacoustic imaging has been shown to offer exclusive applicability across multiple scales with excellent optical contrast and high resolution in deep-tissue observations. Yet, efficient visualization of multi-scale dynamics remained difficult with state-of-the-art systems due to inefficient trade-offs between image acquisition time and effective field of view. Herein, we introduce the spiral volumetric optoacoustic tomography technique that provides spectrally enriched high-resolution contrast across multiple spatiotemporal scales. experiments in mice demonstrate a wide range of dynamic imaging capabilities, from three-dimensional high-frame-rate visualization of moving organs and contrast agent kinetics in selected areas to whole-body longitudinal studies with unprecedented image quality. The newly introduced paradigm shift in imaging of multi-scale dynamics adds to the multifarious advantages provided by the optoacoustic technology for structural, functional and molecular imaging.

Limited-view artifacts are commonly present in optoacoustic tomography images, mainly due to practical geometrical and physical constraints imposed by the imaging systems. Herein, a new approach called dynamic particle-enhanced optoacoustic tomography (DPOT) is proposed for improving image contrast and visibility of optoacoustic images under limited-view scenarios. The method is based on a nonlinear combination of a temporal sequence of tomographic reconstructions representing sparsely distributed moving particles. We demonstrate experimental performance by dynamically imaging the flow of suspended microspheres in three dimensions, which shows promise for DPOT applicability in angiographic imaging in living organisms.

Visualization of dynamic functional and molecular events in an unperturbed in vivo environment is essential for understanding the complex biology of living organisms and of disease state and progression. To this end, optoacoustic (photoacoustic) sensing and imaging have demonstrated the exclusive capacity to maintain excellent optical contrast and high resolution in deep-tissue observations, far beyond the penetration limits of modern microscopy. Yet, the time domain is paramount for the observation and study of complex biological interactions that may be invisible in single snapshots of living systems. This review focuses on the recent advances in optoacoustic imaging assisted by smart molecular labeling and dynamic contrast enhancement approaches that enable new types of multiscale dynamic observations not attainable with other bio-imaging modalities. A wealth of investigated new research topics and clinical applications is further discussed, including imaging of large-scale brain activity patterns, volumetric visualization of moving organs and contrast agent kinetics, molecular imaging using targeted and genetically expressed labels, as well as three-dimensional handheld diagnostics of human subjects.

Genetically-encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping of the activity of entire neuronal populations in vivo. Visualization of these powerful activity sensors has to date been limited to depth-restricted microscopic studies due to intense light scattering in the brain. We demonstrate, for the first time, in vivo real-time volumetric optoacoustic monitoring of calcium transients in adult transgenic zebrafish expressing the GCaMP5G calcium indicator. Fast changes in optoacoustic traces associated with GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques.

Calcium transients in the brain of adult zebrafish expressing GCaMP5G were monitored with optoacoustic tomography. Optoacoustics can visualize neural activity at penetration depths and spatio-temporal scales not covered with the existing modalities.

Hybrid optoacoustic and pulse-echo ultrasound imaging is an attractive multi-modal combination owing to the highly complementary contrast of the two techniques. Efficient hybridization is often hampered by significant dissimilarities between their optimal data acquisition and image formation strategies. Herein, we introduce an approach for combined optoacoustic and ultrasound imaging based on a plano-concave detector array design with a non-uniform pitch distribution. The hybrid design optimized for both modalities allows for maintaining an extended field of view for efficient ultrasound navigation while simultaneously providing broad tomographic coverage for optimal optoacoustic imaging performance. Imaging sessions performed in tissue-mimicking phantoms and healthy volunteers demonstrate that the suggested approach renders an enhanced imaging performance as compared with the previously reported hybrid optoacoustic and ultrasound imaging approaches. Thus, it can greatly facilitate clinical translation of the optoacoustic imaging technology by means of its efficient combination with ultrasonography, a well-established clinical imaging modality.

Optical microscopy remains a fundamental tool for modern biological discovery owing to its excellent spatial resolution and versatile contrast in visualizing cellular and sub-cellular structures. Yet, the time domain is paramount for the observation of biological dynamics in living systems. Commonly, acquisition of microscopy data involves scanning of a spherically-or cylindrically-focused light beam across the imaged volume, which significantly limits temporal resolution in 3D. Additional complications arise from intense light scattering of biological tissues, further restraining the effective penetration depth and field of view of optical microscopy techniques. To overcome these limitations, we devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth beyond 25 MHz. We demonstrate fast three-dimensional imaging of freely-swimming zebrafish larvae, achieving 3D imaging speed of 100 volumes per second with isotropic spatial resolution approaching the dimensions of large cells across a field of view exceeding 50mm(3). As opposed to other microscopy techniques based on optical contrast, OMT resolves optical absorption acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Hand-held implementations of recently introduced real-time volumetric tomography approaches represent a promising path toward clinical translation of the optoacoustic technology. To this end, rapid acquisition of optoacoustic image data with spherical matrix arrays has attained exquisite visualizations of three-dimensional vascular morphology and function deep in human tissues. Nevertheless, significant reconstruction inaccuracies may arise from speed of sound (SoS) mismatches between the imaged tissue and the coupling medium used to propagate the generated optoacoustic responses toward the ultrasound sensing elements. Herein, we analyze the effects of SoS variations in three-dimensional hand-held tomographic acquisition geometries. An efficient graphics processing unit (GPU)-based reconstruction framework is further proposed to mitigate the SoS-related image quality degradation without compromising the high-frame-rate volumetric imaging performance of the method, essential for real-time visualization during hand-held scans.

Imaging dynamics in living organisms is essential for the understanding of biological complexity. While multiple imaging modalities are often required to cover both microscopic and macroscopic spatial scales, dynamic phenomena may also extend over different temporal scales, necessitating the use of different imaging technologies based on the trade-off between temporal resolution and effective field of view. Optoacoustic (photoacoustic) imaging has been shown to offer the exclusive capability to link multiple spatial scales ranging from organelles to entire organs of small animals. Yet, efficient visualization of multi-scale dynamics remained difficult with state-of-the-art systems due to inefficient trade-offs between image acquisition and effective field of view. Herein, we introduce a spiral volumetric optoacoustic tomography (SVOT) technique that provides spectrally-enriched high-resolution optical absorption contrast across multiple spatio-temporal scales. We demonstrate that SVOT can be used to monitor various in vivo dynamics, from video-rate volumetric visualization of cardiac-associated motion in whole organs to high-resolution imaging of pharmacokinetics in larger regions. The multi-scale dynamic imaging capability thus emerges as a powerful and unique feature of the optoacoustic technology that adds to the multiple advantages of this technology for structural, functional and molecular imaging.

Limited-view artefacts affect most optoacoustic (photoacoustic) imaging systems due to geometrical constraints that impede achieving full tomographic coverage as well as limited light penetration into scattering and absorbing objects. Indeed, it has been theoretically established and experimentally verified that accurate optoacoustic images can only be obtained if the imaged sample is fully enclosed (< π angular coverage) by the measuring locations. Since in many cases full angular coverage cannot be achieved, the visibility of structures along certain orientations is hampered. These effects are of particular relevance in the case of hand-held scanners with the imaged volume only accessible from one side. Herein, a new approach termed dynamic particle-enhanced optoacoustic tomography (DPOT) is described for accurate structural imaging in limited-view scenarios. The method is based on the non-linear combination of a sequence of tomographic reconstructions representing sparsely distributed moving particles. Good performance of the method is demonstrated in experiments consisting of dynamic visualization of flow of suspended microspheres in three-dimensions. The method is expected to be applicable for improving accuracy of angiographic optoacoustic imaging in living organisms.

Due to modeling and experimental imperfections, multispectral optoacoustic tomography images are often afflicted with negative values, which are further amplified when propagating into the spectrally unmixed images of chromophore concentrations. Since negative values have no physical meaning, accuracy can potentially be improved by imposing non-negativity constraints on the initial reconstructions and the unmixing steps. Herein, we compare several non-negative constrained approaches with reconstruction and spectral unmixing performed separately or combined in a single inverse step. The quantitative performance and sensitivity of the different approaches in detecting small amounts of spectrally-distinct chromophores are studied in tissue-mimicking phantoms and mouse experiments.

Accurate extraction of physical and biochemical parameters from optoacoustic images is often impeded due to the use of unrigorous inversion schemes, incomplete tomographic detection coverage or other experimental factors that cannot be readily accounted for during the image acquisition and reconstruction process. For instance, inaccurate assumptions in the physical forward model may lead to negative optical absorption values in the reconstructed images. Any artifacts present in the single wavelength optoacoustic images can be significantly aggravated when performing a two-step reconstruction consisting in acoustic inversion and spectral unmixing aimed at rendering the distributions of spectrally-distinct absorbers. We investigate a number of algorithmic strategies with non-negativity constraints imposed at the different phases of the reconstruction process. Performance is evaluated in cross-sectional multispectral optoacoustic tomography (MSOT) recordings from tissue-mimicking phantoms and in vivo mice embedded with varying concentrations of contrast agents. Additional in vivo validation is subsequently performed with molecular imaging data involving subcutaneous tumors labeled with genetically-expressed iRFP proteins and organ perfusion by optical contrast agents. It is shown that constrained reconstruction is essential for reducing the critical image artifacts associated with inaccurate modeling assumptions. Furthermore, imposing the non-negativity constraint directly on the unmixed distribution of the probe of interest was found to maintain the most robust and accurate reconstruction performance in all experiments.

In order to achieve real-time image rendering, optoacoustic tomography reconstructions are commonly done with back-projection algorithms due to their simplicity and low computational complexity. However, model-based algorithms have been shown to attain more accurate reconstruction performance due to their ability to model arbitrary detection geometries, transducer shapes and other experimental factors. The high computational complexity of the model-based schemes makes it challenging to be implemented for real time inversion. Herein, we introduce a novel discretization method for model-based optoacoustic tomography that enables its efficient parallel implementation on graphics processing units with extremely low memory overhead. We demonstrate that, when employing a tomographic scanner with 256 detectors, the new method achieves model-based optoacoustic inversion at 20 frames per second for a 200 × 200 image grid.

Optimal optoacoustic tomographic sampling is often hindered by the frequency-dependent directivity of ultrasound sensors, which can only be accounted for with an accurate three-dimensional (3D) model. Herein, we introduce a 3D modelbased reconstruction method applicable to optoacoustic imaging systems employing detection elements with arbitrary size and shape. The computational complexity and memory requirements are mitigated by introducing an efficient graphics processing unit (GPU)-based implementation of the iterative inversion. On-the-fly calculation of the entries of the model-matrix via a small look-up table avoids otherwise unfeasible storage of matrices typically occupying more than 300GB of memory. Superior imaging performance of the suggested method with respect to standard optoacoustic image reconstruction methods is first validated quantitatively using tissue-mimicking phantoms. Significant improvements in the spatial resolution, contrast to noise ratio and overall 3D image quality are also reported in real tissues by imaging the finger of a healthy volunteer with a hand-held volumetric optoacoustic imaging system.

PURPOSE: In a pilot study, we introduce fast handheld Multi-Spectral Optoacoustic Tomography (MSOT) of the breast at 28 wavelengths, aiming to identify high-resolution optoacoustic (photoacoustic) patterns of breast cancer and non-cancerous breast tissue. EXPERIMENTAL DESIGN: We imaged 10 female patients aged 48-81 years with malignant non-specific breast cancer or invasive lobular carcinoma. Three healthy volunteers aged 31-36 years were also imaged. Fast-MSOT was based on unique single-frame-per-pulse (SFPP) image acquisition employed to improve the accuracy of spectral differentiation over using a small number of wavelengths. Breast tissue was illuminated at the 700 - 970 nm spectral range over 0.56 seconds total scan time. MSOT data were guided by ultrasonography and X-ray mammography or MRI. RESULTS: The extended spectral range allowed the computation of oxygenated hemoglobin (HBO2), deoxygenated hemoglobin (HB), total blood volume (TBV), lipid and water contributions, allowing first insights into in-vivo high-resolution breast tissue MSOT cancer patterns. TBV and Hb/HBO2 images resolved marked differences between cancer and control tissue, manifested as a vessel rich tumor periphery with highly heterogeneous spatial appearance compared to healthy tissue. We observe significant TBV variations between different tumors and between tumors over healthy tissues. Water and fat lipid layers appear disrupted in cancer vs. healthy tissue; however offer weaker contrast compared to TBV images. CONCLUSION: In contrast to optical methods, MSOT resolves physiological cancer features with high resolution and revealed patterns not offered by other radiological modalities. The new features relate to personalized and precision medicine potential.

Ultrasound waves propagating in water or soft biological tissue are strongly reflected when encountering the skull, which limits the use of ultrasound-based techniques in transcranial imaging and therapeutic applications. Current knowledge on the acoustic properties of the cranial bone is restricted to far-field observations, leaving its near-field unexplored. We report on the existence of skull-guided acoustic waves, which was herein confirmed by near-field measurements of optoacoustically-induced responses in ex-vivo murine skulls immersed in water. Dispersion of the guided waves was found to reasonably agree with the prediction of a multilayered flat plate model. We observed a skull-guided wave propagation over a lateral distance of at least 3 mm, with a half-decay length in the direction perpendicular to the skull ranging from 35 to 300 μm at 6 and 0.5 MHz, respectively. Propagation losses are mostly attributed to the heterogenous acoustic properties of the skull.. It is generally anticipated that our findings may facilitate and broaden the application of ultrasound-mediated techniques in brain diagnostics and therapy.

Scanning optoacoustic microscopy operates in two distinct regimes optical resolution microscopy relies on a focused illumination and acoustic resolution microscopy that forms images by focusing the received acoustic field. Recently, a number of approaches have been proposed that combine those two modes of operation to create a highly scalable technique that can image at multiple penetration scales by gradually exchanging microscopic optical resolution in superficial tissues with ultrasonic resolution at diffuse (macroscopic) depths. However, scanning microscopy schemes commonly employ acquisition geometries that impede the use of synthetic aperture techniques to achieve meaningful images due to non-stationary illumination patterns and strong non-uniformity of the excitation light field. Here we present a Weighted Synthetic Aperture Focusing Technique (W-SAFT) as a universal framework that effectively accounts for the non-uniform distribution of both the excitation light field and spatial sensitivity field of the detector. As a result, W-SAFT maintains optical resolution performance at superficial depths while improving the acoustic resolving capacity for deeper tissues. The dynamic range of the optoacoustic data is compressed using a general fluence decay term applied to the W-SAFT operator, allowing a more uniform visualization of the entire imaged volume. Our three-dimensional algorithm makes use of the sample's surface to account for the heterogeneity produced when scanning a finite-size light beam. We tested a GPU implementation of W-SAFT with numerical simulations and showcase its performance on experimental data acquired from targets embedded in tissue mimicking phantoms.

The skull bone, a curved solid multilayered plate protecting the brain, constitutes a big challenge for the use of ultrasound-mediated techniques in neuroscience. Ultrasound waves incident from water or soft biological tissue are mostly reflected when impinging on the skull. To this end, skull properties have been characterized for both high-intensity focused ultrasound (HIFU) operating in the narrowband far-field regime and optoacoustic imaging applications. Yet, no study has been conducted to characterize the near-field of water immersed skulls. We used the thermoelastic effect with a 532 nm pulsed laser to trigger a wide range of broad-band ultrasound modes in a mouse skull. In order to capture the waves propagating in the near-field, a thin hydrophone was scanned in close proximity to the skull's surface. While Leaky pseudo-Lamb waves and grazing-angle bulk water waves are clearly visible in the spatio-temporal data, we were only able to identify skull-guided acoustic waves after dispersion analysis in the wavenumber-frequency space. The experimental data was found to be in a reasonable agreement with a flat multilayered plate model.

Fluorescence lifetime imaging has been shown to serve as a valuable tool for interrogating and diagnosis of biological tissue at a mesoscopic level. The ability to analyze fluorescence decay curves to extract lifetime values in real-time is crucial for clinical translation and applications such as tumor margin delineation or intracoronary imaging of atherosclerotic plaques. In this work, we compare the performance of two popular non-parametric (fit-free) methods for determining lifetime values from fluorescence decays in real-time-the Phasor approach and Laguerre deconvolution. We demonstrate results from simulated and experimental data to compare the accuracy and speed of both methods and their dependence on noise and model parameters.

Objective—Neutrophils accumulate in early atherosclerotic lesions and promote lesion growth. In this study, we evaluated an elastase-specific near-infrared imaging agent for molecular imaging using hybrid fluorescence molecular tomography/x-ray computed tomography. Approach and Results—Murine neutrophils were isolated from bone marrow and incubated with the neutrophil-targeted near-infrared imaging agent Neutrophil Elastase 680 FAST for proof of principle experiments, verifying that the elastase-targeted fluorescent agent is specifically cleaved and activated by neutrophil content after lysis or cell stimulation. For in vivo experiments, low-density lipoprotein receptor–deficient mice were placed on a Western-type diet and imaged after 4, 8, and 12 weeks by fluorescence molecular tomography/x-ray computed tomography. Although this agent remains silent on injection, it produces fluorescent signal after cleavage by neutrophil elastase. After hybrid fluorescence molecular tomography/x-ray computed tomography imaging, mice were euthanized for whole-body cryosectioning and histological analyses. The in vivo fluorescent signal in the area of the aortic arch was highest after 4 weeks of high-fat diet feeding and decreased at 8 and 12 weeks. Ex vivo whole-body cryoslicing confirmed the fluorescent signal to locate to the aortic arch and to originate from the atherosclerotic arterial wall. Histological analysis demonstrated the presence of neutrophils in atherosclerotic lesions. Conclusions—This study provides evidence that elastase-targeted imaging can be used for in vivo detection of early atherosclerosis. This imaging approach may harbor potential in the clinical setting for earlier diagnosis and treatment of atherosclerosis.  

Fluorescence molecular imaging (FMI) has shown potential to detect and delineate cancer during surgery or diagnostic endoscopy. Recent progress on imaging systems has allowed sensitive detection of fluorescent agents even in video rate mode. However, lack of standardization in fluorescence imaging challenges the clinical application of FMI, since the use of different systems may lead to different results from a given study, even when using the same fluorescent agent. In this work, we investigate the use of a composite fluorescence phantom, employed as an FMI standard, to offer a comprehensive method for validation and standardization of the performance of different imaging systems. To exclude user interaction, all phantom features are automatically extracted from the acquired epi-illumination color and fluorescence images, using appropriately constructed templates. These features are then employed to characterize the performance and compare different cameras to each other. The proposed method could serve as a framework toward the calibration and benchmarking of FMI systems, to facilitate their clinical translation.

Lack of standardization in fluorescence imaging challenges its clinical translation. We investigate the use of a composite phantom to perform standardization, which could serve as a framework toward the benchmarking of fluorescence imaging systems.

Visualization of whole brain activity during epileptic seizures is essential for both fundamental research into the disease mechanisms and the development of efficient treatment strategies. It has been previously discussed that pathological synchronization originating from cortical areas may reinforce backpropagating signaling from the thalamic neurons, leading to massive seizures through enhancement of high frequency neural activity in the thalamocortical loop. However, the study of deep brain neural activity is challenging with the existing functional neuroimaging methods due to lack of adequate spatiotemporal resolution or otherwise insufficient penetration into subcortical areas. To investigate the role of thalamocortical activity during epileptic seizures, we developed a new functional neuroimaging framework based on spatiotemporal correlation of volumetric optoacoustic hemodynamic responses with the concurrent electroencephalogram recordings and anatomical brain landmarks. The method is shown to be capable of accurate three-dimensional mapping of the onset, spread, and termination of the epileptiform events in a 4-aminopyridine acute model of focal epilepsy. Our study is the first to demonstrate entirely noninvasive real-time visualization of synchronized epileptic foci in the whole mouse brain, including the neocortex and subcortical structures, thus opening new vistas in systematic studies toward the understanding of brain signaling and the origins of neurological disorders.

Genetically encoded calcium indicators are powerful brain activity-sensors. Here, we demonstrate noninvasive volumetric optoacoustic neuroimaging of rapid calcium transients in living GCaMP6f-expressing mice exposed to electrical hindpaw stimulation.

Thalamocortical activity in epilepsy was studied in mice using an optoacoustic functional neuroimaging framework correlating hemodynamic responses with concurrent EEG-readings, demonstrating noninvasive real-time visualization of deep epileptic foci.

Multi-Spectral Optoacoustic Tomography (MSOT) merges the power of high-resolution imaging at tissue depths of several millimeters to centimeters with the advantages of optical imaging, in large part by exploiting spectral detection of endogenous molecules in tissue or exogenous photoabsorbing probes. Current advances in fast-tuning laser technology, image reconstruction and spectral detection schemes have yielded real-time optoacoustic (photoacoustic) imaging spanning applications from microscopy to human imaging. This progress has generated an unprecedented need for imaging probes and reporter gene approaches optimized for optoacoustic signal generation. New classes of probes are emerging and create new opportunities for visualizing morphological and pathophysiological features in vivo, in a non-invasive manner. Here we review recent progress in optoacoustic probes and discuss applications and challenges for biological imaging as well as prospects for clinical translation.

Optoacoustic imaging is a rapidly expanding field for the diagnosis, characterization, and treatment evaluation of cancer. However, the availability of tumor specific exogenous contrast agents is still limited. Here, we report on a small targeted contrast agent for optoacoustic imaging using a black hole quencher® (BHQ) dye. The sonophore BHQ-1 exhibited strong, concentration-dependent, optoacoustic signals in phantoms, demonstrating its ideal suitability for optoacoustic imaging. After labeling BHQ-1 with cyclic RGD-peptide, BHQ-1-cRGD specifically bound to αvβ3-integrin expressing glioblastoma cell spheroids in vitro. The excellent optoacoustic properties of BHQ-1-cRGD could furthermore be proven in vivo. Together with this emerging imaging modality, our sonophore labeled small peptide probe offers new possibilities for non-invasive detection of molecular structures with high resolution in vivo and furthers the specificity of optoacoustic imaging. Ultimately, the discovery of tailor-made sonophores might offer new avenues for various molecular optoacoustic imaging applications, similar to what we see with fluorescence imaging.

Recent epidemiology studies highlighted the detrimental health effects of exposure to low dose and low dose rate ionizing radiation (IR): nuclear industry workers studies have shown increased leukaemia and solid tumour risks following cumulative doses of <100 mSv and dose rates of <10 mGy per year; paediatric patients studies have reported increased leukaemia and brain tumours risks after doses of 30–60 mGy from computed tomography scans. Questions arise, however, about the impact of even lower doses and dose rates where classical epidemiological studies have limited power but where subsets within the large cohorts are expected to have an increased risk. Further progress requires integration of biomarkers or bioassays of individual exposure, effects and susceptibility to IR. The European DoReMi (Low Dose Research towards Multidisciplinary Integration) consortium previously reviewed biomarkers for potential use in IR epidemiological studies. Given the increased mechanistic understanding of responses to low dose radiation the current review provides an update covering technical advances and recent studies. A key issue identified is deciding which biomarkers to progress. A roadmap is provided for biomarker development from discovery to implementation and used to summarise the current status of proposed biomarkers for epidemiological studies. Most potential biomarkers remain at the discovery stage and for some there is sufficient evidence that further development is not warranted. One biomarker identified in the final stages of development and as a priority for further research is radiation specific mRNA transcript profiles.

Optoacoustic tomography based on insufficient spatial sampling of ultrasound waves leads to loss of contrast and artifacts on the reconstructed images. Compared to reconstructions based on L2-norm regularization, sparsity-based reconstructions may improve contrast and reduce image artifacts but at a high computational cost, which has so far limited their use to 2D optoacoustic tomography. Here we propose a fast, sparsity-based reconstruction algorithm for 3D optoacoustic tomography, based on gradient descent with Barzilai-Borwein line search (L1-GDBB). Using simulations and experiments, we show that the L1-GDBB offers fourfold faster reconstruction than the previously reported L1-norm regularized reconstruction based on gradient descent with backtracking line search. Moreover, the new algorithm provides higher-quality images with fewer artifacts than the L2-norm regularized reconstruction and the back-projection reconstruction.

A hybrid optical and acoustic resolution optoacoustic endoscopy is proposed. Laser light is transmitted to tissue by two types of illumination for optical and acoustic resolution imaging respectively. An unfocused ultrasound detector is used for recording optoacoustic signals. The endoscopy probe attains 3.6 mm diameter and is fully encapsulated into a catheter system. We examine the performance of the hybrid endoscope with phantoms and tissue sample, which shows that the hybrid endoscopy can obtain optical resolution in superficial microscopic imaging and ultrasonic tomography reconstruction resolution when imaging at greater depths. © (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Sparse recovery algorithms have shown great potential to reconstruct images with limited view datasets in optoacoustic tomography, with a disadvantage of being computational expensive. In this paper, we improve the fast convergent Split Augmented Lagrangian Shrinkage Algorithm (SALSA) method based on least square QR (LSQR) formulation for performing accelerated reconstructions. Further, coherence factor is calculated to weight the final reconstruction result, which can further reduce artifacts arising in limited-view scenarios and acoustically heterogeneous mediums. Several phantom and biological experiments indicate that the accelerated SALSA method with coherence factor (ASALSA-CF) can provide improved reconstructions and much faster convergence compared to existing sparse recovery methods.

The non-coding transcriptome, in particular microRNAs (miRNA), influences cellular survival after irradiation. However, the underlying mechanisms of radiation-induced miRNA expression changes and consequently target expression changes are poorly understood. In this study, we show that ionizing radiation decreases expression of the miR-23a~27a~24-2 cluster through transcriptional regulation by promoter methylation and at the post-transcriptional level by reduced processing through AGO-phosphorylation. Furthermore, we demonstrate that all three mature cluster miRNAs reduce apoptosis by increasing expression of the common target protein XIAP. These findings link a temporal succession of transcriptional and post-transcriptional regulatory mechanisms of the miR~23a~24-2~27a cluster, enabling a dynamic stress response and assuring cellular survival after radiation exposure.

Label-free multispectral optoacoustic tomography (MSOT) has recently shown superior performance in visualizing the morphology of human vasculature, especially of smaller vessels, compared to ultrasonography. Herein, we extend these observations towards MSOT interrogation of macrovascular endothelial function. We employed a real-time handheld MSOT scanner to assess flow-mediated dilatation (FMD), a technique used to characterize endothelial function. A data processing scheme was developed to quantify the dimensions and diameter changes of arteries in humans and determine wall distensibility parameters. By enabling high-resolution delineation of the blood-vessel wall in a cross-sectional fashion, the findings suggest MSOT as a capable alternative to ultrasonography for clinical FMD measurements.

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multi-scale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labelled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

Multispectral optoacoustic mesoscopy (MSOM) has been recently introduced for cancer imaging, it has the potential for high resolution imaging of cancer development in vivo, at depths beyond the diffusion limit. Based on spectral features, optoacoustic imaging is capable of visualizing angiogenesis and imaging cancer heterogeneity of malignant tumors through endogenous hemoglobin. However, high-resolution structural and functional imaging of whole tumor mass is limited by modest penetration and image quality, due to the insufficient capability of ultrasound detectors and the twodimensional scan geometry. In this study, we introduce a novel multi-spectral optoacoustic mesoscopy (MSOM) for imaging subcutaneous or orthotopic tumors implanted in lab mice, with the high-frequency ultrasound linear array and a conical scanning geometry. Detailed volumetric images of vasculature and oxygen saturation of tissue in the entire tumors are obtained in vivo, at depths up to 10 mm with the desirable spatial resolutions approaching 70μm. This unprecedented performance enables the visualization of vasculature morphology and hypoxia conditions has been verified with ex vivo studies. These findings demonstrate the potential of MSOM for preclinical oncological studies in deep solid tumors to facilitate the characterization of tumor’s angiogenesis and the evaluation of treatment strategies.

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. Owing to the large field of view and fast acquisition speed of our newly developed SPIM system, volumetric image stacks from entire intact samples of pig ovaries have been rendered demonstrating clearly discernible follicular features like follicle diameters (70 μm-2.5 mm), size of developing Cumulus oophorus complexes (COC) (40 μm-110 μm), and follicular wall thicknesses (90 μm-120 μm). The observation of clearly distinguishable COCs protruding into the follicular antrum was also shown possible, and correlation with the developmental stage of the follicles was determined. Follicles of all developmental stages were identified, and even the small primordial follicle clusters forming the egg nest could be observed. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

Extraction of murine cardiac functional parameters on a beat-by-beat basis remains challenging with the existing imaging modalities. Novel methods enabling in vivo characterization of functional parameters at a high temporal resolution are poised to advance cardiovascular research and provide a better understanding of the mechanisms underlying cardiac diseases. We present a new approach based on analyzing contrast-enhanced optoacoustic (OA) images acquired at high volumetric frame rate without using cardiac gating or other approaches for motion correction. Acute myocardial infarction was surgically induced in murine models, and the method was modified to optimize for acquisition of artifact-free optoacoustic data. Infarcted hearts could be differentiated from healthy controls based on a significantly higher pulmonary transit time (PTT: infarct 2.07 s vs. healthy 1.34 s), while no statistically significant difference was observed in the heart rate (318 bpm vs. 309 bpm). In combination with the proven ability of optoacoustics to track targeted probes within the injured myocardium, our method is capable of depicting cardiac anatomy, function, and molecular signatures on a beat-by-beat basis, both with high spatial and temporal resolution, thus providing new insights into the study of myocardial ischemia.

Extraction of murine cardiac functional parameters on a beat-by-beat basis is limited with the existing imaging modalities due to insufficient three-dimensional temporal resolution. Faster volumetric imaging methods enabling in vivo characterization of functional parameters are poised to advance cardiovascular research and provide a better understanding of the mechanisms underlying cardiac diseases. We present a new approach based on analyzing contrast-enhanced optoacoustic (OA) images acquired at high volumetric frame rate without using cardiac gating or other approaches for motion correction. We apply an acute murine myocardial infarction model optimized for acquisition of artifact-free optoacoustic imaging data to study cardiovascular hemodynamics. Infarcted hearts (n = 21) could be clearly differentiated from healthy controls (n = 9) based on a significantly higher pulmonary transit time (PTT) (2.25 [2.00-2.41] s versus 1.34 [1.25-1.67] s, p = 0.0235), while no statistically significant difference was observed in the heart rate (318 [252-361] bpm versus 264 [252-320] bpm, p = 0.3129). Nevertheless, nonlinear heartbeat dynamics was stronger in the healthy hearts, as evidenced by the third harmonic component in the heartbeat spectra. MRI data acquired from the same mice further revealed that the PTT increases with the size of infarction and similarly increases with reduced ejection fraction. Moreover, an inverse relationship between infarct PTT and time post-surgery was found, which suggests the occurrence of cardiac healing. In combination with the proven ability of optoacoustics to track targeted probes within the injured myocardium, our method can depict cardiac anatomy, function, and molecular signatures, with both high spatial and temporal resolution. Volumetric four-dimensional optoacoustic characterization of cardiac dynamics with supreme temporal resolution can capture cardiovascular dynamics on a beat-by-beat basis in mouse models of myocardial ischemia.

Photoacoustic imaging (PAI) is a new biomedical imaging modality based on light triggered ultrasound emission. For in vivo application, materials with gobd photoacustic response to illumination in the near-infrared spectrum and suited tissue delivery strategies are needed. We developed polymeric, near-infrared responsive nanomaterials tuned for in vivo application based on oxazoline block copolymer chemistry by living cationic polymerization and a related functional transformation, loaded with a new pho'tonic material, hydrophobized phthalocyanine Zinc complex (H-PcZn), that was efficiently encapsulated into the nanoparticles by self-assembly. The resulting nanoparticles P-NPs and N-NPs bear positive, and negative surface charge, respectively. After physicochemical characterization, applicability of the two nanoparticles as photoacoustic contrast agents was evaluated in vitro and in phantom experiments, where they exhibited excellent PAI contrast. In vivo distribution and visualization of P-NPs and N-NPs following i.v. injection imaged by PAI was confirmed by cryosection fluorescence analysis and showed that the Materials accumulated in tissues within 1 h with differential tissue distribution. This pilot study thus describes synthesis of a novel polymeric photoacoustic nanosystem and demonstrates its potential for multimodal, photoacoustic in vivo imaging and for fluorescence imaging.

Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC) model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF) probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib) for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future.

The high complementarity of ultrasonography and optoacoustic tomography has prompted the development of combined approaches that utilize the same transducer array for detecting both optoacoustic and pulse-echo ultrasound responses from tissues. Yet, due to the fundamentally different physical contrast and image formation mechanisms, the development of detection technology optimally suited for image acquisition in both modalities remains a major challenge. Herein, we introduce a multi-segment detector array approach incorporating array segments of linear and concave geometry to optimally support both ultrasound and optoacoustic image acquisition. The various image rendering strategies are tested and optimized in numerical simulations and calibrated tissue-mimicking phantom experiments. We subsequently demonstrate real-time hybrid optoacoustic ultrasound (OPUS) image acquisition in a healthy volunteer. The new approach enables the acquisition of highquality anatomical data by both modalities complemented by functional information on blood oxygenation status provided by the multispectral optoacoustic tomography.

Magnetic cell sorting provides a valuable complementary mechanism to fluorescent techniques, especially if its parameters can be fine-tuned. In addition, there has recently been growing interest in studying naturally occurring magnetic cells and genetic engineering of cells to render them magnetic in order to control molecular processes via magnetic fields. For such approaches, contamination-free magnetic separation is an essential capability. We here present a robust and tunable microfluidic sorting system in which magnetic gradients of up to 1700 T/m can be applied to cells flowing through a sorting channel by reversible magnetization of ferrofluids. Visual control of the sorting process allowed us to optimize sorting efficiencies for a large range of sizes and magnetic moments of cells. Using automated quantification based on imaging of fluorescent markers, we showed that macrophages containing phagocytosed magnetic nanoparticles, with cellular magnetic dipole moments on the order of 10 fAm(2), could be sorted with an efficiency of 90 +/- 1%. Furthermore, we successfully sorted intrinsically magnetic magnetotactic bacteria with magnetic moments of 0.1 fAm(2). In distinction to column-based magnetic sorting devices, microfluidic systems can prevent sample contact with superparamagnetic material. This ensures contamination-free separation of naturally occurring or bioengineered magnetic cells and is essential for downstream characterization of their properties.

Optoacoustic (photoacoustic) imaging enables high-resolution optical imaging at depths well beyond optical microscopy, revolutionizing optical interrogation of tissues. Operation in the near-infrared (NIR) is nevertheless necessary to capitalize on the technology potential and reach depths of several centimeters. Using Flash NanoPrecipitation for highly-scalable single-step encapsulation of hydrophobic hexacene at self-quenching concentrations, we propose quenched fluorescence-dye nanoparticles as a potent alternative to NIR metal nanoparticles for strong optoacoustic signal generation. Comprehensive hexacene-based nanoparticle characterization was based on a 5-step approach that examined the physicochemical features (Step 1), optoacoustic signal generation (Step 2), stability (Step 3), biocompatibility (Step 4) and spectral sensitivity (Step 5). Using this characterization framework we showcase the discovery of two nanoparticle formulations, QH2-50 nm and QH2-100 nm that attain superior stability characteristics and optimal optoacoustic properties compared to gold standards commonly employed for near-infrared optoacoustics. We discuss encapsulation and self-quenching (ESQ) of organic dyes as a promising strategy to generate optimal optoacoustic particles.

PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) expression is transiently elevated following low dose irradiation typically encountered in the workplace and from natural sources. This long non-coding RNA recruits epigenetic silencers for cis-acting repression of its neighbouring Methionine adenosyltransferase 2A gene. It now emerges that PARTICLE operates as a trans-acting mediator of DNA and histone lysine methylation. Chromatin immunoprecipitation sequencing (ChIP-seq) and immunological evidence established elevated PARTICLE expression linked to increased histone 3 lysine 27 trimethylation. Live-imaging of dbroccoli-PARTICLE revealing its dynamic association with DNA methyltransferase 1 was confirmed by flow cytometry, immunoprecipitation and direct competitive binding interaction through electrophoretic mobility shift assay. Acting as a regulatory docking platform, the long non-coding RNA PARTICLE serves to interlink epigenetic modification machineries and represents a compelling innovative component necessary for gene silencing on a global scale.

Generated by Quaking (QKI), circular RNAs (circRNAs) are newly recognised non-coding RNA (ncRNA) members characterised by tissue specificity, increased stability and enrichment within exosomes. Studies have shown that ionizing radiation (IR) can influence ncRNA transcription. However, it is unknown whether circRNAs or indeed QKI are regulated by IR. Microarray circRNA profiling and next generation sequencing revealed that circRNA expression was altered by low and medium dose exposure sourced predominantly from genes influencing the p53 pathway. CircRNAs KIRKOS-71 and KIRKOS-73 transcribed from the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor (a p53 regulator) responded within hours to IR. KIRKOS-71 and KIRKOS-73 were present in exosomes yet exhibited differential transcript clearance between irradiated cell lines. Dual-quasar labelled probes and in-situ hybridization demonstrated the intercellular distribution of KIRKOS-71 and KIRKOS-73 predominantly within the perinucleus. QKI knockdown removed nuclear expression of these circRNAs with no significant effect on cytosolic KIRKOS-71 and KIRKOS-73. Distinct QKI transcription between cell lines and its augmented interaction with KIRKOS-71 and KIRKOS-73 was noted post IR. This foremost study provides evidence that QKI and circRNAs partake in the cellular irradiation response. KIRKOS-71 and KIRKOS-73 as stable secreted circRNAs may afford vital characteristics worth syphoning as promising diagnostic radiotherapy biomarkers.

The long non-coding RNA PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) partakes in triple helix (triplex) formation, is transiently elevated following low dose irradiation and regulates transcription of its neighbouring gene - Methionine adenosyltransferase 2A. It now emerges that PARTICLE triplex sites are predicted in many different genes across all human chromosomes. In silico analysis identified additional regions for PARTICLE triplexes at >1600 genomic locations. Multiple PARTICLE triplexes are clustered predominantly within the human and mouse tumor suppressor WW Domain Containing Oxidoreductase (WWOX) gene. Surface plasmon resonance diffraction and electrophoretic mobility shift assays were consistent with PARTICLE triplex formation within human WWOX with high resolution imaging demonstrating its enrichment at this locus on chromosome 16. PARTICLE knockdown and over-expression resulted in inverse changes in WWOX transcripts levels with siRNA interference eliminating PARTICLEs elevated transcription to irradiation. The evidence for a second functional site of PARTICLE triplex formation at WWOX suggests that PARTICLE may form triplex-mediated interactions at multiple positions in the human genome including remote loci. These findings provide a mechanistic explanation for the ability of lncRNAs to regulate the expression of numerous genes distributed across the genome.

Breakage of the fragile site FRA16D disrupts the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor gene in osteosarcoma. However, the frequency of breakage is not sufficient to explain the rate of WWOX loss in pathogenesis. The involvement of non-coding RNA transcripts is proposed due to their accumulation at fragile sites, where they are advocated to influence specific chromosomal regions associated with malignancy. The long ncRNA PARTICLE (promoter of MAT2A antisense radiation-induced circulating long non-coding RNA) is transiently elevated in response to irradiation and influences epigenetic silencing modification within WWOX. It now emerges that elevated PARTICLE levels are significantly associated with FRA16D non-breakage in OS patients. Although not associated with overall survival, high PARTICLE levels were found to be significantly linked to metastasis free outcome. The transcription of both PARTICLE and WWOX are transiently responsive to exposure to low doses of radiation in osteosarcoma cell lines. Herein, a relationship between WWOX and PARTICLE transcription is suggested in human osteosarcoma cell lines representing alternative genetic backgrounds. PARTICLE over-expression ameliorated WWOX promoter activity in U2OS harboring FRA16D non-breakage. It can be concluded that the lncRNA PARTICLE influences the WWOX tumor suppressor and in the absence of WWOX FRA16D breakage, it is associated with OS metastasis-free survival.

Model organisms such as zebrafish play an important role for developmental biologists and experimental geneticists. Still, as they grow into their post-embryonic stage of development it becomes more and more difficult to image them because of high light scattering inside biological tissue. Optoacoustic mesoscopy based on spherically focused, high frequency, ultrasound detectors offers an alternative, where it relies on the focusing capabilities of the ultrasound detectors in generating the image rather than on the focusing of light. Nonetheless, because of the limited numerical aperture the resolution is not isotropic, and many structures, especially elongated ones, such as blood vessels and other organs, are either invisible, or not clearly identifiable on the final image. Herein, based on high frequency ultrasound detectors at 100 MHz and 50 MHz we introduce multi orientation (view) optoacoustic mesoscopy. We collect a rich amount of signals from multiple directions and combine them using a weighted sum in the Fourier domain and a Wiener deconvolution into a single high resolution three-dimensional image. The new system achieves isotropic resolutions on the order of 10 μm in-plane, 40 μm axially, and SNR enhancement of 15 dB compared to the single orientation case. To showcase the system we imaged a juvenile zebrafish ex vivo, which is too large to image using optical microscopic techniques, the reconstructed images show unprecedented performance in terms of SNR, resolution, and clarity of the observed structures. Using the system we see the inner organs of the zebrafish, the pigmentation, and the vessels with unprecedented clarity.

It has long been held that the rise of neurons as a specialized cell type also marked the onset of the grand evolutionary journey for chemical synapses. Research over recent decades has shown, however, that the most dynamic chapters of synaptic history have been ‘written’ out of the context of neurobiology and neuronal evolution, dating back to the early metazoa and unicellular living forms. Here, I consider and discuss emerging evidence suggesting the exaptive origin of chemical synapses, via tinkering and neo-functionalization of already existent junctional morphologies and constituents of primeval paracrine signalling. Through combination and collateral use of long-established structures and functions, a remarkable enrichment of regulatory and control mechanisms of complex living organisms was achieved, without large-scale reorganization of the genome, with tremendous impact on the evolution and life on our planet.

With the central ability to visualize a variety of endogenous chromophores and biomarkers or exogenous contrast agents, optoacoustic (photoacoustic) imaging empowers new experimental capabilities for investigating brain mechanisms and functions. Here, the operational principles of optoacoustic neuroimaging are reviewed in conjunction with recent advances enabling high-resolution and real-time observation, which extend beyond the reach of optical imaging methods. Multiple implementations of optoacoustics for monitoring hemodynamics and neuro-vascular responses in the brain are showcased. The unique capabilities of optoacoustic imaging for multi-spectral cellular and molecular sensing are discussed with reference to recent application for visualizing healthy and diseased brains. Outstanding challenges in the field are considered in the context of current and future applications of optoacoustic neuroimaging for basic and translational neuroscience research. In pushing the boundaries of brain imaging, optoacoustic methods afford major insights into the neuronal mechanisms of brain functions and organization of behavior.

Lack of haptic feedback during laser surgery hampers controlling the incision depth, leading to a high risk of undesired tissue damage. Here we present a new feedback sensing method that accomplishes non-contact realtime monitoring of laser ablation procedures by detecting shock waves emanating from the ablation spot with air-coupled transducers. Experiments in soft and hard tissue samples attained high reproducibity in real-time depth estimation of the laser-induced cuts. The advantages derived from the non-contact nature of the suggested monitoring approach are expected to greatly promote the general applicability of laser-based surgeries.

Photothermal therapy and ablation are commonplace medical procedures employed for treatment of tumors, vascular and brain abnormalities as well as other disorders that require selective destruction of tissues. Yet, accurate mapping of the dynamic temperature field distribution in the treated region represents an unmet clinical need, strongly affecting the clinical outcome of these interventions. We introduce a fast three-dimensional temperature mapping method based on real-time optoacoustic sensing of the treated region coupled with a thermal-diffusion-based model of heat distribution in tissues. Deviations of the optoacoustic temperature readings provided at 40  ms intervals remained below 10% in tissue-mimicking phantom experiments for temperature elevations above 3 °C, as validated by simultaneous thermocouple measurements. Performance of the new method to dynamically estimate the volumetric temperature distribution was further showcased in post-mortem mouse imaging experiments. The newly discovered capacity to non-invasively measure the temperature map in an entire treated volume with both high spatial and temporal resolutions holds potential for improving safety and efficacy of light-based therapeutic interventions.

Ablation and photothermal therapy are widely employed medical protocols where the selective destruction of tissue is a necessity as in cancerous tissue removal or vascular and brain abnormalities. Tissue denaturation takes place when the temperature reaches a threshold value while the time of exposure determines the lesion size. Therefore, the spatio-temporal distribution of temperature plays a crucial role in the outcome of these clinical interventions. We demonstrate fast volumetric temperature mapping with optoacoustic tomography based on real-time optoacoustic readings from the treated region. The performance of the method was investigated in tissue-mimicking phantom experiments. The new ability to non-invasively measure temperature volumetrically in an entire treated region with high spatial and temporal resolutions holds potential for improving safety and efficacy of thermal ablation and to advance the general applicability of laser-based therapy.

Re-excision rates for breast cancer lumpectomy procedures are currently nearly 25% due to surgeons relying on inaccurate or incomplete methods of evaluating specimen margins. The objective of this study was to determine if cancer could be automatically detected in breast specimens from mastectomy and lumpectomy procedures by a classification algorithm that incorporated parameters derived from fluorescence lifetime imaging (FLIm). This study generated a database of co-registered histologic sections and FLIm data from breast cancer specimens (N=20) and a support vector machine (SVM) classification algorithm able to automatically detect cancerous, fibrous, and adipose breast tissue. Classification accuracies were greater than 97% for automated detection of cancerous, fibrous, and adipose tissue from breast cancer specimens. The classification worked equally well for specimens scanned by hand or with a mechanical stage, demonstrating that the system could be used during surgery or on excised specimens. The ability of this technique to simply discriminate between cancerous and normal breast tissue, in particular to distinguish fibrous breast tissue from tumor, which is notoriously challenging for optical techniques, leads to the conclusion that FLIm has great potential to assess breast cancer margins. Identification of positive margins before waiting for complete histologic analysis could significantly reduce breast cancer re-excision rates.

Frequency characteristics of ultrasound detectors used in optoacoustic tomography have a major impact on imaging performance. It is common practice to select transducers based on their sensitivity at the central frequency and under normal incidence. However, the bandwidth and angular sensitivity play an equally important role in establishing the quality and accuracy of the reconstructed images. Here, we developed a calibrated optoacoustic characterization method specifically tailored for broadband measurements of the angular transducer sensitivity (directivity). Ultrawideband omnidirectional optoacoustic responses were generated by uniformly illuminating thin absorbing sutures with nanosecond laser pulses and characterized with a needle hydrophone. This calibrated optoacoustic source was used to characterize the frequency dependence of the angular response by a conventional piezoelectric transducer (PZT) and a capacitive micromachined ultrasonic transducer (cMUT) with similar size and central frequency. Furthermore, both transducers had no preamplification electronics directly attached to the detection elements. While the PZT presented a 7.8 dB sensitivity advantage at normal incidence, it was able to provide detectable signal-to-noise levels only at incidence angles of up to 20 deg whereas the cMUT maintained reasonable sensitivity levels and broadband response at incidence angles of 40 deg and beyond. We further experimentally showcase a reduction in the limited-view image artifacts resulting from the broader acceptance angle of the cMUT.

Many neurological disorders are linked to abnormal activation or pathological alterations of the vasculature in the affected brain region. Obtaining simultaneous morphological and physiological information of neurovasculature is very challenging due to the acoustic distortions and intense light scattering by the skull and brain. In addition, the size of cerebral vasculature in murine brains spans an extended range from just a few microns up to about a millimeter, all to be recorded in 3D and over an area of several dozens of mm2. Numerous imaging techniques exist that excel at characterizing certain aspects of this complex network but are only capable of providing information on a limited spatiotemporal scale. We present a hybrid ultrasound and dual-wavelength optoacoustic microscope, capable of rapid imaging of murine neurovasculature in-vivo, with high spatial resolution down to 12 μm over a large field of view exceeding 50mm2. The dual wavelength imaging capability allows for the visualization of functional blood parameters through an intact skull while pulse-echo ultrasound biomicroscopy images are captured simultaneously by the same scan head. The flexible hybrid design in combination with fast high-resolution imaging in 3D holds promise for generating better insights into the architecture and function of the neurovascular system.

Optoacoustic imaging is a rapidly developing area of biomedical imaging due its combination of rich optical contrast and ultrasound depth penetration. Just like conventional pulse-echo ultrasound imaging, optoacoustic tomography relies on the use of ultrasound detector arrays with a large number of elements. The precise knowledge of the transducer's sensitivity is crucial for the prediction of its performance for a given imaging task. Sensitivity characteristics such as the central frequency and bandwidth are routinely characterized. However, this characterization is typically performed solely under normal incidence since the measurement of the angle and frequency depended sensitivity (directivity) is difficult and time consuming with existing ultrasound characterization methods. We present a simple and fast characterization method for broadband directivity measurements of the angular transducer sensitivity based on the optoacoustic effect. The method utilizes a thin absorbing suture in order to generate omnidirectional and broadband optoacoustic signals, which are calibrated using a needle hydrophone. We applied this method to characterize and compare the directivity of a conventional piezoelectric (PZT) transducer to the directivity of a capacitive micromachined ultrasonic (cMUT) transducer. Both technologies showed a similar broadband response at normal incidence and the PZT transducer displayed a more than two times larger signal to noise ratio at normal incidence. However, the cMUT transducer's sensitivity was significantly less angle-depended and outperformed the PZT's sensitivity for angles larger than 20°.

SPIE-OSA. We present an optoacoustic microscope, combining structural and functional opticalresolution optoacoustic and ultrasound pulse-echo imaging. The system was applied to image Zebrafish larvae and complex vascular networks in the murine brain and ear.

Objective. Inclusion body myositis (IBM) is characterized by a combination of inflammatory and degenerative changes affecting muscle. While the primary cause of IBMis unknown, genetic factors may influence disease susceptibility. To determine genetic factors contributing to the etiology of IBM, we conducted the largest genetic association study of the disease to date, investigating immune-related genes using the Immunochip. Methods. A total of 252 Caucasian patients with IBM were recruited from 11 countries through the Myositis Genetics Consortium and compared with 1,008 ethnically matched controls. Classic HLA alleles and amino acids were imputed using SNP2HLA. Results. The HLA region was confirmed as the most strongly associated region in IBM (p=3.58 x 10(-33)). HLA imputation identified 3 independent associations (with HLA-DRB1*03: 01, DRB1*01: 01, and DRB1*13: 01), although the strongest association was with amino acid positions 26 and 11 of the HLA-DRB1 molecule. No association with anti-cytosolic 50-nucleotidase 1A-positive status was found independent ofHLA-DRB1*03: 01. There was no association of HLA genotypes with age at onset of IBM. Three non-HLA regions reached suggestive significance, including the chromosome 3 p21.31 region, an established risk locus for autoimmune disease, where a frameshift mutation in CCR5 is thought to be the causal variant. Conclusion. This is the largest, most comprehensive genetic association study to date in IBM. The data confirm that HLA is the most strongly associated region and identifies novel amino acid associations that may explain the risk in this locus. These amino acid associations differentiate IBM from polymyositis and dermatomyositis and may determine properties of the peptide-binding groove, allowing it to preferentially bind autoantigenic peptides. A novel suggestive association within the chromosome 3 p21.31 region suggests a role for CCR5.

Optoacoustic (photoacoustic) dermoscopy offers two principal advantages over conventional optical imaging applied in dermatology. First, it yields high-resolution cross-sectional images of the skin at depths not accessible to other non-invasive optical imaging methods. Second, by resolving absorption spectra at multiple wavelengths, it enables label-free three-dimensional visualization of morphological and functional features. However, the relation of pulse energy to generated bandwidth and imaging depth remains poorly defined. In this study, we apply computer models to investigate the optoacoustic frequency response generated by simulated skin. We relate our simulation results to experimental measurements of the detection bandwidth as a function of optical excitation energy in phantoms and human skin. Using raster-scan optoacoustic mesoscopy (RSOM), we further compare the performance of two broadband ultrasonic detectors (bandwidth of 20-180 MHz and 10-90 MHz) in acquiring optoacoustic readouts. Based on the findings of this study, we propose energy ranges required for skin imaging with considerations of laser safety standards.

Raster-scan optoacoustic mesoscopy (RSOM), also termed photoacoustic mesoscopy, offers novel insights into vascular morphology and pathophysiological biomarkers of skin inflammation in vivo at depths unattainable by other optical imaging methods. Using ultra-wideband detection and focused ultrasound transducers, RSOM can achieve axial resolution of 4 micron and lateral resolution of 20 micron to depths of several millimeters. However, motion effects may deteriorate performance and reduce the effective resolution. To provide high-quality optoacoustic images in clinical measurements, we developed a motion correction algorithm for RSOM. The algorithm is based on observing disruptions of the ultrasound wave front generated by the vertical movement of the melanin layer at the skin surface. From the disrupted skin surface, a smooth synthetic surface is generated, and the offset between the two surfaces is used to correct for the relative position of the ultrasound detector. We test the algorithm in measurements of healthy and psoriatic human skin and achieve effective resolution up to 5-fold higher than before correction. We discuss the performance of the correction algorithm and its implications in the context of multispectral mesoscopy.

Carotid atheromatosis is causally related to stroke, a leading cause of disability and death. We present the analysis of a human carotid atheroma using a novel hybrid microscopy system that combines optical-resolution optoacoustic (photoacoustic) microscopy and several non-linear optical microscopy modalities (second and third harmonic generation, as well as, two-photon excitation fluorescence) to achieve a multimodal examination of the extracted tissue within the same imaging framework. Our system enables the label-free investigation of atheromatous human carotid tissue with a resolution of about 1 μm and allows for the congruent interrogation of plaque morphology and clinically relevant constituents such as red blood cells, collagen, and elastin. Our data reveal mutual interactions between blood embeddings and connective tissue within the atheroma, offering comprehensive insights into its stage of evolution and severity, and potentially facilitating the further development of diagnostic tools, as well as treatment strategies.

Hypothalamic neural circuits are recognised as primary sites of the neuromodulator effect of homeostatic food intake, whereas changes in ventral tegmental area (VTA), hippocampus and amygdala have been implicated in the hedonic, cognitive and emotional aspects of eating. Here, we discuss synaptic transmission and plasticity within brain circuits governing appetite and food intake behaviour, focusing on the metabolic hormones ghrelin and leptin. We discuss functional changes within these circuitries and critically assess the applicability of electrophysiological measurements using in vitro multielectrode array (MEA) systems to identify novel appetite modulators. Stringent validation of functional assays to screen neuroactive substrates is of crucial importance for the discovery of novel food intake modulators, with major implications for the nutraceutical food industry and drug development.

The addition of optoacoustic sensing to optical microscopy may supplement fluorescence contrast with label-free measurements of optical absorption, enhancing biological observation. However, the physical dimensions of many optoacoustic systems have restricted the implementation of hybrid optical and optoacoustic (O2A) microscopy to imaging thin samples in transmission mode or to ex-vivo investigations. Here we describe a miniaturized optoacoustic sensor, based on a

BACKGROUND: The role of local alterations in endothelial functional integrity in atherosclerosis remains incompletely understood. This study used nanoparticle-enhanced optical molecular imaging to probe in vivo mechanisms involving impaired endothelial barrier function in experimental atherothrombosis. METHODS AND RESULTS: Atherosclerosis was induced in rabbits (n=31) using aortic balloon injury and high-cholesterol diet. Rabbits received ultrasmall superparamagnetic iron oxide nanoparticles (CLIO) derivatized with a near-infrared fluorophore (CyAm7) 24 hours before near-infrared fluorescence imaging. Rabbits were then either euthanized (n=9) or underwent a pharmacological triggering protocol to induce thrombosis (n=22). CLIO-CyAm7 nanoparticles accumulated in areas of atheroma (P<0.05 versus reference areas). On near-infrared fluorescence microscopy, CLIO-CyAm7 primarily deposited in the superficial intima within plaque macrophages, endothelial cells, and smooth muscle cells. Nanoparticle-positive areas further exhibited impaired endothelial barrier function as illuminated by Evans blue leakage. Deeper nanoparticle deposition occurred in areas of plaque neovascularization. In rabbits subject to pharmacological triggering, plaques that thrombosed exhibited significantly higher CLIO-CyAm7 accumulation compared with nonthrombosed plaques (P<0.05). In thrombosed plaques, nanoparticles accumulated preferentially at the plaque-thrombus interface. Intravascular 2-dimensional near-infrared fluorescence imaging detected nanoparticles in human coronary artery-sized atheroma in vivo (P<0.05 versus reference segments). CONCLUSIONS: Plaques that exhibit impaired in vivo endothelial permeability in cell-rich areas are susceptible to subsequent thrombosis. Molecular imaging of nanoparticle deposition may help to identify biologically high-risk atheroma.

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

The development of suitable contrast agents can significantly enhance the efficiency of modern imaging and treatment techniques, such as thermoacoustic (TA) tomography and radio-frequency (RF) hyperthermia of cancer. Here, we examine the heating of aqueous suspensions of silicon (Si) and gold (Au) nanoparticles (NPs) under RF irradiation in the MHz frequency range. The heating rate of aqueous suspensions of Si NPs exhibited non-monotonic dependency on the electrical conductivity of the suspension. The experimental results were explained by the mathematical model considering oscillating solvated ions as the main source of Joule heating. These ions could be the product of the dissolution of Si NPs or organic coating of Au NPs. Thus, the ions governed the conductivity of the suspensions, which in turn governs both the heating rate and the near-field RF TA response. The model predicted the contrast in different tissues taking into account both Joule heating and dielectric losses.

Optoacoustic microscopy (OAM) is a hybrid imaging method that can achieve high spatial resolution at superficial depths through use of focused illumination; it can be adapted for imaging with ultrasonic resolution at much greater depths where the excitation light is diffuse. These two distinct modes of operation can be further combined to create a highly scalable technique that can image at multiple penetration scales by gradually exchanging microscopic optical resolution in superficial tissue layers with ultrasonic resolution at diffuse (macroscopic) depths. However, OAM commonly employs scanning acquisition geometries that impede the effective use of synthetic aperture focusing techniques due to varying illumination patterns and non-uniformity of the excitation light field. Here we present a universal framework for scanning optoacoustic microscopy that uses a weighted synthetic aperture focusing technique (W-SAFT) to create a uniform imaging sensitivity across microscopic, mesoscopic, and macroscopic penetration regimes. Robust performance of the new multi-scale reconstruction methodology is showcased with simulations and synthetic phantoms, and validated with experimental data acquired from a highly scattering juvenile zebrafish specimen. It is shown that consideration of the light fluence is vital for maintaining the optically dictated lateral resolution at ballistic depths while optimizing the resolution of out-of-focus ultrasonic data; additionally, the dynamic-range compression facilitates the visualization across the entire imaged volume. The newly introduced W-SAFT reconstruction framework is universally applicable to a wide palette of scanning-based optoacoustic imaging techniques employing non-uniform and/or varying illumination, such as acoustic resolution and hybrid focus microscopy, raster-scan optoacoustic mesoscopy, as well as tomographic approaches using scanning of focused array transducers.

A key feature of optoacoustic imaging is the ability to illuminate tissue at multiple wavelengths and therefore record images with a spectral dimension. While optoacoustic images at single wavelengths reveal morphological features, in analogy to ultrasound imaging or X-ray imaging, spectral imaging concedes sensing of intrinsic chromophores and externally administered agents that can reveal physiological, cellular and subcellular functions. Nevertheless, identification of spectral moieties within images obtained at multiple wavelengths requires spectral unmixing techniques, which present a unique mathematical problem given the three-dimensional nature of the optoacoustic images. Herein we discuss progress with spectral unmixing techniques developed for multispectral optoacoustic tomography. We explain how different techniques are required for accurate sensing of intrinsic tissue chromophores such as oxygenated and deoxygenated haemoglobin versus extrinsically administered photo-absorbing agents and nanoparticles. Finally, we review recent developments that allow accurate quantification of blood oxygen saturation (sO2) by transforming and solving the sO2 estimation problem from the spatial to the spectral domain.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

We present an optoacoustic (photoacoustic) microscopy (OAM) imaging system that uses a pi-shifted Fiber Bragg Grating (pi-FBG) as ultrasound (US) sensor. The sensor has an ultra-small footprint and hence allows for the detection of optoacoustic signals in close proximity to their origin. The interrogation of the pi-FBG is performed by a broadband pulsed laser, enabling a high sensitivity of the sensor as well as the elimination of ambient noise. We characterize the pi-FBG in terms of axial and lateral resolution as well as its bandwidth and find that its performance is comparable to US sensors that are based on the piezoelectric effect. We demonstrate the system’s capabilities by images taken from ex vivo zebrafish and mouse ear samples. The results presented herein highlight that pi-FBGs are a promising tool for the comprehensive label-free optoacoustic imaging of biomedical samples.

We have further developed the skin imaging optoacoustic mesoscopy technology building the first broadband, high frequency, raster scan handheld system. The apparatus is designed to provide cross sectional images that contain rich depth dependent information. A compact fixed illumination scheme fulfills the imaging and size demands of the scanner. The system capabilities haves been characterized using experiments with phantoms and healthy subjects.

Fluorescence imaging has been considered for over a half-century as a modality that could assist surgical guidance and visualization. The administration of fluorescent molecules with sensitivity to disease biomarkers and their imaging using a fluorescence camera can outline pathophysiological parameters of tissue invisible to the human eye during operation. The advent of fluorescent agents that target specific cellular responses and molecular pathways of disease has facilitated the intraoperative identification of cancer with improved sensitivity and specificity over nonspecific fluorescent dyes that only outline the vascular system and enhanced permeability effects. With these new abilities come unique requirements for developing phantoms to calibrate imaging systems and algorithms. We briefly review herein progress with fluorescence phantoms employed to validate fluorescence imaging systems and results. We identify current limitations and discuss the level of phantom complexity that may be required for developing a universal strategy for fluorescence imaging calibration. Finally, we present a phantom design that could be used as a tool for interlaboratory system performance evaluation.

Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer's disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75(NTR)) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin-biotin technology with anti-p75(NTR) antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75(NTR) for targeted transduction of vectors to BFCNs in vivo.

Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.

Diversity of the design and alignment of illumination and ultrasonic transducers empower the fine scalability and versatility of optoacoustic imaging. In this study, we implement an innovative high-resolution optoacoustic mesoscopy for imaging the vasculature and tissue oxygenation within subcutaneous and orthotopic cancerous implants of mice in vivo through acquisition of tomographic projections over 180° at a central frequency of 24 MHz. High-resolution volumetric imaging was combined with multispectral functional measurements to resolve the exquisite inner structure and vascularization of the entire tumor mass using endogenous and exogenous optoacoustic contrast. Evidence is presented for constitutive hypoxemia within the carcinogenic tissue through analysis of the hemoglobin absorption spectra and distribution. Morphometric readouts obtained with optoacoustic mesoscopy have been verified with high-resolution ultramicroscopic studies. The findings described herein greatly extend the applications of optoacoustic mesoscopy toward structural and multispectral functional measurements of the vascularization and hemodynamics within solid tumors in vivo and are of major relevance to basic and preclinical oncological studies in small animal models.

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM-DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO2 ). Unlike conventional photoacoustic spectroscopy, WM-DPAS measures simultaneously two signals induced from square-wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy- and oxy-hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two-wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM-DPAS to detect very small changes in total hemoglobin concentration (CHb ) and oxygenation levels, thereby identifying pre-malignant tumors before they are anatomically apparent. The non-invasive nature also makes WM-DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM-DPAS can be tuned for different applications such as quick cancer screening and accurate StO2 quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM-DPAS theory has been validated with sheep blood phantom measurements. Sensitivity comparison between conventional single-ended signal and differential signal.

Fluorescence diffuse optical tomography (fDOT) provides 3D images of fluorescence distributions in biological tissue, which represent molecular and cellular processes. The image reconstruction problem is highly ill-posed and requires regularisation techniques to stabilise and find meaningful solutions. Quadratic regularisation tends to either oversmooth or generate very noisy reconstructions, depending on the regularisation strength. Edge preserving methods, such as anisotropic diffusion regularisation (AD), can preserve important features in the fluorescence image and smooth out noise. However, AD has limited ability to distinguish an edge from noise. We propose a patch-based anisotropic diffusion regularisation (PAD), where regularisation strength is determined by a weighted average according to the similarity between patches around voxels within a search window, instead of a simple local neighbourhood strategy. However, this method has higher computational complexity and, hence, we wavelet compress the patches (PAD-WT) to speed it up, while simultaneously taking advantage of the denoising properties of wavelet thresholding. Furthermore, structural information can be incorporated into the image reconstruction with PAD-WT to improve image quality and resolution. In this case, the weights used to average voxels in the image are calculated using the structural image, instead of the fluorescence image. The regularisation strength depends on both structural and fluorescence images, which guarantees that the method can preserve fluorescence information even when it is not structurally visible in the anatomical images. In part 1, we tested the method using a denoising problem. Here, we use simulated and in vivo mouse fDOT data to assess the algorithm performance. Our results show that the proposed PAD-WT method provides high quality and noise free images, superior to those obtained using AD.

We report on a new method for mapping light fluence distribution deep in a scattering tissues based on real-time optoacoustic tomographic acquisition of temporal data from reversibly switchable fluorescent proteins (RSFPs).

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically-dense breast. Even when combined with ultrasound or magnetic resonance imaging, X-Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high-resolution functional and anatomical breast angiography using volumetric hand-held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular-dominated dense breasts revealed the feasibility of rendering three-dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real-time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow-up of carcinomas in radiographically-dense breasts. Volumetric handheld optoacoustic tomography scanner uses safe pulses of near-infrared light to render three-dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.

Optoacoustic techniques rely on ultrasound transmission between optical absorbers within tissues and the measurement location. Much like in echography, commonly used piezoelectric transducers require either direct contact with the tissue or through a liquid coupling medium. The contact nature of this detection approach then represents a disadvantage of standard optoacoustic systems with respect to other imaging modalities (including optical techniques) in applications where non-contact imaging is needed, e.g. in open surgeries or when burns or other lesions are present in the skin. Herein, non-contact optoacoustic imaging using raster-scanning of a spherically-focused piezoelectric air-coupled ultrasound transducer is demonstrated. When employing laser fluence levels not exceeding the maximal permissible human exposure, it is shown possible to attain detectable signals from objects as small as 1 mm having absorption properties representative of blood at near-infrared wavelengths with a relatively low number of averages. Optoacoustic images from vessel-mimicking tubes embedded in an agar phantom are further showcased. The initial results indicate that the air-coupled ultrasound detection approach can be potentially made suitable for non-contact biomedical imaging with optoacoustics.

Synthetic and genetically encoded chromo- and fluorophores have become indispensable tools for biomedical research enabling a myriad of applications in imaging modalities based on biomedical optics. The versatility offered by the optoacoustic (photoacoustic) contrast mechanism enables to detect signals from any substance absorbing light, and hence these probes can be used as optoacoustic contrast agents. While contrast versatility generally represents an advantage of optoacoustics, the strong background signal generated by light absorption in endogeneous chromophores hampers the optoacoustic capacity to detect a photo-absorbing agent of interest. Increasing the optoacoustic sensitivity is then determined by the capability to differentiate specific features of such agent. For example, multispectral optoacoustic tomography (MSOT) exploits illuminating the tissue at multiple optical wavelengths to spectrally resolve (unmix) the contribution of different chromophores. Herein, we present an alternative approach to enhance the sensitivity and specificity in the detection of optoacoustic contrast agents. This is achieved with photoswitchable probes that change optical absorption upon illumination with specific optical wavelengths. Thereby, temporally unmixed MSOT (tuMSOT) is based on photoswitching the compounds according to defined schedules to elicit specific time-varying optoacoustic signals, and then use temporal unmixing algorithms to locate the contrast agent based on their particular temporal profile. The photoswitching kinetics is further affected by light intensity, so that tuMSOT can be employed to estimate the light fluence distribution in a biological sample. The performance of the method is demonstrated herein with the reversibly switchable fluorescent protein Dronpa and its fast-switching fatigue resistant variant Dronpa-M159T.

Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience. We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains. It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm with respective spatial resolution of 35-200 μm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains. The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics; in combination with optoacoustics' well-established capacity for resolving vascular hemodynamics, it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.

Wavefront shaping based on optoacoustic (photoacoustic) feedback has recently emerged as a promising tool to control the light distribution in optically-scattering media. In this approach, the phase of a short-pulsed light beam is spatially-modulated to create constructive light interference (focusing) at specific locations in the speckle pattern of the scattered wavefield. The optoacoustic signals generated by light absorption provide a convenient feedback mechanism to optimize the phase mask of the spatial light modulator in order to achieve the desired light intensity distribution. The optimization procedure can be done by directly considering the acquired signals or the reconstructed images of the light absorption distribution. Recently, our group has introduced a volumetric (three-dimensional) optoacoustic wavefront shaping platform that enables monitoring the distribution of light absorption in an entire volume with frame rates of tens of Hz. With this approach, it is possible to simultaneously control the volumetric light distribution through turbid media. Experiments performed with absorbing microparticles distributed in a three-dimensional region showcase the feasibility of enhancing the light intensity at specific points, where the size of particles is also essential to maximize the signal enhancement. The advantages provided by optoacoustic imaging in terms of spatial and temporal resolution anticipate new capabilities of wavefront shaping techniques in biomedical optics.

Similar to pulse-echo ultrasound, optoacoustic imaging encodes the location of optical absorbers by the time-of-flight of ultrasound waves. Yet, signal generation mechanisms are fundamentally different for the two modalities, leading to significant distinction between the optimum image formation strategies. While interference of back-scattered ultrasound waves with random phases causes speckle noise in ultrasound images, speckle formation is hindered by the strong correlation between the optoacoustic responses corresponding to individual sources. However, visibility of structures is severely hampered when attempting to acquire optoacoustic images under limited-view tomographic geometries. In this tutorial article, we systematically describe the basic principles of optoacoustic signal generation and image formation for objects ranging from individual sub-resolution absorbers to a continuous absorption distribution. The results are of relevance for the proper interpretation of optoacoustic images acquired under limited-view scenarios and may also serve as a basis for optimal design of tomographic acquisition geometries and image formation strategies.

BACKGROUND: Smart Health is known as a concept that enhances networking, intelligent data processing and combining patient data with other parameters. Open data models can play an important role in creating a framework for providing interoperable data services that support the development of innovative Smart Health applications profiting from data fusion and sharing. METHODS: This article describes a model-driven engineering approach based on standardized clinical information models and explores its application for the development of interoperable electronic health record systems. The following possible model-driven procedures were considered: provision of data schemes for data exchange, automated generation of artefacts for application development and native platforms that directly execute the models. The applicability of the approach in practice was examined using the openEHR framework as an example. RESULTS: A comprehensive infrastructure for model-driven engineering of electronic health records is presented using the example of the openEHR framework. It is shown that data schema definitions to be used in common practice software development processes can be derived from domain models. The capabilities for automatic creation of implementation artefacts (e.g., data entry forms) are demonstrated. Complementary programming libraries and frameworks that foster the use of open data models are introduced. Several compatible health data platforms are listed. They provide standard based interfaces for interconnecting with further applications. CONCLUSION: Open data models help build a framework for interoperable data services that support the development of innovative Smart Health applications. Related tools for model-driven application development foster semantic interoperability and interconnected innovative applications.

We interrogated the application and imaging features obtained by non-invasive and handheld optoacoustic imaging of the thyroid in-vivo. Optoacoustics can offer complementary contrast to ultrasound, by resolving optical absorption-based and offering speckle-free imaging. In particular we inquired whether vascular structures could be better resolved using optoacoustics. For this reason we developed a compact handheld version of real-time multispectral optoacoustic tomography (MSOT) using a detector adapted to the dimensions and overall geometry of the human neck. For delivering high-fidelity performance, a curved ultrasound array was employed. The feasibility of handheld thyroid MSOT was assessed on healthy human volunteers at single wavelength. The results were contrasted to ultrasound and Doppler ultrasound images obtained from the same volunteers. Imaging findings demonstrate the overall MSOT utility to accurately retrieve optical features consistent with the thyroid anatomy and the morphology of surrounding structures.

Analytical (closed-form) inversion schemes have been the standard approach for image reconstruction in optoacoustic tomography due to their fast reconstruction abilities and low memory requirements. Yet, the need for quantitative imaging and artifact reduction has led to the development of more accurate inversion approaches, which rely on accurate forward modeling of the optoacoustic wave generation and propagation. In this way, multiple experimental factors can be incorporated, such as the exact detection geometry, spatio-temporal response of the transducers, and acoustic heterogeneities. The modelbased inversion commonly results in very large sparse matrix formulations that require computationally extensive and memory demanding regularization schemes for image reconstruction, hindering their effective implementation in real-time imaging applications. Herein, we introduce a new discretization procedure for efficient model-based reconstructions in two-dimensional optoacoustic tomography that allows for parallel implementation on a graphics processing unit (GPU) with a relatively low numerical complexity. By on-the-fly calculation of the model matrix in each iteration of the inversion procedure, the new approach results in imaging frame rates exceeding 10Hz, thus enabling real-time image rendering using the model-based approach.

We describe a novel discretization procedure for model-based inversion in twodimensional optoacoustic tomography. This procedure can be efficiently implemented on a graphic-processing-unit and enables model-based image reconstruction at a frame rate exceeding 10 Hz.

A functional cancer theranostic nanoplatform is developed, specifically tailored toward the optoacoustic modality by combining gold nanorods with DNA nanostructures (D-AuNR). DNA origami is used as an efficient delivery vehicle owing to its prominent tumor-targeting property. The D-AuNR hybrids display an enhanced tumor diagnostic sensitivity by improved optoacoustic imaging and excellent photothermal therapeutic properties in vivo.

OBJECTIVES: Intravital imaging within heterogenic solid tumours is important for understanding blood perfusion profiles responsible for establishment of multiple parameters within the tumour mass, such as hypoxic and nutrition gradients, cell viability, proliferation and drug response potentials. METHODS: Herein, we developed a method based on a volumetric multispectral optoacoustic tomography (vMSOT) for cancer imaging in preclinical models and explored its capacity for three-dimensional imaging of anatomic, vascular and functional tumour profiles in real time. RESULTS: In contrast to methods based on cross-sectional (2D) image acquisition as a basis for 3D rendering, vMSOT has attained concurrent observations from the entire tumour volume at 10 volumetric frames per second. This truly four dimensional imaging performance has enabled here the simultaneous assessment of blood oxygenation gradients and vascularization in solid breast tumours and revealed different types of blood perfusion profiles in-vivo. CONCLUSION: The newly introduced capacity for high-resolution three-dimensional tracking of fast tumour perfusion suggests vMSOT as a powerful method in preclinical cancer research and theranostics. As the imaging setup can be equally operated in both stationary and handheld mode, the solution is readily translatable for perfusion monitoring in a clinical setting. KEY POINTS: • vMSOT visualizes 3D anatomic, vascular and functional tumour profiles in real time. • Three types of blood perfusion profiles are revealed in breast tumour model. • The method is readily adaptable to operate in a handheld clinical mode.

It has been well recognized that the presence of a skull imposes harsh restrictions on the use of ultrasound and optoacoustic techniques in the study, treatment and modulation of the brain function. We propose a rigorous modeling and experimental methodology for estimating the insertion loss and the elastic constants of the skull over a wide range of frequencies and incidence angles. A point-source-like excitation of ultrawideband acoustic radiation was induced via the absorption of nanosecond duration laser pulses by a 20 μm diameter microsphere. The acoustic waves transmitted through the skull are recorded by a broadband, spherically focused ultrasound transducer. A coregistered pulse-echo ultrasound scan is subsequently performed to provide accurate skull geometry to be fed into an acoustic transmission model represented in an angular spectrum domain. The modeling predictions were validated by measurements taken from a glass cover-slip and ex vivo adult mouse skulls. The flexible semi-analytical formulation of the model allows for seamless extension to other transducer geometries and diverse experimental scenarios involving broadband acoustic transmission through locally flat solid structures. It is anticipated that accurate quantification and modeling of the skull transmission effects would ultimately allow for skull aberration correction in a broad variety of applications employing transcranial detection or transmission of high frequency ultrasound.

The acoustically-mismatched skull bone poses significant challenges for the application of ultrasonic and optical techniques in neuroimaging, still typically requiring invasive approaches using craniotomy or skull thinning. Optoacoustic imaging partially circumvents the acoustic distortions due to the skull because the induced wave is transmitted only once as opposed to the round trip in pulse-echo ultrasonography. To this end, the mouse brain has been successfully imaged transcranially by optoacoustic scanning microscopy. Yet, the skull may adversely affect the lateral and axial resolution of transcranial brain images. In order to accurately characterize the complex behavior of the optoacoustic signal as it traverses through the skull, one needs to consider the ultrawideband nature of the optoacoustic signals. Here the insertion loss of murine skull has been measured by means of a hybrid optoacoustic-ultrasound scanning microscope having a spherically focused PVDF transducer and pulsed laser excitation at 532 nm of a 20 μm diameter absorbing microsphere acting as an optoacoustic point source. Accurate modeling of the acoustic transmission through the skull is further performed using a Fourier-domain expansion of a solid-plate model, based on the simultaneously acquired pulse-echo ultrasound image providing precise information about the skull's position and its orientation relative to the optoacoustic source. Good qualitative agreement has been found between the a solid-plate model and experimental measurements. The presented strategy might pave the way for modeling skull effects and deriving efficient correction schemes to account for acoustic distortions introduced by an adult murine skull, thus improving the spatial resolution, effective penetration depth and overall image quality of transcranial optoacoustic brain microscopy.

Chronic venous insufficiency (CVI) is one of the most common medical conditions with reported prevalence estimates as high as 30% in the adult population. Although conservative management with compression therapy may improve the symptoms associated with CVI, healing often demands invasive procedures. Besides established surgical methods like vein stripping or bypassing, endovenous laser therapy (ELT) emerged as a promising novel treatment option during the last 15 years offering multiple advantages such as less pain and faster recovery. Much of the treatment success hereby depends on monitoring of the treatment progression using clinical imaging modalities such as Doppler ultrasound. The latter however do not provide sufficient contrast, spatial resolution and three-dimensional imaging capacity which is necessary for accurate online lesion assessment during treatment. As a consequence, incidence of recanalization, lack of vessel occlusion and collateral damage remains highly variable among patients. In this study, we examined the capacity of volumetric optoacoustic tomography (VOT) for real-time monitoring of ELT using an ex-vivo ox foot model. ELT was performed on subcutaneous veins while optoacoustic signals were acquired and reconstructed in real-time and at a spatial resolution in the order of 200μm. VOT images showed spatio-temporal maps of the lesion progression, characteristics of the vessel wall, and position of the ablation fiber’s tip during the pull back. It was also possible to correlate the images with the temperature elevation measured in the area adjacent to the ablation spot. We conclude that VOT is a promising tool for providing online feedback during endovenous laser therapy.

Accurate visualization of biological events occurring on a sub-second scale requires high frame rate acquisition of image data from living tissues. Yet,fast imaging performance commonly comes at the cost of limited field-of-view and reduced image quality. Here,we report on a small-animal optoacoustic tomographic imaging concept based on scanning of a spherical detection array. The suggested approach delivers whole-body images of unparalleled quality while retaining real-time volumetric imaging capability within selected regions at the whole organ scale. Imaging performance was tested in tissue-mimicking phantoms and living animals,attaining nearly isotropic three-dimensional spatial resolution in the range of 250-500 µm across fields of view covering the entire mouse body. The system maintained high volumetric imaging rates of 100 frames per second within volumes of up to 1.5 cm3,which further allowed visualizing the fast motion of a beating mouse heart without gating the acquisition. The newly introduced approach is ideally suited for acquisition of both real-time and whole-body volumetric image data,thus offering powerful capacities for simultaneous anatomical,functional,and molecular imaging with optoacoustics.

Visualizing anatomical and functional features of hair follicle development in their unperturbed environment is key in understanding complex mechanisms of hair pathophysiology and in discovery of novel therapies. Of particular interest is in-vivo visualization of the intact pilosebaceous unit, vascularization of the hair bulb and evaluation of the hair cycle particularly in humans. Furthermore, non-invasive visualization of the sebaceous glands could offer crucial insight into pathophysiology of follicle-related diseases, dry or seborrhoic skin, in particular by combining in-vivo imaging with other phenotyping, genotyping and microbial analyses. The available imaging techniques are limited in their ability for deep tissue in-vivo imaging of hair follicles and lipid-rich sebaceous glands in their entirety without biopsy. We developed a non-invasive, painless and risk-free volumetric multispectral optoacoustic tomography (vMSOT) method for deep tissue three-dimensional visualization of whole hair follicles and surrounding structures with high spatial resolution below 70μm. Herein we demonstrate on-the-fly assessment of key morphometric parameters of follicles and lipid content as well as functional oxygenation parameters of the associated capillary bed. The ease of handheld operation and versatility of the newly-developed approach poise it as an indispensable tool for early diagnosis of disorders of the pilosebaceous unit and surrounding structures, and for monitoring the efficacy of cosmetic and therapeutic interventions.

Multispectral tissue imaging based on optical cameras and continuous-wave tissue illumination is commonly used in medicine and biology. Surprisingly, there is a characteristic absence of a critical look at the quantities that can be uniquely characterized from optically diffuse matter by multispectral imaging. Here, we investigate the fundamental question of uniqueness in epi-illumination measurements from turbid media obtained at multiple wavelengths. By utilizing an analytical model, tissue-mimicking phantoms, and an in vivo imaging experiment we show that independent of the bands employed, spectral measurements cannot uniquely retrieve absorption and scattering coefficients. We also establish that it is, nevertheless, possible to uniquely quantify oxygen saturation and the Mie scattering power-a previously undocumented uniqueness condition.

In optoacoustic tomography, detectors with relatively large areas are often employed to achieve high detection sensitivity. However, spatial-averaging effects over large detector areas may lead to attenuation of high acoustic frequencies and, subsequently, loss of fine features in the reconstructed image. Model-based reconstruction algorithms improve image resolution in such cases by correcting for the effect of the detector's aperture on the detected signals. However, the incorporation of the detector's geometry in the optoacoustic model leads to a significant increase of the model matrix memory cost, which hinders the application of inversion and analysis tools such as singular value decomposition (SVD). We demonstrate the use of the wavelet-packet framework for optoacoustic systems with finite-aperture detectors. The decomposition of the model matrix in the wavelet-packet domain leads to sufficiently smaller model matrices on which SVD may be applied. Using this methodology over an order of magnitude reduction in inversion time is demonstrated for numerically generated and experimental data. Additionally, our framework is demonstrated for the analysis of inversion stability and reveals a new, nonmonotonic dependency of the system condition number on the detector size. Thus, the proposed framework may assist in choosing the optimal detector size in future optoacoustic systems.

BACKGROUND: Optimum cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is essential for the curative treatment of peritoneal carcinomatosis of colorectal origin. At present, surgeons depend on visual inspection and palpation for tumour detection. Improved detection of tumour tissue using molecular fluorescence-guided surgery could not only help attain a complete cytoreduction of metastatic lesions, but might also prevent overtreatment by avoiding resection of benign lesions. METHODS: For this non-randomised, single-centre feasibility study, we enrolled patients with colorectal peritoneal metastases scheduled for cytoreductive surgery and HIPEC. 2 days before surgery, 4·5 mg of the near-infrared fluorescent tracer bevacizumab-IRDye800CW was administered intravenously. The primary objectives were to determine the safety and feasibility of molecular fluorescence-guided surgery using bevacizumab-IRDye800CW. Molecular fluorescence-guided surgery was deemed safe if no allergic or anaphylactic reactions were recorded and no serious adverse events were attributed to bevacizumab-IRDye800CW. The technique was deemed feasible if bevacizumab-IRDye800CW enabled detection of fluorescence signals intraoperatively. Secondary objectives were correlation of fluorescence with histopathology by back-table imaging of the fresh surgical specimen and semi-quantitative ex-vivo analyses of formalin-fixed paraffin embedded (FFPE) tissue on all peritoneal lesions. Additionally, VEGF-α staining and fluorescence microscopy was done. This study is registered with the Netherlands Trial Registry, number NTR4632. FINDINGS: Between July 3, 2014, and March 2, 2015, seven patients were enrolled in the study. One patient developed an abdominal sepsis 5 days postoperatively and another died from an asystole 4 days postoperatively, most probably due to a cardiovascular thromboembolic event. However, both serious adverse events were attributed to the surgical cytoreductive surgery and HIPEC procedure. No serious adverse events related to bevacizumab-IRDye800CW occurred in any of the patients. Intraoperatively, fluorescence was seen in all patients. In two patients, additional tumour tissue was detected by molecular fluorescence-guided surgery that was initially missed by the surgeons. During back-table imaging of fresh surgical specimens, a total of 80 areas were imaged, marked, and analysed. All of the 29 non-fluorescent areas were found to contain only benign tissue, whereas tumour tissue was detected in 27 of 51 fluorescent areas (53%). Ex-vivo semi-quantification of 79 FFPE peritoneal lesions showed a tumour-to-normal ratio of 6·92 (SD 2·47). INTERPRETATION: Molecular fluorescence-guided surgery using the near-infrared fluorescent tracer bevacizumab-IRDye800CW is safe and feasible. This technique might be of added value for the treatment of patients with colorectal peritoneal metastases through improved patient selection and optimisation of cytoreductive surgery. A subsequent multicentre phase 2 trial is needed to make a definitive assessment of the diagnostic accuracy and the effect on clinical decision making of molecular fluorescence-guided surgery.

High fidelity optoacoustic (photoacoustic) tomography requires dense spatial sampling of optoacoustic signals using point acoustic detectors. However, in practice, spatial resolution of the images is often limited by limited sampling either due to coarse multi-element arrays or time in raster scan measurements. Herein, we investigate a method that integrates information from multiple optoacoustic images acquired at sub-diffraction steps into one high resolution image by means of an iterative registration algorithm. Experimental validations performed in target phantoms and ex-vivo tissue samples confirm that the suggested approach renders significant improvements in terms of optoacoustic image resolution and quality without introducing significant alterations into the signal acquisition hardware or inversion algorithms.

We propose the implementation of hybrid optical and acoustic resolution optoacoustic endoscopy. Laser light is transmitted to tissue by two types of illumination for achieving optical and acoustic resolution imaging. A 20 MHz ultrasound detector is used for recording optoacoustic signals. The endoscopy probe attains a 3.6 mm diameter and is fully encapsulated into a catheter system. We validate the imaging performance of the hybrid endoscope on phantoms and ex vivo, and discuss the necessity for the extended resolution and depth range of endoscopy achieved.

In optoacoustic imaging, the resolution and image quality in a certain imaging position usually cannot be enhanced without changing the imaging configuration. Post-reconstruction image processing methods offer a new possibility to improve image quality and resolution. We have developed a geometrical super-resolution (GSR) method which uses information from spatially separated frames to enhance resolution and contrast in optoacoustic images. The proposed method acquires several low resolution images from the same object located at different positions inside the imaging plane. Thereafter, it applies an iterative registration algorithm to integrate the information in the acquired set of images to generate a single high resolution image. Herein, we present the method and evaluate its performance in simulation and phantom experiments, and results show that geometrical super-resolution techniques can be a promising alternative to enhance resolution in optoacoustic imaging.

Optoacoustic technique has been shown to resolve anatomical, functional and molecular features at depths that go beyond the reach of epi-illumination optical microscopy offering new opportunities for endoscopic imaging. Herein, we interrogate the merits of optoacoustic endoscopy implemented by translating a sound detector in linear or curved geometries. The linear and curved detection geometries are achieved by employing an intravascular ultrasound transducer (IVUS) within a plastic guide shaped to a line or a curve. This concept could be used together with optical endoscopes to yield hybrid optical and optoacoustic imaging.

Sparse recovery algorithms have shown great potential to accurately reconstruct images using limited-view optoacoustic (photoacoustic) tomography data sets, but these are computationally expensive. In this paper, we propose an improvement of the fast converging Split Augmented Lagrangian Shrinkage Algorithm method based on least square QR inversion for improving the reconstruction speed. We further show image fidelity improvement when using a coherence factor to weight the reconstruction result. Phantom and in vivo measurements show that the accelerated Split Augmented Lagrangian Shrinkage Algorithm method with coherence factor weighting offers images with reduced artifacts and provides faster convergence compared with existing sparse recovery algorithms.

Ions provide a more advantageous dose distribution than photons for external beam radiotherapy, due to their so-called inverse depth dose deposition and, in particular a characteristic dose maximum at their end-of-range (Bragg peak). The favorable physical interaction properties enable selective treatment of tumors while sparing surrounding healthy tissue, but optimal clinical use requires accurate monitoring of Bragg peak positioning inside tissue. We introduce ionoacoustic tomography based on detection of ion induced ultrasound waves as a technique to provide feedback on the ion beam profile. We demonstrate for 20 MeV protons that ion range imaging is possible with submillimeter accuracy and can be combined with clinical ultrasound and optoacoustic tomography of similar precision. Our results indicate a simple and direct possibility to correlate, in-vivo and in real-time, the conventional ultrasound echo of the tumor region with ionoacoustic tomography. Combined with optoacoustic tomography it offers a well suited pre-clinical imaging system.

The interaction of magnetic nanoparticles and electromagnetic fields can be determined through electrical signal induction in coils due to magnetization. However, the direct measurement of instant electromagnetic energy absorption by magnetic nanoparticles, as it relates to particle characterization or magnetic hyperthermia studies, has not been possible so far. We introduce the theory of magnetoacoustics, predicting the existence of second harmonic pressure waves from magnetic nanoparticles due to energy absorption from continuously modulated alternating magnetic fields. We then describe the first magnetoacoustic system reported, based on a fiber-interferometer pressure detector, necessary for avoiding electric interference. The magnetoacoustic system confirmed the existence of previously unobserved second harmonic magnetoacoustic responses from solids, magnetic nanoparticles, and nanoparticle-loaded cells, exposed to continuous wave magnetic fields at different frequencies. We discuss how magnetoacoustic signals can be employed as a nanoparticle or magnetic field sensor for biomedical and environmental applications.

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments. (a) Experimental setup for hybrid acoustic and optical resolution optoacoustic microscopy. (b) Transcranial scan of an adult mouse brain using the optical resolution mode. Scale bar is 375 µm.

cattering within biological samples limits the imaging depth and the resolution in microscopy. We present a prior and regularization approach for blind deconvolution algorithms to correct the influence of scattering to increase the imaging depth and resolution. The effect of the prior is demonstrated on a three-dimensional image stack of a zebrafish embryo captured with a selective plane illumination microscope. Blind deconvolution algorithms model the recorded image as a convolution between the distribution of fluorophores and a point spread function (PSF). Our prior uses image information from adjacent z-planes to estimate the unknown blur in tissue. The increased size of the PSF due to the cascading effect of scattering in deeper tissue is accounted for by a depth adaptive regularizer model. In a zebrafish sample, we were able to extend the point in depth, where scattering has a significant effect on the image quality by around 30  μm.

Surgical success depends on the accuracy with which disease and vital tissue can be intraoperatively detected. However, the dominant visualization approach, i.e., human vision, does not see under the tissue surface and operates on low contrast between sites of disease, such as cancer, and the surrounding tissue. Intraoperative fluorescence imaging is emerging as a highly effective method to improve surgical vision and offers the potential to be intergrated seamlessly into the normal workflow of the operating room without causing disruption or undue delay. We review and compare two critical fluorescence imaging directions: one that uses nonspecific fluorescence dyes, addressing tissue perfusion and viability, and one that uses targeted agents, interrogating pathophysiological features of disease. These two approaches present detection sensitivity challenges that may differ by orders of magnitude and require different detection strategies. Nevertheless, fluorescence imaging provides the surgeon with previously unavailable real-time feedback that improves surgical precision and can become essential for interventional decision-making.

The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Co-culture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or non-supportive stroma co-cultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knock-down in supportive stroma impaired HSC survival, while ectopic expression of the Dpt gene or protein in non-supportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood islet model and a syngeneic murine transplantation model. Indeed, Del-1 pretreatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with18F-fluorodeoxyglucose ([18F]-FDG PET).13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using13CMRSI.

Purpose: to provide proof of principle of safety, breast tumor-specific uptake and positive tumor margin assessment of the systemically administered near-infrared fluorescent (NIRF) tracer bevacizumab-IRDye800CW targeting vascular endothelial growth factor (VEGF)-A in breast cancer patients. Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab-IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and co-registration of tumor tissue and healthy tissue. Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared to those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two out of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level. Conclusions: Our study shows that systemic administration of the bevacizumab-IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor-margin uptake as evaluated by a systematic validation methodology. The findings are a step towards a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in tumor tissue.

Introduction It is estimated that more than 382 million people suffer from diabetes across the globe, most of which are between the age of 40 and 59 years. ICT can play a key role in better management of diabetes and in patient empowerment. Patient empowerment involves patients to a greater extent in their own healthcare process and disease management becomes an integrated part of their daily life. Self-management opens the possibility for patients to contribute to their own healthcare as well as to be more in control of their disease. Objectives The objective of our study was to explore the impact of an ICT-based patient empowerment framework in diabetes self-management. Methods A modular patient empowerment framework that fosters diabetes self-management was designed and implemented. The framework incorporates expert knowledge in the form of clinical guidelines, and it supports patients in the specification of personalized activities that are based on medical recommendations and personal goals, and in the collection of observations of daily living. The usability and usefulness of the proposed framework were assessed in a pilot study with the participation of 60 patients and 12 health professionals. Results The study revealed that a patient empowerment approach based on self-management ICT tools is useful and accepted by both the patients and the physicians. For those patients who were already disciplined in their disease management the piloted solution served as a facilitator for data logging. For the rest, it served as an incentive for better adherence to disease management principles. The ICT tools prompted many patients into becoming more physically active and into making dietary habits' adjustments. However, this impact proved to be tightly correlated with the sociocultural background of the subjects. The study also demonstrated that even in patient-centric self-management interventions the physicians still have a key role to play. However, the acceptance of such interventions by the healthcare professionals depends not only on the level of impact in their patients’ disease management but also on the level of impact in their workflow. Conclusions It is evident that a patient empowerment approach based on self-management ICT tools is useful and accepted by patients and physicians. Further, there are clear indications that ICT frameworks such as the one presented in this paper support patients in behavioral changes and in better disease management. Finally, it was realized that self-management solutions should be built around the objective not only to educate and guide patients in disease self-management, but also to assist them in exploring the decision space and to provide insight and explanations about the impact of their own values on the decision.

In vertebrates, neurotrophic signaling plays an important role in neuronal development, neural circuit formation, and neuronal plasticity, but its evolutionary origin remains obscure. We found and validated nucleotide sequences encoding putative neurotrophic ligands (neurotrophin, NT) and receptors (Trk and p75) in two annelids, Platynereis dumerilii (Errantia) and Capitella teleta (Sedentaria, for which some sequences were found recently by Wilson, 2009). Predicted protein sequences and structures of Platynereis neurotrophic molecules reveal a high degree of conservation with the vertebrate counterparts; some amino acids signatures present in the annelid Trk sequences are absent in the basal chordate amphioxus, reflecting secondary loss in the cephalochordate lineage. In addition, expression analysis of NT, Trk, and p75 during Platynereis development by whole-mount mRNA in situ hybridization supports a role of these molecules in nervous system and circuit development. These annelid data corroborate the hypothesis that the neurotrophic signaling and its involvement in shaping neural networks predate the protostome-deuterostome split and were present in bilaterian ancestors.

Post-transcriptional regulation of gene expression plays a crucial role in many bacterial pathways. In particular, the translation of mRNA can be regulated by trans-acting, small, non-coding RNAs (sRNAs) or mRNA-binding proteins, each of which has been successfully treated theoretically using two-component models. An important system that includes a combination of these modes of post-transcriptional regulation is the Colicin E2 system. DNA damage, by triggering the SOS response, leads to the heterogeneous expression of the Colicin E2 operon including the cea gene encoding the toxin colicin E2, and the cel gene that codes for the induction of cell lysis and release of colicin. Although previous studies have uncovered the system’s basic regulatory interactions, its dynamical behavior is still unknown. Here, we develop a simple, yet comprehensive, mathematical model of the colicin E2 regulatory network, and study its dynamics. Its post-transcriptional regulation can be reduced to three hierarchically ordered components: the mRNA including the cel gene, the mRNA-binding protein CsrA, and an effective sRNA that regulates CsrA. We demonstrate that the stationary state of this system exhibits a pronounced threshold in the abundance of free mRNA. As post-transcriptional regulation is known to be noisy, we performed a detailed stochastic analysis, and found fluctuations to be largest at production rates close to the threshold. The magnitude of fluctuations can be tuned by the rate of production of the sRNA. To study the dynamics in response to an SOS signal, we incorporated the LexA-RecA SOS response network into our model. We found that CsrA regulation filtered out short-lived activation peaks and caused a delay in lysis gene expression for prolonged SOS signals, which is also seen in experiments. Moreover, we showed that a stochastic SOS signal creates a broad lysis time distribution. Our model thus theoretically describes Colicin E2 expression dynamics in detail and reveals the importance of the specific regulatory components for the timing of toxin release.

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. The large field of view and fast acquisition speed of our SPIM system enables rendering of volumetric image stacks from intact whole porcine ovarian follicles, clearly visualizing follicular features including follicle volume and average diameter (70 μm–2.5 mm), their spherical asymmetry parameters, size of developing cumulus oophorus complexes (40 μm–110 μm), and follicular wall thickness (90 μm–120 μm). Follicles at all developmental stages were identified. A distribution of the theca thickness was measured for each follicle, and a relationship between these distributions and the stages of follicular development was discerned. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

The concept of sparsity is extensively exploited in the fields of data acquisition and image processing, contributing to better signal-to-noise and spatio-temporal performance of the various imaging methods. In the field of optoacoustic tomography, the image reconstruction problem is often characterized by computationally extensive inversion of very large datasets, for instance when acquiring volumetric multispectral data with high temporal resolution. In this article we seek to accelerate accurate model-based optoacoustic inversions by identifying various sources of sparsity in the forward and inverse models as well as in the single- and multi-frame representation of the projection data. These sources of sparsity are revealed through appropriate transformations in the signal, model and image domains and are subsequently exploited for expediting image reconstruction. The sparsity-based inversion scheme was tested with experimental data, offering reconstruction speed enhancement by a factor of 40 to 700 times as compared with the conventional iterative model-based inversions while preserving similar image quality. The demonstrated results pave the way for achieving real-time performance of model-based reconstruction in multi-dimensional optoacoustic imaging.

Nuclear imaging plays a critical role in asthma research but is limited in its readings of biology due to the short-lived signals of radio-isotopes. We employed hybrid fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) for the assessment of asthmatic inflammation based on resolving cathepsin activity and matrix metalloproteinase activity in dust mite, ragweed, and Aspergillus species-challenged mice. The reconstructed multimodal fluorescence distribution showed good correspondence with ex vivo cryosection images and histological images, confirming FMT-XCT as an interesting alternative for asthma research.

Segmentation of biomedical images is essential for studying and characterizing anatomical structures as well as for detection and evaluation of tissue pathologies. Segmentation has been further shown to enhance the reconstruction performance in many tomographic imaging modalities by accounting for heterogeneities in the excitation field and tissue properties in the imaged region. This is particularly relevant in optoacoustic tomography, where discontinuities in the optical and acoustic tissue properties, if not properly accounted for, may result in deterioration of the imaging performance. Efficient segmentation of optoacoustic images is often hampered by the relatively low intrinsic contrast of large anatomical structures, which is further impaired by the limited angular coverage of some commonly employed tomographic imaging configurations. Herein, we analyze the performance of active contour models for boundary segmentation in cross-sectional optoacoustic tomography. The segmented mask is employed to construct a two compartment model for the acoustic and optical parameters of the imaged tissues, which is subsequently used to improve accuracy of the image reconstruction routines. The performance of the suggested segmentation and modeling approach are showcased in tissuemimicking phantoms and small animal imaging experiments.

We review the history of photoacoustics from the discovery in 1880 that modulated light produces acoustic waves to the current time, when the pulsed variant of the discovery is fast developing into a powerful biomedical imaging modality. We trace the meandering and fascinating passage of the effect along several conceptual and methodological trajectories to several variants of the method, each with its set of proposed applications. The differences in mechanisms between the intensity modulated effect and the pulsed version are described in detail. We also learn the several names given to the effect, and trace the modern-day divide in nomenclature.

We introduce hyperpolarizable13C-labeled probes that identify multiple biologically important divalent metals via metal-specific chemical shifts. These features enable NMR measurements of calcium concentrations in human serum in the presence of magnesium. In addition, signal enhancement through dynamic nuclear polarization (DNP) increases the sensitivity of metal detection to afford measuring micromolar concentrations of calcium as well as simultaneous multi-metal detection by chemical shift imaging. The hyperpolarizable13C-MRI sensors presented here enable sensitive NMR measurements and MR imaging of multiple divalent metals in opaque biological samples.

Photoacoustic imaging (PAI) is an attractive imaging modality that can volumetrically map the distribution of photoabsorbing molecules with deeper tissue penetration than multiphoton microscopy. To enable dynamic sensing of divalent cations via PAI, we have engineered a new reversible near-infrared probe that is more sensitive to calcium as compared to other biologically relevant cations. The metallochromic compound showed a strong reduction of its peak absorbance at 765 nm upon addition of calcium ions that was translated into robust signal changes in photoacoustic images. Therefore, the heptamethine cyanine dye will be an attractive scaffold to create a series of metallochromic sensors for molecular PAI.

The imaging performance of fluorescence molecular tomography (FMT) improves when information from the underlying anatomy is incorporated into the inversion scheme, in the form of priors. The requirement for incorporation of priors has recently driven the development of hybrid FMT systems coupled to other modalities, such as X-ray CT and MRI. A critical methodological aspect in this case relates to the particular method selected to incorporate prior information obtained from the anatomical imaging modality into the FMT inversion. We propose herein a new approach for utilizing prior information, which preferentially minimizes residual errors associated with measurements that better describe the anatomical segments considered. This preferential minimization was realized using a weighted least square (WLS) approach, where the weights were optimized using a Mamdani-type fuzzy inference system. The method of priors introduced herein was deployed as a two-step structured regularization approach and was verified with experimental measurements from phantoms as well as ex vivo and in vivo animal studies. The results demonstrate accurate performance and minimization of reconstruction bias, without requiring user input for setting the regularization parameters. As such, the proposed method offers significant progress in incorporation of anatomical priors in FMT and, as a result, in realization of the full potential of hybrid FMT.

A handheld approach to optoacoustic imaging is essential for the clinical translation. The first 2- and 3-dimensional handheld multispectral optoacoustic tomography (MSOT) probes featuring real-time unmixing have recently been developed. Imaging performance of both probes was determined in vitro and in a brain melanoma metastasis mouse model in vivo. T1-weighted MR images were acquired for anatomical reference. The limit of detection of melanoma cells in vitro was significantly lower using the 2D than the 3D probe. The signal decrease was more profound in relation to depth with the 3D versus the 2D probe. Both approaches were capable of imaging the melanoma tumors qualitatively at all time points. Quantitatively, the 2D approach enabled closer anatomical resemblance of the tumor compared to the 3D probe, particularly at depths beyond 3 mm. The 3D probe was shown to be superior for rapid 3D imaging and, thus, holds promise for more superficial target structures.

Purpose To study whether multispectral optoacoustic tomography (MSOT) can serve as a label-free imaging modality for the detection of lymph node micrometastases and in-transit metastases from melanoma on the basis of the intrinsic contrast of melanin in comparison to fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT). Materials and Methods The study was approved by the institutional animal care and use committee. Sequential MSOT was performed in a mouse B16F10 melanoma limb lymph node metastasis model (n = 13) to survey the development of macro-, micro- and in-transit metastases (metastases that are in transit from the primary tumor site to the local nodal basin) in vivo. The in vitro limit of detection was assessed in a B16F10 cell phantom. Signal specificity was determined on the basis of a simultaneous lymphadenitis (n = 4) and 4T1 breast cancer lymph metastasis (n = 2) model. MSOT was compared with intravenous FDG PET/CT. The diagnosis was assessed with histologic examination. Differences in the signal ratio (metastatic node to contralateral limb) between the two modalities were determined with the two-tailed paired t test. Results The mean signal ratios acquired with MSOT in micrometastases (2.5 ± 0.3, n = 6) and in-transit metastases (8.3 ± 5.8, n = 4) were higher than those obtained with FDG PET/CT (1.1 ± 0.5 [P < .01] and 1.3 ± 0.6 [P < .05], respectively). MSOT was able to help differentiate even small melanoma lymph node metastases from the other lymphadenopathies (P < .05 for both) in vivo, whereas FDG PET/CT could not (P > .1 for both). In vitro, the limit of detection was at an approximate cell density of five cells per microliter (P < .01). Conclusion MSOT enabled detection of melanoma lymph node micrometastases and in-transit metastases undetectable with FDG PET/CT and helped differentiate melanoma metastasis from other lymphadenopathies.

Expanding usage of small animal models in biomedical research necessitates development of technologies for structural, functional, or molecular imaging that can be readily integrated in the biological laboratory. Herein, we consider dual multispectral optoacoustic (OA) and ultrasound tomography based on curved ultrasound detector arrays and describe the performance achieved for hybrid morphological and physiological brain imaging of mice in vivo. We showcase coregistered hemodynamic parameters resolved by OA tomography under baseline conditions and during alterations of blood oxygen saturation. As an internal reference, we provide imaging of abdominal organs. We illustrate the performance advantages of hybrid curved detector ultrasound and OA tomography and discuss immediate and long-term implications of our findings in the context of animal and human studies.

We used raster-scan optoacoustic mesoscopy (RSOM) at 50 MHz, and at 100 MHz, to monitor tumor growth, and tumor angiogenesis, which is a central hallmark of cancer, in-vivo. In this study we compared the performance, and the effect of the 50 MHz, and the 100 MHz frequencies on the quality of the final image. The system is based on a reflection-mode implementation of RSOM. The detectors used are custom made, ultrawideband, and spherically focused. The use of such detectors enables light coupling from the same side as the detector, thus reflection-mode. Light is in turn coupled using a fiber bundle, and the detector is raster scanned in the xy-plane. Subsequently, to retrieve small features, the raw data are reconstructed using a multi-bandwidth, beamforming reconstruction algorithm. Comparison of the system performance at the different frequencies shows as expected a higher resolution in case of the 100 MHz detector compared to the 50 MHz. On the other hand the 50 MHz has a better SNR, can detect features from deeper layers, and has higher angular acceptance. Based on these characteristics the 50 MHz detector was mostly used. After comparing the performance we monitored the growth of B16F10 cells, melanin tumor, over the course of 9 days. We see correspondence between the optoacoustic measurements and the cryoslice validations. Additionally, in areas close to the tumor we see sprouting of new vessels, starting at day 4-5, which corresponds to tumor angiogenesis.

Whole-body optical imaging of post-embryonic stage model organisms is a challenging and long sought-after goal. It requires a combination of high-resolution performance and high-penetration depth. Optoacoustic (photoacoustic) mesoscopy holds great promise, as it penetrates deeper than optical and optoacoustic microscopy while providing high-spatial resolution. However, optoacoustic mesoscopic techniques only offer partial visibility of oriented structures, such as blood vessels, due to a limited angular detection aperture or the use of ultrasound frequencies that yield insufficient resolution. We introduce 360° multi orientation (multi-projection) raster scan optoacoustic mesoscopy (MORSOM) based on detecting an ultra-wide frequency bandwidth (up to 160 MHz) and weighted deconvolution to synthetically enlarge the angular aperture. We report unprecedented isotropic in-plane resolution at the 9-17 μm range and improved signal to noise ratio in phantoms and opaque 21-day-old Zebrafish. We find that MORSOM performance defines a new operational specification for optoacoustic mesoscopy of adult organisms, with possible applications in the developmental biology of adulthood and aging.

The diversity of pore-forming subunits of KV1 channels (KV1.1-KV1.8) affords their physiological versatility and predicts a range of functional impairments resulting from genetic aberrations. Curiously, identified so far human neurological conditions associated with dysfunctions of KV1 channels have been linked exclusively to mutations in the KCNA1 gene encoding for the KV1.1 subunit. The absence of phenotypes related to irregularities in other subunits, including the prevalent KV1.2 subunit of neurons is highly perplexing given that deletions of corresponding kcna2 gene in mouse models precipitate symptoms reminiscent to those of KV1.1 knockouts. Herein, we critically evaluate the molecular and biophysical characteristics of the KV1.1 protein in comparison with others and discuss their role in the greater penetrance of KCNA1 mutations in humans leading to the neurological signs of episodic ataxia type 1 (EA1). Future research and interpretation of emerging data should afford new insight towards a better understanding of the role of KV1.1 in integrative mechanisms of neurons and synaptic functions under normal and disease conditions.

In addition to safeguarding the central nervous system (CNS) from the vast majority of pathogens and toxins, transvascular barriers impose immense challenges to the delivery of beneficial cargo. A few toxins and neurotropic viruses capable of penetrating the brain have proved to be potentially valuable for neuron targeting and enhanced transfer of restorative medicine and therapeutic genes. Here we review molecular concepts and implications of the highly neurotropic tetanus toxin (TeTx) and botulinum neurotoxins (BoNTs) and their ability to infiltrate and migrate throughout neurons. We discuss recent applications of their detoxified variants as versatile nanovehicles for retroaxonal delivery of therapeutics to motor neurons and synapses. Continued advances in research on these remarkable agents in preclinical trials might facilitate their future use for medical benefit.

Lack of haptic feedback during laser surgery makes it difficult to control the incision depth, leading to high risk of undesired tissue damage. Here, we present a new feedback sensing method that accomplishes noncontact real-time monitoring of laser ablation procedures by detecting shock waves emanating from the ablation spot with air-coupled transducers. Experiments in soft and hard tissue samples attained high reproducibility in real-time depth estimation of the laser-induced cuts. The advantages derived from the noncontact nature of the suggested monitoring approach are expected to advance the general applicability of laser-based surgeries.

Activation and invasion of the vascular endothelium by Staphylococcus aureus (S. aureus) is a major cause of sepsis and endocarditis. For endothelial cell invasion, S. aureus triggers actin polymerization via Cdc42, N-WASp and Arp2/3 complex to assemble a phagocytic cup-like structure. Here we show that after stimulating actin polymerization staphylococci recruit Cdc42GAP which deactivates Cdc42 and terminates actin polymerization in the phagocytic cups. Cdc42GAP is delivered to the invading bacteria on recycling endocytic vesicles in concert with exocyst complex. When Cdc42GAP recruitment by staphylococci was prevented by blocking recycling endocytic vesicles or exocyst complex or when Cdc42 was constitutively activated, phagocytic cup closure was impaired and endothelial cell invasion was inhibited. Thus, to complete invasion of the endothelium staphylococci reorient recycling endocytic vesicles to recruit Cdc42GAP which terminates Cdc42-induced actin polymerization in phagocytic cups. Analogical mechanisms may govern other Cdc42-dependent cell functions.

Li et al (Research Articles, 28 August 2015, aab3500) purport to present solutions to long-standing challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods' reliability to visualize specimen features at sub-100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.

Colloidal clusters of magnetic iron oxide nanocrystals (MIONs), particularly in the condensed pattern (co-CNCs), have emerged as new superstructures to improve further the performance of MIONs in applications pertaining to magnetic manipulation (drug delivery) and magnetic resonance imaging (MRI). Exploitation of the advantages they represent and their establishment in the area of nanomedicine demands a particular set of assets. The present work describes the development and evaluation of MION-based co-CNCs featuring for the first time such assets: High magnetization, as well as magnetic content and moment, high relaxivities (r2 = 400 and [Formula: see text] ) and intrinsic loss power (2.3 nH m(2) kgFe(-1)) are combined with unprecedented colloidal stability and structural integrity, stealth and drug-loading properties. The reported nanoconstructs are endowed with additional important features such as cost-effective synthesis and storage, prolonged self-life and biocompatibility. It is finally showcased with in vivo multispectral optoacoustic tomography how these properties culminate in a system suitable for targeting breast cancer and for forceful in vivo manipulation with low magnetic field gradients.

Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three-dimensional detection of pathophysiological parameters within skin. It was recently shown that single-wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three-dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15-125 MHz. We employ a per-pulse tunable laser to inherently co-register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label-free visualization of physiological biomarkers in skin in vivo. Cross-sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50-90%. All scale bars: 250 µm.

The absorption of visible light by human skin is governed by a number of natural chromophores: Eumelanin, pheomelanin, oxyhemoglobin, and deoxyhemoglobin are the major absorbers in the visible range in cutaneous tissue. Label-free quantification of these tissue chromophores is an important step of optoacoustic (photoacoustic) imaging towards clinical application, since it provides relevant information in diseases. In tumor cells, for instance, there are metabolic changes (Warburg effect) compared to healthy cells, leading to changes in oxygenation in the environment of tumors. In malignant melanoma changes in the absorption spectrum have been observed compared to the spectrum of nonmalignant nevi. So far, optoacoustic imaging has been applied to human skin mostly in single-wavelength mode, providing anatomical information but no functional information. In this work, we excited the tissue by a tunable laser source in the spectral range from 413-680 nm with a repetition rate of 50 Hz. The laser was operated in wavelengthsweep mode emitting consecutive pulses at various wavelengths that allowed for automatic co-registration of the multispectral datasets. The multispectral raster-scan optoacoustic mesoscopy (MSOM) system provides a lateral resolution of <60 μm independent of wavelength. Based on the known absorption spectra of melanin, oxyhemoglobin, and deoxyhemoglobin, three-dimensional absorption maps of all three absorbers were calculated from the multispectral dataset. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.

We present a multi-scale imaging system that integrates five optoacoustic and multi-photon modalities into the same device. The hybrid microscope offers a unique zoom-in ability by allowing for optoacoustic microscopy and mesoscopy scans of the sample within the same imaging framework. Furthermore, by combining several label-free modalities, we are able to visualize a broad range of anatomical features, taking advantage of their complementary contrast mechanisms. We characterize the spatial resolution and relative orientation of the different sub-modalities and demonstrate the system's performance by the imaging of several model organisms ex vivo. The presented ability to dynamically vary scanning volume and resolution together with its multi-contrast and label-free imaging capabilities make the hybrid microscope a promising tool for comprehensive biological imaging.

Purpose To investigate whether multispectral optoacoustic tomography (MSOT) developed for deep-tissue imaging in humans could enable the clinical assessment of major blood vessels and microvasculature. Materials and Methods The study was approved by the Institutional Review Board of the University Medical Center Groningen (CCMO-NL-43587) and registered in the Dutch National Trial Registry (NTR4125). The authors designed a real-time handheld optoacoustic scanner for human use, based on a concave 8-MHz transducer array, attaining 135° angular coverage. They applied a single-pulse-frame (SPF) sequence, which enabled motion insensitive optoacoustic imaging during handheld operation. SPF optoacoustic imaging was applied to imaging arteries and microvascular landmarks in the lower extremities of 10 healthy volunteers. The diameters selected microvessels were determined by measuring the full width at half maximum through the vessels in the MSOT images. Duplex ultrasonography was performed on the same landmarks in seven of the 10 volunteers for subjective comparison to the corresponding optoacoustic images. Results Optoacoustic imaging resolved blood vessels as small as 100 µm in diameter and within 1 cm depth. Additionally, MSOT provided images reflecting hemoglobin oxygen saturation in blood vessels, clearly identifying arteries and veins, and was able to identify pulsation in arteries during imaging. Larger blood vessels, specifically the tibialis posterior and the dorsalis pedis arteries, were also visualized with MSOT. Conclusion Handheld MSOT was found to be capable of clinical vascular imaging, providing visualization of major blood vessels and microvasculature and providing images of hemoglobin oxygen saturation and pulsation.

Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular-guided endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR) targeted fluorescent tracers during ex vivo colonoscopy with a NIR endoscopy platform. METHODS: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples: 48 sessile serrated adenomas/polyps (SSA/P), 70 sporadic high-grade dysplastic (HGD) adenomas, 19 hyperplastic polyps (HP) and tissue derived from patients with Lynch syndrome (LS): 78 low-grade dysplastic (LGD) adenomas, 57 HGD adenomas and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116luc tumors received intravenously bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW or sodium chloride. Three days later, 8 resected HCT116luc tumors (2-5 mm) were stitched into one freshly resected human colon specimen and followed by an ex vivo molecular-guided colonoscopy procedure. RESULTS: Immunohistochemistry showed high VEGF-A expression in 79-96% and high EGFR expression in 51-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions compared to the adjacent normal colon crypts. During ex vivo molecular-guided endoscopy all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing respectively stromal and cell membrane fluorescence. CONCLUSION: VEGF-A is a promising target for molecular-guided fluorescence endoscopy as it showed a high protein expression, especially in SSA/P and LS. We demonstrate the feasibility to visualize small tumors real-time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field 'red-flag' technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

Statistical sub-pixel detection via the adaptive matched filter (AMF) has been shown to improve the molecular imaging sensitivity and specificity of optoacoustic (photoacoustic) imaging. Applied to multispectral optoacoustic tomography (MSOT), AMF assumes that the spatially-varying tissue spectra follow a multivariate Gaussian distribution, that the spectrum of the target molecule is precisely known and that the molecular target lies in "low probability" within the data. However, when these assumptions are violated, AMF may result in considerable performance degradation. The objective of this work is to develop a robust statistical detection framework that is appropriately suited to the characteristics of MSOT molecular imaging. Using experimental imaging data, we perform a statistical characterization of MSOT tissue images and conclude to a detector that is based on the t-distribution. More importantly, we introduce a method for estimating the covariance matrix of the background-tissue statistical distribution, which enables robust detection performance independently of the molecular target size or intensity. The performance of the statistical detection framework is assessed through simulations and experimental in vivo measurements and compared to previously used methods.

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.

BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVES: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets.

Visual observation of Cumulus Oocyte Complexes provides only limited information about its functional competence, whereas the molecular evaluations methods are cumbersome or costly. Image analysis of mammalian oocytes can provide attractive alternative to address this challenge. However, it is complex, given the huge number of oocytes under inspection, subjective nature of the features inspected for identification. Supervised machine learning methods like random forest with annotations from expert biologists can make the analysis task standardized and reduces inter-subject variability. We present a semiautomatic framework for predicting the class an oocyte belongs to, based on multi-object parametric segmentation on the acquired microscopic image followed by a feature based classification using random forests.

In photoacoustic imaging, the angular reception performance of ultrasonic transducers is a critical parameter to be considered for system designers. The quantitative comparison between cMUT and PZT emphasizes the difference between the transducer requirements and specifications between conventional ultrasound and photoacoustic imaging. In this present work, we show significant benefits of cMUT based array transducers over conventional PZT arrays for the improvement of quality in photoacoustic imaging systems. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Optical and optoacoustic (photoacoustic) microscopy have been recently joined in hybrid implementations that resolve extended tissue contrast compared to each modality alone. Nevertheless, the application of the hybrid technique is limited by the requirement to combine an optical objective with ultrasound detection collecting signal from the same micro-volume. We present an all-optical optoacoustic microscope based on a pi-phase-shifted fiber Bragg grating (π-FBG) with coherence-restored pulsed interferometry (CRPI) used as the interrogation method. The sensor offers an ultra-small footprint and achieved higher sensitivity over piezoelectric transducers of similar size. We characterize the spectral bandwidth of the ultrasound detector and interrogate the imaging performance on phantoms and tissues. We show the first optoacoustic images of biological specimen recorded with π-FBG sensors. We discuss the potential uses of π-FBG sensors based on CRPI.

Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

PURPOSE: Range verification in ion beam therapy relies to date on nuclear imaging techniques which require complex and costly detector systems. A different approach is the detection of thermoacoustic signals that are generated due to localized energy loss of ion beams in tissue (ionoacoustics). Aim of this work was to study experimentally the achievable position resolution of ionoacoustics under idealized conditions using high frequency ultrasonic transducers and a specifically selected probing beam. METHODS: A water phantom was irradiated by a pulsed 20 MeV proton beam with varying pulse intensity and length. The acoustic signal of single proton pulses was measured by different PZT-based ultrasound detectors (3.5 and 10 MHz central frequencies). The proton dose distribution in water was calculated by Geant4 and used as input for simulation of the generated acoustic wave by the matlab toolbox k-WAVE. RESULTS: In measurements from this study, a clear signal of the Bragg peak was observed for an energy deposition as low as 10(12) eV. The signal amplitude showed a linear increase with particle number per pulse and thus, dose. Bragg peak position measurements were reproducible within ±30 μm and agreed with Geant4 simulations to better than 100 μm. The ionoacoustic signal pattern allowed for a detailed analysis of the Bragg peak and could be well reproduced by k-WAVE simulations. CONCLUSIONS: The authors have studied the ionoacoustic signal of the Bragg peak in experiments using a 20 MeV proton beam with its correspondingly localized energy deposition, demonstrating submillimeter position resolution and providing a deep insight in the correlation between the acoustic signal and Bragg peak shape. These results, together with earlier experiments and new simulations (including the results in this study) at higher energies, suggest ionoacoustics as a technique for range verification in particle therapy at locations, where the tumor can be localized by ultrasound imaging. This acoustic range verification approach could offer the possibility of combining anatomical ultrasound and Bragg peak imaging, but further studies are required for translation of these findings to clinical application.

There is a need for contrast agents for non-invasive diagnostic imaging of tumors. Herein, Multispectral Optoacoustic Tomography (MSOT) was employed to evaluate phthalocyanines commonly used in photodynamic therapy as photoacoustic contrast agents. We studied the photoacoustic activity of three water-soluble phthalocyanine photosensitizers: phthalocyanine tetrasulfonic acid (PcS4), Zn(II) phthalocyanine tetrasulfonic acid (ZnPcS4) and Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) in phantom and in tumor-bearing mice to investigate the biodistribution and fate of the phthalocyanines in the biological tissues. PcS4 was observed to grant good contrast between the different reticuloendothelial organs and accumulate in the tumor within an hour of post-administration. ZnPcS4 and AlPcS4 offered little contrast in photoacoustic signals between the organs. PcS4 is a promising photoacoustic contrast agent and can be exploited as a photodiagnostic agent.

Conjugated polymers (CPs) are upcoming optical contrast agents in view of their unique optical properties and versatile synthetic chemistry. Biofunctionalization of these polymer-based nanoparticles enables molecular imaging of biological processes. In this work, we propose the concept of using a biofunctionalized CP for noninvasive photoacoustic (PA) molecular imaging of breast cancer. In particular, after verifying the PA activity of a CP nano­particle (CP dots) in phantoms and the targeting efficacy of a folate-functionalized version of the same (folate-CP dots) in vitro, we systemically administered the probe into a folate receptor-positive (FR+ve) MCF-7 breast cancer xenograft model to demonstrate the possible application of folate-CP dots for imaging FR+ve breast cancers in comparison to CP dots with no folate moieties. We observed a strong PA signal at the tumor site of folate-CP dots-administered mice as early as 1 hour after administration as a result of the active targeting of the folate-CP dots to the FR+ve tumor cells but a weak PA signal at the tumor site of CP-dots-administered mice as a result of the passive accumulation of the probe by enhanced permeability and retention effect. We also observed that folate-CP dots produced ∼4-fold enhancement in the PA signal in the tumor, when compared to CP dots. These observations demonstrate the great potential of this active-targeting CP to be used as a contrast agent for molecular PA diagnostic imaging in various biomedical applications.

Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0±0.6cm(-1) at 716nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.

Lack of sensory feedback during laser surgery prevents surgeons from discerning the exact location of the incision, which increases duration and complexity of the treatment. In this study we demonstrate a new method for monitoring of laser ablation procedures. Real-time tracking of the exact three dimensional (3D) lesion profile is accomplished by detection of shock waves emanating from the ablation spot and subsequent reconstruction of the incision location using time-of-flight data obtained from multiple acoustic detectors. Here, incisions of up to 9 mm in depth, created by pulsed laser ablation of fresh bovine tissue samples, were successfully monitored in real time. It was further observed that, by utilizing as little as 12 detection elements, the incision profile can be characterized with accuracy below 0.5 mm in all three dimensions and in good agreement with histological examinations. The proposed method holds therefore promise for delivering high precision real-time feedback during laser surgeries.

We investigated in vitro and in vivo the optoacoustic responses of a silicon naphthalocyanine (SiNc), considered herein as a reporter molecule for optoacoustic imaging, elucidating its efficiency for optoacoustic (photoacoustic) signal generation and examined the in vivo performance achieved. METHODS: SiNc solutions were prepared using Cremophor E.L. in water and evaluated for light absorbing and photoacoustic contrast generating properties. Photostability and singlet oxygen generation were investigated under pulsed laser illumination and validated using photoabsorbance. HT-29 mice tumor models were used to assess the biodistribution of the compound and its performance as an optoacoustic contrast agent in vivo. RESULTS: SiNc was found to generate superior optoacoustic signals compared to the commonly used Indocyanine Green (ICG). Multispectral optoacoustic tomography (MSOT) of mouse tumors efficiently resolved the biodistribution of SiNc and the underlying perfusion parameters in vivo. In addition, we demonstrate how light-triggered SiNc reactions with molecular oxygen can be potentially sensed and discuss the relation of these measurements to the biochemical process involved in photothermal treatment. CONCLUSION: SiNc appears to be a promising family of contrast agent for optoacoustic imaging. Further development possibilities promise to expand its use in purely contrast generation settings, as well as its photodynamic therapy application.

Optical mesoscopy extends the capabilities of biological visualization beyond the limited penetration depth achieved by microscopy. However, imaging of opaque organisms or tissues larger than a few hundred micrometers requires invasive tissue sectioning or chemical treatment of the specimen for clearing photon scattering, an invasive process that is regardless limited with depth. We developed previously unreported broadband optoacoustic mesoscopy as a tomographic modality to enable imaging of optical contrast through several millimeters of tissue, without the need for chemical treatment of tissues. We show that the unique combination of three-dimensional projections over a broad 500 kHz-40 MHz frequency range combined with multi-wavelength illumination is necessary to render broadband multispectral optoacoustic mesoscopy (2B-MSOM) superior to previous optical or optoacoustic mesoscopy implementations.

In the field of medical diagnostics, biomedical photoacoustics (PA) is a non-invasive hybrid optical-ultrasonic imaging modality. Due to the unique hybrid capability of optical and acoustic imaging, PA imaging has risen to the frontiers of medical diagnostic procedures such as human breast cancer detection. While conventional PA imaging has been mainly carried out by a high-power pulsed laser, an alternative technology, the frequency domain biophotoacoustic radar (FD-PAR) is under intensive development. It utilizes a continuous wave optical source with the laser intensity modulated by a frequency-swept waveform for acoustic wave generation. The small amplitude of the generated acoustic wave is significantly compensated by increased signal-to-noise ratio (several orders of magnitude) using matched-filter and pulse compression correlation processing in a manner similar to radar systems. The current study introduces the theory of a novel FD-PAR modality for ultra-sensitive characterization of functional information for breast cancer imaging. The newly developed theory of wavelength-modulated differential PA spectroscopy (WM-DPAS) detection has been introduced to address angiogenesis and hypoxia monitoring, two well-known benchmarks of breast tumor formation. Based on the WM-DPAS theory, this modality efficiently suppresses background absorptions and is expected to detect very small changes in total hemoglobin concentration and oxygenation levels, thereby identifying pre-malignant tumors before they are anatomically apparent. An experimental system design for the WM-DPAS is presented and preliminary single-ended laser experimental results were obtained and compared to a limiting case of the developed theoretical formalism.

© 2015 Society of Photo-Optical Instrumentation Engineers (SPIE). Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

BACKGROUND: In the past decade, there has been a major drive towards clinical translation of optical and, in particular, fluorescence imaging in surgery. In surgical oncology, radical surgery is characterized by the absence of positive resection margins, a critical factor in improving prognosis. Fluorescence imaging provides the surgeon with reliable and real-time intraoperative feedback to identify surgical targets, including positive tumour margins. It also may enable decisions on the possibility of intraoperative adjuvant treatment, such as brachytherapy, chemotherapy or emerging targeted photodynamic therapy (photoimmunotherapy). METHODS: This article reviews the use of optical imaging for intraoperative guidance and decision-making. RESULTS: Image-guided cancer surgery has the potential to be a powerful tool in guiding future surgical care. Photoimmunotherapy is a theranostic concept (simultaneous diagnosis and treatment) on the verge of clinical translation, and is highlighted as an effective combination of image-guided surgery and intraoperative treatment of residual disease. Multispectral optoacoustic tomography, a technique complementary to optical image-guided surgery, is currently being tested in humans and is anticipated to have great potential for perioperative and postoperative application in surgery. CONCLUSION: Significant advances have been achieved in real-time optical imaging strategies for intraoperative tumour detection and margin assessment. Optical imaging holds promise in achieving the highest percentage of negative surgical margins and in early detection of micrometastastic disease over the next decade.

Focusing light through turbid media represents a highly fascinating challenge in modern biophotonics. The unique capability of opto-acoustics for high-resolution imaging of light absorption contrast in deep tissues can provide a natural and efficient feedback to control light delivery in a scattering medium. While the basic feasibility of using opto-acoustic readings as a feedback mechanism for wavefront shaping has been recently reported, the suggested approaches may require long acquisition times, making them challenging to be translated into realistic tissue environments. In an attempt to significantly accelerate dynamic wavefront shaping capabilities, we present here a feedback-based approach using real-time three-dimensional opto-acoustic imaging assisted with genetic-algorithm-based optimization. The new technique offers robust performance in the presence of noisy measurements and can simultaneously control the scattered wave field in an entire volumetric region.

Functional imaging of mouse models of cardiac health and disease provides a major contribution to our fundamental understanding of the mammalian heart. However, imaging murine hearts presents significant challenges due to their small size and rapid heart rate. Here we demonstrate the feasibility of high-frame-rate, noninvasive optoacoustic imaging of the murine heart. The temporal resolution of 50 three-dimensional frames per second provides functional information at important phases of the cardiac cycle without the use of gating or other motion-reduction methods. Differentiation of the blood oxygenation state in the heart chambers was enabled by exploiting the wavelength dependence of optoacoustic signals. Real-time volumetric tracking of blood perfusion in the cardiac chambers was also evaluated using indocyanine green. Taken together, the newly-discovered capacities offer a unique tool set for in-vivo structural and functional imaging of the whole heart with high spatio-temporal resolution in all three dimensions.

Non-contact optoacoustic imaging employing raster-scanning of a spherically focused air-coupled ultrasound transducer is showcased herein. Optoacoustic excitation with laser fluence within the maximal permissible human exposure limits in the visible and near-infrared spectra is applied to objects with characteristic dimensions smaller than 1 mm and absorption properties representative of the whole blood at near-infrared wavelengths, and these signals are shown to be detectable without contact to the sample using an air-coupled transducer with reasonable signal averaging. Optoacoustic images of vessel-mimicking tubes embedded in an agar phantom captured with this non-contact sensing technique are also showcased. These initial results indicate that an air-coupled ultrasound detection approach can be suitable for non-contact biomedical imaging with optoacoustics.

Discerning the accurate distribution of chromophores and biomarkers by means of optoacoustic imaging is commonly challenged by the highly heterogeneous excitation light patterns resulting from strong spatial variations of tissue scattering and absorption. Here we used the light-fluence dependent switching kinetics of reversibly switchable fluorescent proteins (RSFPs), in combination with real-time acquisition of volumetric multi-spectral optoacoustic data to correct for the light fluence distribution deep in scattering media. The new approach allows for dynamic fluence correction in time-resolved imaging, e.g., of moving organs, and can be extended to work with a large palette of available synthetic and genetically encoded photochromic substances for multiplexed wavelength-specific fluence normalization.

The recently demonstrated control over light distribution through turbid media based on real-time three-dimensional optoacoustic feedback has offered promising prospects to interferometrically focus light within scattering objects. Nevertheless, the focusing capacity of the feedback-based approach is strongly conditioned by the number of optical modes (speckle grains) enclosed in the volume that can be resolved with the optoacoustic imaging system. In this Letter, we experimentally tested the light intensity enhancement achieved with optoacoustic feedback measurements from different sizes of absorbing microparticles. The importance of the obtained results is discussed in the context of potential signal enhancement at deep locations within a scattering medium where the effective speckle grain sizes approach the minimum values dictated by optical diffraction.

Wavefront shaping techniques have recently evolved as a promising tool to control the light distribution in optically-scattering media. These techniques are based on spatially-modulating the phase of an incident light beam to create positive interference (focusing) at specific locations in the speckle pattern of the scattered wavefield. The optimum phase distribution (mask) of the spatial light modulator that allows focusing at the target location(s) is determined iteratively by monitoring the light intensity at such target. In this regard, optoacoustic (photoacoustic) imaging may provide the convenient advantage of simultaneous feedback information on light distribution in an entire region of interest. Herein, we showcase that volumetric optoacoustic images can effectively be used as a feedback mechanism in an iterative optimization algorithm allowing controlling the light distribution after propagation through a scattering sample. Experiments performed with absorbing microparticles distributed in a three-dimensional region showcase the feasibility of enhancing the light intensity at specific points. The advantages provided by optoacoustic imaging in terms of spatial and temporal resolution anticipate new capabilities of wavefront shaping techniques in biomedical optics.

Multispectral optoacoustic tomography offers unprecedented capabilities in biological research and newly-developed systems prompt the clinical translation of this modality. By exciting tissues at multiple optical wavelengths, the distribution of spectrally-distinctive absorbers can be resolved with high resolution in deep tissues, thus enabling reading important biological parameters such as blood oxygenation or the biodistribution of photo-absorbing agents. Multispectral three-dimensional optoacoustic imaging generally comes at the expense of slow acquisition times, which limits the dynamic imaging capabilities of this modality. Recently, the feasibility of multispectral three-dimensional imaging in real time (five dimensional imaging) has been showcased. Two different illumination strategies can be used for this purpose. The first approach is based on tuning the wavelength of the laser on a per-pulse basis, which enables acquisition of large multispectral datasets on a very short time. The second approach is based on properly synchronizing the light beams from two (or more) laser sources. The performances of these two approaches are compared and discussed herein based on experiments with mice and human volunteers.CCC.

Three-dimensional hand-held optoacoustic imaging comes with important advantages that prompt the clinical translation of this modality, with applications envisioned in cardiovascular and peripheral vascular disease, disorders of the lymphatic system, breast cancer, arthritis or inflammation. Of particular importance is the multispectral acquisition of data by exciting the tissue at several wavelengths, which enables functional imaging applications. However, multispectral imaging of entire three-dimensional regions is significantly challenged by motion artefacts in concurrent acquisitions at different wavelengths. A method based on acquisition of volumetric datasets having a microsecond-level delay between pulses at different wavelengths is described in this work. This method can avoid image artefacts imposed by a scanning velocity greater than 2 m/s, thus, does not only facilitate imaging influenced by respiratory, cardiac or other intrinsic fast movements in living tissues, but can achieve artifact-free imaging in the presence of more significant motion, e.g. abrupt displacements during handheld-mode operation in a clinical environment.

We report on an optoacoustic imaging system capable of acquiring volumetric multispectral optoacoustic data in real time. The system is based on simultaneous acquisition of optoacoustic signals from 256 different tomographic projections by means of a spherical matrix array. Thereby, volumetric reconstructions can be done at high frame rate, only limited by the pulse repetition rate of the laser. The developed tomographic approach presents important advantages over previously reported systems that use scanning for attaining volumetric optoacoustic data. First, dynamic processes, such as the biodistribution of optical biomarkers, can be monitored in the entire volume of interest. Second, out-of-plane and motion artifacts that could degrade the image quality when imaging living specimens can be avoided. Finally, real-time 3D performance can obviously save time required for experimental and clinical observations. The feasibility of optoacoustic imaging in five dimensions, i.e. real time acquisition of volumetric datasets at multiple wavelengths, is reported. In this way, volumetric images of spectrally resolved chromophores are rendered in real time, thus offering an unparallel imaging performance among the current bio-imaging modalities. This performance is subsequently showcased by video-rate visualization of in vivo hemodynamic changes in mouse brain and handheld visualization of blood oxygenation in deep human vessels. The newly discovered capacities open new prospects for translating the optoacoustic technology into highly performing imaging modality for biomedical research and clinical practice with multiple applications envisioned, from cardiovascular and cancer diagnostics to neuroimaging and ophthalmology.

The inversion accuracy in optoacoustic tomography depends on a number of parameters, including the number of detectors employed, discrete sampling issues or imperfectness of the forward model. These parameters result in ambiguities on the reconstructed image. A common ambiguity is the appearance of negative values, which have no physical meaning since optical absorption can only be higher or equal than zero. We investigate herein algorithms that impose non-negative constraints in model-based optoacoustic inversion. Several state-of-the-art non-negative constrained algorithms are analyzed. Furthermore, an algorithm based on the conjugate gradient method is introduced in this work. We are particularly interested in investigating whether positive restrictions lead to accurate solutions or drive the appearance of errors and artifacts. It is shown that the computational performance of non-negative constrained inversion is higher for the introduced algorithm than for the other algorithms, while yielding equivalent results. The experimental performance of this inversion procedure is then tested in phantoms and small animals, showing an improvement in image quality and quantitativeness with respect to the unconstrained approach. The study performed validates the use of non-negative constraints for improving image accuracy compared to unconstrained methods, while maintaining computational efficiency.

In optoacoustic tomography, images representing the light absorption distribution are reconstructed from the measured acoustic pressure waves at several locations around the imaged sample. Most reconstruction algorithms typically yield negative absorption values due to modelling inaccuracies and imperfect measurement conditions. Those negative optical absorption values have no physical meaning and their presence hinders image quantification and interpretation of biological information. We investigate herein the performance of optimization methods that impose non-negative constraints in model-based optoacoustic inversion. Specifically, we analyze the effects of the non-negative restrictions on image quality and accuracy as compared to the unconstrained approach. An efficient algorithm based on the projected quasi-Newton scheme and the limitedmemory Broyden-Fletcher-Goldfarb-Shannon method is used for the non-negative constrained inversion. We showcase that imposing non-negative constraints in model-based reconstruction leads to a quality increase in cross-sectional tomographic optoacoustic images.CCC.

Unlike near-infrared spectroscopy, multispectral opto-acoustic tomography (MSOT) has the potential to offer high-resolution imaging assessment of hemodynamics and blood saturation levels in muscle. However motion artifacts impede the real-time applications of the technique. We developed fast-MSOT with motion tracking that reduces motion artifacts. We used this algorithm to follow blood oxygenation level changes associated with muscle exercise in the muscle and the skin of healthy volunteers.

Nanosized contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. However, many solid nanoparticles exhibit a great potential to induce toxicity, hindering their use for clinical applications. On the other hand, near-infrared (NIR) dyes have also been used for extensive biological applications, but show some limitations due to their poor aqueous stability, tendency to aggregation and rapid elimination from the body. An alternative proposed in this work to overcome these limitations is the use of NIR dye-loaded nanoparticles. Here we introduce nanoparticles constructed with poly(D,L-lactide-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer widely used for biomedical applications, attached to the polycation polyethyleneimine (PEI) to obtain positively charged nanoparticles. The in vivo biodistribution of the cationic PEI-PLGA nanoparticles was investigated after administration through three different routes (intravenous, intraperitoneal and subcutaneous) using multispectral optoacoustic tomography (MSOT). The prepared nanoparticles exhibited good colloidal stability and adequate optical properties for optoacoustic imaging. The in vivo biodistribution assays indicated a strong accumulation of the particles in the liver and spleen, and retention in these organs for at least 24 h. Therefore, these nanoparticles could find promising applications in MSOT due to a sharp and characteristic optoacoustic spectrum and high optoacoustic signal generation, and become a promising building block for theranostic strategies.

Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex-vivo and Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) in-vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex-vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions at all ages studied. The panel of immunofluorescence experiments demonstrated that pulmonary lesions, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be non-invasively detected in-vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

Pulse-echo ultrasound and optoacoustic imaging possess very different, yet highly complementary, advantages of mechanical and optical contrast in living tissues. Integration of pulse-echo ultrasound with optoacoustic imaging may therefore significantly enhance the potential range of clinical applications. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition and image reconstruction approaches. We report on a new method for hybrid three-dimensional optoacoustic and pulse-echo ultrasound imaging based on passive generation of ultrasound with a spherical optical absorber, thus avoiding the hardware complexity of active ultrasound generation. The proposed approach allows for acquisition of complete hybrid datasets with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at a rate of 10 volumetric frames per second. Real time image rendering for both modalities is enabled by using parallel GPU-based implementation of the reconstruction algorithms. Performance is first characterized in tubing phantoms followed by in vivo measurements in healthy human volunteers, confirming general clinical applicability of the method. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Endovenous laser therapy (ELT) was introduced in clinical practice for treating incompetent veins about fifteen years ago. Despite the considerable clinical evidence collected so far, no rigorous guidelines are yet available regarding the optimal energy deposition protocols while incidence of recanalization, lack of vessel occlusion and collateral damage remains variable among patients. Online monitoring and feedback-based control over the lesion progression may improve clinical outcomes. Yet the currently employed monitoring tools, such as Doppler ultrasound, often do not provide sufficient contrast as well as three-dimensional imaging capacity for accurate lesion assessment during thermal treatments. Here we investigate on the utility of volumetric optoacoustic tomography for real-time monitoring of the ELT procedures. Experiments performed in subcutaneous veins of an ox foot model revealed the accurate spatio-temporal maps of the lesion progression and characteristics of the vessel wall. Optoacoustic images further correlated with the temperature elevation measured in the area adjacent to the coagulation spot and made it possible to track the position of the fiber tip during its pull back in real time and in all three dimensions. Overall, we showcase that volumetric optoacoustic tomography is a promising tool for providing online feedback during endovenous laser therapy.

The fast heart rate (∼7 Hz) of the mouse makes cardiac imaging and functional analysis difficult when studying mouse models of cardiovascular disease, and cannot be done truly in real-time and 3D using established imaging modalities. Optoacoustic imaging, on the other hand, provides ultra-fast imaging at up to 50 volumetric frames per second, allowing for acquisition of several frames per mouse cardiac cycle. In this study, we combined a recently-developed 3D optoacoustic imaging array with novel analytical techniques to assess cardiac function and perfusion dynamics of the mouse heart at high, 4D spatiotemporal resolution. In brief, the heart of an anesthetized mouse was imaged over a series of multiple volumetric frames. In another experiment, an intravenous bolus of indocyanine green (ICG) was injected and its distribution was subsequently imaged in the heart. Unique temporal features of the cardiac cycle and ICG distribution profiles were used to segment the heart from background and to assess cardiac function. The 3D nature of the experimental data allowed for determination of cardiac volumes at ∼7-8 frames per mouse cardiac cycle, providing important cardiac function parameters (e.g. stroke volume, ejection fraction) on a beat-by-beat basis, which has been previously unachieved by any other cardiac imaging modality. Furthermore, ICG distribution dynamics allowed for the determination of pulmonary transit time and thus additional quantitative measures of cardiovascular function. This work demonstrates the potential for optoacoustic cardiac imaging and is expected to have a major contribution toward future preclinical studies of animal models of cardiovascular health and disease.

Wide-field targeted fluorescence evolves as a promising approach for interventional guidance. We present an overview of the key developments from our laboratory and discuss their potential to shift the surgical and endoscopic imaging paradigm.

Oxidative-based diseases including diabetes, chronic renal failure, cardiovascular diseases and neurological disorders are accompanied by accumulation of advanced glycation endproducts (AGE). Therefore, AGE-associated changes in tissue optical properties could yield a viable pathological indicator for disease diagnostics and monitoring. We investigated whether skin glycation could be detected based on absorption changes associated with AGE accumulation using spectral optoacoustic measurements and interrogated the optimal spectral band for skin glycation determination. Glycated and non-glycated skin was optoacoustically measured at multiple wavelengths in the visible region. The detected signals were spectrally processed and compared to measurements of skin autofluorescence and to second harmonic generation multiphoton microscopy images. Optoacoustic measurements are shown to be capable of detecting skin glycolysis based on AGE detection. A linear dependence was observed between optoacoustic intensity and the progression of skin glycation. The findings where corroborated by autofluorescence observations. Detection sensitivity is enhanced by observing normalised tissue spectra. This result points to a ratiometric method for skin glycation detection, specifically at 540 nm and 620 nm. We demonstrate that optoacoustic spectroscopy could be employed to detect AGE accumulation, and possibly can be employed as a non-invasive quick method for monitoring tissue glycation.

A student chapter can be considered to be a miniature enterprise; however without the latter's major financial risks. Involvement in the student chapter of a professional society like IEEE at undergraduate level plays a pivotal role in the overall professional development of the student by keeping the students informed about the various career possibilities. A student chapter shapes the hitherto naive students into industry ready professionals and to suitable candidates for some of the best grad schools worldwide. This assertion has been discussed in-depth taking the example of IEEE EMBS Student Branch chapter of VIT University. It has been described how the entire process, - starting from inception of an idea to its materialization in to an activity, has shaped the volunteers and participants into better professionals.

AIMS: This study aimed at testing whether drinking volume and episodic heavy drinking (EHD) frequency in Germany are polarizing between consumption levels over time. Polarization is defined as a reduction in alcohol use among the majority of the population, while a subpopulation with a high intake level maintains or increases its drinking or its EHD frequency. The polarization hypothesis was tested across and within socio-economic subgroups. METHOD: Analyses were based on seven cross-sectional waves of the Epidemiological Survey of Substance Abuse (ESA) conducted between 1995 and 2012 (n = 7833-9084). Overall polarization was estimated based on regression models with time by consumption level interactions; the three-way interaction with socio-economic status (SES) was consecutively introduced to test the stability of effects over socio-economic strata. Interactions were interpreted by graphical inspection. RESULTS: For both alcohol use indicators, declines over time were largest in the highest consumption level. This was found within all SES groups, but was most pronounced at low and least pronounced at medium SES. CONCLUSION: The results indicate no polarization but convergence between consumption levels. Socio-economic status groups differ in the magnitude of convergence which was lowest in medium SES. The overall decline was strongest for the highest consumption level of low SES.

Current functional neuroimaging methods are not adequate for high-resolution whole-brain visualization of neural activity in real time. Here, we show imaging of fast hemodynamic changes in deep mouse brain using fully noninvasive acquisition of five-dimensional optoacoustic data from animals subjected to oxygenation stress. Multispectral video-rate acquisition of three-dimensional tomographic data enables simultaneous label-free assessment of multiple brain hemodynamic parameters, including blood oxygenation, total hemoglobin, cerebral blood volume, oxygenized and deoxygenized hemoglobin, in real time. The unprecedented results indicate that the proposed methodology may serve as a powerful complementary, and potentially superior, method for functional neuroimaging studies in rodents.

Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging.

We present a technique for fully noninvasive acquisition of real-time volumetric multispectral optoacoustic data from whole mouse brain. The neuroimaging capacity of the new methodology is demonstrated here by simultaneous label-free assessment of multiple stimulus-evoked hemodynamic responses, including blood oxygenation, total hemoglobin, cerebral blood volume, oxygenized and deoxygenized hemoglobin.

Wide-field targeted fluorescence evolves as a promising approach for interventional guidance. We present an overview of the key developments from our laboratory and discuss their potential to shift the surgical and endoscopic imaging paradigm.

Model-based optoacoustic reconstruction can incorporate the shape of transducers. However, the accompanying memory cost will hinder it for high resolution performance. The propose method provides over an order of magnitude reduction in inversion time in experiments. Additionally, it also suits for the analysis of inversion stability.

PURPOSE: With recent advancement in hardware of optoacoustic imaging systems, highly detailed cross-sectional images may be acquired at a single laser shot, thus eliminating motion artifacts. Nonetheless, other sources of artifacts remain due to signal distortion or out-of-plane signals. The purpose of image reconstruction algorithms is to obtain the most accurate images from noisy, distorted projection data. METHODS: In this paper, the authors use the model-based approach for acoustic inversion, combined with a sparsity-based inversion procedure. Specifically, a cost function is used that includes the L1 norm of the image in sparse representation and a total variation (TV) term. The optimization problem is solved by a numerically efficient implementation of a nonlinear gradient descent algorithm. TV-L1 model-based inversion is tested in the cross section geometry for numerically generated data as well as for in vivo experimental data from an adult mouse. RESULTS: In all cases, model-based TV-L1 inversion showed a better performance over the conventional Tikhonov regularization, TV inversion, and L1 inversion. In the numerical examples, the images reconstructed with TV-L1 inversion were quantitatively more similar to the originating images. In the experimental examples, TV-L1 inversion yielded sharper images and weaker streak artifact. CONCLUSIONS: The results herein show that TV-L1 inversion is capable of improving the quality of highly detailed, multiscale optoacoustic images obtained in vivo using cross-sectional imaging systems. As a result of its high fidelity, model-based TV-L1 inversion may be considered as the new standard for image reconstruction in cross-sectional imaging.

The optoacoustic (photoacoustic) technique has been shown to resolve anatomical, functional, and molecular features at depths that go beyond the reach of epi-illumination optical microscopy, offering new opportunities for endoscopic imaging. In this Letter, we investigate the merits of optoacoustic endoscopy implemented by translating a sound detector in linear or curved geometries. The linear and curved detection geometries are achieved by employing an intravascular ultrasound transducer within a plastic guide shaped to a line or a curve. This concept could be used together with optical endoscopes to yield hybrid optical and optoacoustic imaging.

Optoacoustic imaging has been widely used for in vivo disease diagnosis and therapy monitoring. Acquisition hardware, analysis, and contrast agents have been subject to much innovation, creating access to an ever-growing range of biomedical applications. In this review, a broad overview of optoacoustic theory, instrumentation and data processing is provided, together with the various categories of contrast agents that have been developed. In addition, the application of these techniques and contrast agents in preclinical and clinical imaging applications will be discussed in detail, ranging from imaging of cancer and various organs like skin, brain and breast to sentinel lymph node mapping. Finally under conclusions, we highlighted future perspectives in this field, in the context of instrumentation and software development, as well as advances in clinical translation.

There is growing interest in genetically expressed reporters for in vivo studies of bacterial colonization in the context of infectious disease research, studies of the bacterial microbiome or cancer imaging and treatment. To empower non-invasive high-resolution bacterial tracking with deep tissue penetration, we herein use the genetically controlled biosynthesis of the deep-purple pigment Violacein as a photobleaching-resistant chromophore label for in vivo optoacoustic (photoacoustic) imaging in the near-infrared range. We demonstrate that Violacein-producing bacteria can be imaged with high contrast-to-noise in strongly vascularized xenografted murine tumors and further observe that Violacein shows anti-tumoral activity. Our experiments thus identify Violacein as a robust bacterial label for non-invasive optoacoustic imaging with high potential for basic research and future theranostic applications in bacterial tumor targeting.

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

Unveiling mechanisms driving specification, recruitment and regeneration of melanophores is key in understanding melanin-related disorders. This study reports on the applicability of a hybrid focus optoacoustic microscope (HFOAM) for volumetric tracking of migratory melanophores in developing zebrafish. The excellent contrast from highly-absorbing melanin provided by the method is shown to be ideal for label-free dynamic visualization of melanophores in their unperturbed environment. We established safe laser energy levels that enable high-contrast longitudinal tracking of the cells over an extended period of developmental time without causing cell toxicity or pigment bleaching. Owing to its hybrid optical and acoustic resolution, the new imaging technique can be seamlessly applied for noninvasive studies of both optically-transparent larval as well as adult stages of the zebrafish model organism, which is not possible using other optical microscopy methods.

One in six couples of reproductive age worldwide are affected at least once by some form of infertility. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely-available assisted reproductive technologies (ART). The identification and isolation of the most-motile sperm with DNA integrity are essential to IVF and ICSI, ultimately affecting treatment consequences and the health of offspring. Recently, microfluidic technologies been developed to sort sperm according to sperm morphology, motility, DNA integrity, and functionality for IVF techniques. There have also been emerging applications in wildlife conservation, high-throughput single-sperm genomics, sperm-driven robotics, and in-home fertility testing. We review a broad range of studies applying the principles of microfluidics to sperm research.

BACKGROUND: When medical data have been successfully recorded or exchanged between systems there appear a need to present the data consistently to ensure that it is clearly understood and interpreted. A standard based user interface can provide interoperability on the visual level. OBJECTIVES: The goal of this research was to develop, implement and evaluate an information model for building user interfaces for archetype based medical data. METHODS: The following types of knowledge were identified as important elements and were included in the information model: medical content related attributes, data type related attributes, user-related attributes, device-related attributes. In order to support flexible and efficient user interfaces an approach that represents different types of knowledge with different models separating the medical concept from a visual concept and interface realization was chosen. We evaluated the developed approach using Guideline for Good Evaluation Practice in Health Informatics (GEP-HI). RESULTS: We developed a higher level information model to complement the ISO 13606 archetype model. This enabled the specification of the presentation properties at the moment of the archetypes' definition. The model allows realizing different users' perspectives on the data. The approach was implemented and evaluated within a functioning EHR system. The evaluation involved 30 patients of different age and IT experience and 5 doctors. One month of testing showed that the time required reading electronic health records decreased for both doctors (from average 310 to 220 seconds) and patients (from average 95 to 39 seconds). Users reported a high level of satisfaction and motivation to use the presented data visualization approach especially in comparison with their previous experience. CONCLUSION: The introduced information model allows separating medical knowledge and presentation knowledge. The additional presentation layer will enrich the graphical user interface's flexibility and will allow an optimal presentation of medical data considering the different users' perspectives and different media used for data presentation.

AIMS: Trends in morbidity and mortality, fully or partially attributable to alcohol, for adults aged 18-64 were assessed for Germany. METHODS: The underestimation of population exposure was corrected by triangulating survey data with per capita consumption. Alcohol-attributable fractions by sex and two age groups were estimated for major disease categories causally linked to alcohol. Absolute numbers, population rates and proportions relative to all hospitalizations and deaths were calculated. RESULTS: Trends of 100% alcohol-attributable morbidity and mortality over thirteen and eighteen years, respectively, show an increase in rates of hospitalizations and a decrease in mortality rates. Comparisons of alcohol-attributable morbidity including diseases partially caused by alcohol revealed an increase in hospitalization rates between 2006 and 2012. The proportion of alcohol-attributable hospitalizations remained constant. Rates of alcohol-attributable mortality and the proportion among all deaths decreased. CONCLUSIONS: The increasing trend in mortality due to alcohol until the mid-1990s has reversed. The constant proportion of all hospitalizations that were attributable to alcohol indicates that factors such as improved treatment and easier health care access may have influenced the general increase in all-cause morbidity. To further reduce alcohol-related mortality, efforts in reducing consumption and increasing treatment utilization are needed.

Intravital imaging of large specimens is intrinsically challenging for postembryonic studies. Selective plane illumination microscopy (SPIM) has been introduced to volumetrically visualize organisms used in developmental biology and experimental genetics. Ideally suited for imaging transparent samples, SPIM can offer high frame rate imaging with optical microscopy resolutions and low phototoxicity. However, its performance quickly deteriorates when applied to opaque tissues. To overcome this limitation, SPIM optics were merged with optical and optoacoustic (photoacoustic) readouts. The performance of this hybrid imaging system was characterized using various phantoms and by imaging a highly scattering ex vivo juvenile zebrafish. The results revealed the system's enhanced capability over that of conventional SPIM for high-resolution imaging over extended depths of scattering content. The approach described here may enable future visualization of organisms throughout their entire development, encompassing regimes in which the tissue may become opaque.

Indocyanine green (ICG) is an FDA-approved, strongly photo-absorbent/fluorescent probe that has been incorporated into a clinically-relevant PEGylated liposome as a flexible optoacoustic contrast agent platform. This study describes the engineering of targeted PEGylated liposome-ICG using the anti-MUC-1 "humanized" monoclonal antibody (MoAb) hCTM01 as a tumour-specific theranostic system. We aimed to visualise non-invasively the tumour accumulation of these MoAb-targeted liposomes over time in tumour-bearing mice using multispectral optoacoustic tomography (MSOT). Preferential accumulation of targeted PEGylated liposome-ICG was studied after intravenous administration in comparison to non-targeted PEGylated liposome-ICG using both fast growing (4T1) and slow growing (HT-29) MUC-1 positive tumour models. Monitoring liposomal ICG in the tumour showed that both targeted and non-targeted liposome-ICG formulations preferentially accumulated into the tumour models studied. Rapid accumulation was observed for targeted liposomes at early time points mainly in the periphery of the tumour volume suggesting binding to available MUC-1 receptors. In contrast, non-targeted PEGylated liposomes showed accumulation at the centre of the tumour at later time points. In an attempt to take this a step further, we successfully encapsulated the anticancer drug, doxorubicin (DOX) into both targeted and non-targeted PEGylated liposome-ICG. The engineering of DOX-loaded targeted ICG liposome systems present a novel platform for combined tumour-specific therapy and diagnosis. This can open new possibilities in the design of advanced image-guided cancer therapeutics.

Efficient segmentation of optoacoustic images has importance in enhancing the diagnostic and quantification capacity of this modality. It may also aid in improving the tomographic reconstruction accuracy by accounting for heterogeneous optical and acoustic tissue properties. In particular, when imaging through complex biological tissues, the real acoustic properties often deviate considerably from the idealized assumptions of homogenous conditions, which may lead to significant image artifacts if not properly accounted for. Although several methods have been proposed aiming at estimating and accounting for the complex acoustic properties in the image domain, accurate delineation of structures is often hindered by low contrast of the images and other artifacts produced due to incomplete tomographic coverage and heuristic assignment of the tissue properties during the reconstruction process. In this letter, we propose instead a signal domain analysis approach that retrieves acoustic properties of the object to be reconstructed from characteristic features of the detected optoacoustic signals prior to image reconstruction. Performance of the proposed method is first tested in simulation and experiment using two-dimensional tissue-mimicking phantoms. Significant improvements in the segmentation abilities and overall reconstructed image quality are further showcased in experimental cross-sectional data acquired from a human finger.

In this article, we present a novel scheme for segmenting the image boundary (with the background) in optoacoustic small animal in vivo imaging systems. The method utilizes a multiscale edge detection algorithm to generate a binary edge map. A scale dependent morphological operation is employed to clean spurious edges. Thereafter, an ellipse is fitted to the edge map through constrained parametric transformations and iterative goodness of fit calculations. The method delimits the tissue edges through the curve fitting model, which has shown high levels of accuracy. Thus, this method enables segmentation of optoacoutic images with minimal human intervention, by eliminating need of scale selection for multiscale processing and seed point determination for contour mapping.

The speed of sound (SoS) in the imaged sample and in the coupling medium is an important parameter in optoacoustic tomography that must be specified in order to accurately restore maps of local optical absorbance. In this work, several hybrid focusing functions are described that successfully determine the most suitable SoS based on post-reconstruction images. The SoS in the coupling medium (water) can be determined from temperature readings. Thereby, this value is suggested to be used as an initial guess for faster SoS calibration in the reconstruction of tissues having a different SoS than water.

Despite the ancient discovery of the basic physical phenomenon underlying optoacoustic imaging and tomography [1], the lack of suitable laser sources, ultrasound detection technology, data acquisition, and processing capacities has long hindered the realization of efficient imaging devices. In fact, the first high-quality images from living animals were obtained about a decade ago (Figure 1), which was followed by an exponential growth of technical developments in instrumentation, algorithms, and biomedical applications surrounding this fascinating field. The ability of optoacoustics to probe optical contrast along a wide domain of penetration scales while maintaining excellent spatiotemporal resolution representative of ultrasound imaging, as shown in Figure 2, is unparalleled among the other optical imaging modalities [2], [3].

Implementation of hybrid imaging using optoacoustic tomography (OAT) and ultrasound (US) brings together the important advantages and complementary features of both methods. However, the fundamentally different physical contrast mechanisms of the two modalities may impose significant difficulties in the optimal tomographic data acquisition and image formation strategies. We investigate the applicability of the commonly applied imaging geometries for acquisition and reconstruction of hybrid optoacoustic tomography and pulse-echo ultrasound (OPUS) images. Optimization of the ultrasound image formation strategy using concave array geometry was implemented using a synthetic aperture method combined with spatial compounding. Experimental validation was performed using a custom-made multiplexer unit executing switching between the two modalities employing the same transducer array. A variety of array probes with different angular coverages were subsequently tested, including arrays for clinical hand-held imaging as well as stationary arrays for tomographic small animal imaging. The results demonstrate that acquisition of OAT data by mere addition of an illumination source to the common US linear array geometry may result in significant limited-view artifacts and overall loss of image quality. On the other hand, unsatisfactory US image quality is achieved with tomographic arrays solely optimized for OAT image acquisition without considering the optimal transmit-receive beamforming parameters. Optimal selection of the array pitch size, tomographic coverage and spatial compounding parameters has achieved here an accurate hybrid imaging performance, which was experimentally showcased in tissuemimicking phantoms, post-mortem mice, and hand-held imaging of a healthy volunteer. The efficient combination of the two modalities in a single imaging device reveals the true power of functional and molecular imaging capacities of OAT in addition to the morphological and functional imaging capabilities of US.

We present a hybrid preclinical imaging scanner that optimally supports image acquisition in both reflection-mode ultrasonography and optoacoustic (OA) tomography modes. The system comprises a quasi-full-ring tomographic geometry capable of the simultaneous dual-mode imaging through entire cross sections of mice with in-plane spatial resolution in the range of 150 and 350 mu m in the respective OA and ultrasound (US) imaging modes with an imaging speed of up to 10 two-dimensional frames per second. Three-dimensional whole-body data is subsequently rendered by rapid scanning of the imaged plane. The system further incorporates rapid laser wavelength tuning for real-time acquisition of multispectral OA data, which enables studies of longitudinal dynamics as well as fast kinetics and biodistribution of contrast agents. In vivo imaging performance is demonstrated by label-free hybrid anatomical scans through living mice, as well as real-time visualization of optical contrast agent perfusion. By setting new standards for wholebody tomographic imaging performance in both the OA and pulse-echo US modes, the developed hybrid imaging approach is expected to benefit numerous applications where the availability of high-quality structural information provided by the tomographic reflection-mode US can ease interpretation of the functional and molecular imaging results attained by the OA modality.

Light offers a range of interactions with tissue that give rise to an extensive list of methods to sense physical, chemical, or biological processes. Combined with using safe and nonionizing radiation, optical imaging is considered as a fundamental tool in the biomedical sciences [1].

The purpose of this work was the development of robust inversion and modelling algorithms as well as numerical frameworks and methods for upcoming applications of fluorescence imaging and tomography. Several algorithms were developed for processing of fluorescence molecular tomography-X-ray CT (FMT-XCT) measurements involving light propagation modeling, data compression and robust inversion, driven by data and anatomical priors. The proposed methods were further expanded to address practical problems in both preclinical and clinical domains. Several preclinical applications were made possible for the first time, such as hybrid FMT-PCCT. In the clinic, we addressed the problem of imaging rheumatoid arthritis (RA), via development of a spatiotemporal analysis algorithm as well as tomographic imaging of synovitis in human hand joint.

Successful detection of rheumatoid arthritis (RA) at the early stages of development can significantly enhance the chances of effective therapy. The early onset of RA is often marked with inflammation of the synovial lining of the joint, a condition known as synovitis. Effective imaging of synovitis is therefore of critical importance. While dynamic, contrast-enhanced magnetic resonance imaging (MRI) is capable of effective imaging of synovitis, it is a costly modality. As an alternative, inexpensive approach, optical imaging post injection of the near-infrared fluorescent dye indocynine green (ICG) has been recently proposed for imaging RA. Evaluation of the obtained optical images is performed via examination by trained human readers. However, optical imaging has yet to achieve the diagnostic accuracy of MRI. In this paper we present a method for automatic evaluation of the fluorescence images and compare its performance with the human-based evaluation. Our method relies on our previous work on spatiotemporal analysis of image sequence with principal component analysis (PCA) to seek synovitis signal components with the help of a segmentation method. The results for a group of 600 joints, obtained from 20 patients, suggest improved diagnostic performance using the automatic approach in comparison to human-based evaluation.

While acute myocardial infarction mortality declines, patients continue to face reinfarction and/or heart failure. The immune system, which intimately interacts with healthy and diseased tissues through resident and recruited leukocytes, is a central interface for a global host response to ischemia. Pathways that enhance the systemic leukocyte supply may be potential therapeutic targets. Pre-clinically, imaging helps to identify immunity's decision nodes, which may serve as such targets. In translating the rapidly-expanding pre-clinical data on immune activity, the difficulty of obtaining multiple clinical tissue samples from involved organs is an obstacle that whole-body imaging can help overcome. In patients, molecular and cellular imaging can be integrated with blood-based diagnostics to assess the translatability of discoveries, including the activation of hematopoietic tissues after myocardial infarction, and serve as an endpoint in clinical trials. In this review, we discuss these concepts while focusing on imaging immune activity in organs involved in ischemic heart disease.

We present the development of novel biomedical imaging methods based on optoacoustics. These novel tools can revolutionize biomedical discovery and clinical healthcare. We focus in particular in multispectral optoacoustic tomography (MSOT). This technology, now recipient of numerous awards and acclaim, shows two important developments: on one hand on how it can break through the limits of conventional optical imaging to shift the paradigm of biomedical observation and on the other how biomedical technology offers a necessary leverage point in scientific discovery but also economic growth, even at moderate investments.

One of the challenges of multispectral optoacoustic tomography (MSOT) is the reconstruction of the images from the projection data. Conventionally, analytical inversion formulae are used owing to their simplicity and numerical efficiency. However, such solutions are often limited to ideal detection scenarios and lead to image artifacts when the system characteristics deviate from the assumed ones. In such cases, image quality may be improved by adopting a model-based approach in which the MSOT system is modeled via a matrix relation, which is subsequently inverted using established algebraic techniques to reconstruct the image. Nonetheless, model-based inversion is usually more computationally demanding than its analytical counterparts owing to the large size of the model matrix. In this paper, we analyze the sparsity that exists in the model matrix and show how it may be exploited for accelerating image reconstruction. In particular, a wavelet-packet framework is presented under which the size of the model matrix may be reduced.

Angiogenesis is a central cancer hallmark, necessary for supporting tumor growth and metastasis. In vivo imaging of angiogenesis is commonly applied, to understand dynamic processes in cancer development and treatment strategies. However, most radiological modalities today assess angiogenesis based on indirect mechanisms, such as the rate of contrast enhancement after contrast agent administration. We studied the performance of raster-scan optoacoustic mesoscopy (RSOM), to directly reveal the vascular network supporting melanoma growth in vivo, at 50 MHz and 100 MHz, through several millimeters of tumor depth. After comparing the performance at each frequency, we recorded, for the first time, high-resolution images of melanin tumor vasculature development in vivo, over a period of several days. Image validation was provided by means of cryo-slice sections of the same tumor after sacrificing the mice. We show how optoacoustic (photoacoustic) mesoscopy reveals a potentially powerful look into tumor angiogenesis, with properties and features that are markedly different than other radiological modalities. This will facilitate a better understanding of tumor's angiogenesis, and the evaluation of treatment strategies.

We developed a reflection-mode, raster-scan optoacoustic mesoscopy system, based on a custom-made ultrasonic detector, with an ultra wide bandwidth of 20-180 MHz. To optimally use this bandwidth, we implemented multi-frequency reconstruction. System characterization reveals a 4 μm axial, and 18 μm transverse resolution, at penetration depths reaching 5 mm. After characterization, the system was applied to image a zebrafish ex vivo, and an excised mouse ear. In the zebrafish, the lateral line, intestines, eyes, and melanocytes are seen, while in the mouse ear, multi-frequency reconstruction recovered the small vessels, otherwise not seen on the image.

We have developed an epi-illumination raster-scan optoacoustic mesoscopy system (RSOM), the new system is capable of imaging model organisms, and vasculature. The newly developed system is based on a custom designed; spherically focused detector with a Characterization of the system shows an isotropic lateral resolution of 18 μm, and an axial resolution of 4 μm. The scan times are on the order of 8 minutes for a field of view of 10×10 mm2. The achieved resolution is slightly degraded up to a depth of 5 mm. After characterizing the system we showcase it's performance on a zebrafish ex vivo, and an excised mouse ear. Additionally, to improve the visibility of small structures we have reconstructed the high frequencies, and the low frequencies separately, and at the end overplayed the two reconstructions using different colors, this way the high frequencies are not masked by the low frequencies which have a higher signal to noise ratio.

In this dissertation we develop, and test systems for higher resolution thermoacoustic imaging, both using RF-based thermoacoustics, and laser-based thermoacoustics. After characterizing the systems for resolution, SNR, and penetration depth, we showcase their performance on selected biological samples, both ex vivo, and in vivo. Finally, we showcase reconstruction improvement, both in resolution, and contrast, based on multi-frequency reconstruction.

INTRODUCTION: Due to lack of reliable imaging contrast from catheter radiofrequency ablation (RFA) lesions, the vast majority of current procedures rely on indirect indicators of ablation activity, resulting in a significant number of arrhythmia reoccurrences after RFA procedures and the need for repeat surgeries. The objective of this work is to develop an accurate method for on-the-fly assessment of the durability and size of lesions formed during RFA procedures. METHOD AND RESULTS: Radiofrequency catheter ablation on freshly-excised porcine ventricular myocardial tissue was optoacoustically monitored by means of pulsed-laser illumination in the near-infrared spectrum. Lesion formation during ablation was captured at a rate of 10 Hz with a 256-detector optoacoustic imaging probe. Post-ablated samples were imaged using multispectral excitation in the wavelength range 740 nm to 860 nm to determine the lesion contrast spectrum. Tomographic reconstruction was performed to generate three-dimensional images of the lesions, which were compared to photographs depicting the final ablated tissue samples. Video-rate three-dimensional tomographic reconstructions depict formation of the lesion with high contrast and spatial resolution. The size and geometry of the lesion was shown to be in excellent agreement with the histological examinations. The wavelength dependence of the lesion contrast shows a contrast peak near 780 nm. CONCLUSION: Deep-tissue three-dimensional monitoring of RFA lesion generation in real time was demonstrated for the first time in this work. The results suggest the potential of optoacoustic monitoring for providing critical feedback on lesion position and size during radiofrequency catheter ablation, improving safety and efficacy of these treatments.

Current radiofrequency cardiac ablation procedures lack real-time lesion monitoring guidance, limiting the reliability and efficacy of the treatment. The objective of this work is to demonstrate that optoacoustic imaging can be applied to develop a diagnostic technique applicable to radiofrequency ablation for cardiac arrhythmia treatment with the capabilities of real-time monitoring of ablated lesion size and geometry. We demonstrate an optoacoustic imaging method using a 256-detector optoacoustic imaging probe and pulsed-laser illumination in the infrared wavelength range that is applied during radiofrequency ablation in excised porcine myocardial tissue samples. This technique results in images with high contrast between the lesion volume and unablated tissue, and is also capable of capturing time-resolved image sequences that provide information on the lesion development process. The size and geometry of the imaged lesion were shown to be in excellent agreement with the histological examinations. This study demonstrates the first deep-lesion real-time monitoring for radiofrequency ablation generated lesions, and the technique presented here has the potential for providing critical feedback that can significantly impact the outcome of clinical radiofrequency ablation procedures. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Diabetes is a serious world-wide medical challenge and there is a recognised need for improved diabetes care outcomes. This paper describes results of the EMPOWER project, to foster the self-management of diabetes patients by integration of existing and new services offered to patients after having been diagnosed with diabetes. The Self-Management Pathway described in this paper helps patients in the specification of personalized activities based on medical recommendations and personal goals, as well as self-monitoring of the results. The whole process is supported by innovative ICT services that motivate patients to change their lifestyle and adhere to defined medication and activity plans. We describe the approach and present the findings of the validation phase in Germany and Turkey.

Purpose: A prior image based temporally constrained reconstruction (PITCR) algorithm was developed for obtaining accurate temperature maps having better volume coverage, and spatial, and temporal resolution than other algorithms for highly undersampled data in magnetic resonance (MR) thermometry. Methods: The proposed PITCR approach is an algorithm that gives weight to the prior image and performs accurate reconstruction in a dynamic imaging environment. The PITCR method is compared with the temporally constrained reconstruction (TCR) algorithm using pork muscle data. Results: The PITCR method provides superior performance compared to the TCR approach with highly undersampled data. The proposed approach is computationally expensive compared to the TCR approach, but this could be overcome by the advantage of reconstructing with fewer measurements. In the case of reconstruction of temperature maps from 16% of fully sampled data, the PITCR approach was 1.57× slower compared to the TCR approach, while the root mean square error using PITCR is 0.784 compared to 2.815 with the TCR scheme. Conclusions: The PITCR approach is able to perform more accurate reconstructions of temperature maps compared to the TCR approach with highly undersampled data in MR guided high intensity focused ultrasound.

This thesis investigates reconstruction techniques for optoacoustic (oa) imaging, implemented both in pulsed and continuous wave (cw) mode. Two approaches to incorporate the transducer geometry into model-based reconstruction are implemented and its benefits showcased in simulations and experiments. The size of the imaging model could be reduced using wavelet packets, thus enabling complex inversion algorithms. Finally, a tomographic oa system applying a cw light source was implemented, including a dedicated reconstruction algorithm providing cross-sectional images.

Imaging of tissue oxygenation is important in several applications associated with patient care. Optical sensing is commonly applied for assessing oxygen saturation but is often restricted to local measurements or else it requires spectral and spatial information at the expense of time. Many methods proposed so far require assumptions on the properties of measured tissue. In this study we investigated a computational method that uses only multispectral information and quantitatively computes tissue oxygen saturation independently of tissue optical properties. The method is based on linear transformations of measurements in three isosbestic points. We investigated the ideal isosbestic point combination out of six isosbestic points available for measurement in the visible and near-infrared region that enable accurate oxygen saturation computation. We demonstrate this method on controlled tissue mimicking phantoms having different optical properties and validated the measurements using a gas analyzer. A mean error of 2.9 ± 2.8% O2 Sat was achieved. Finally, we performed pilot studies in tissues in-vivo by measuring dynamic changes in fingers subjected to vascular occlusion, the vasculature of mouse ears and exposed mouse organs. Selected steps of spectral transformations applied to oxygenation spectra. The original reflectance spectrum M(λ) is transformed in step 1 to overlap with reference spectra (grey) in three isosbestic points, resulting in M″(λ). In step 2, the gradient of M″(λ) is computed resulting in M″grad (λ), which can be used for quantitative oxygenation computation.

Multispectral molecular imaging exhibits the unique potential to simultaneously probe multiple molecules in living objects. This thesis reports on developments realized on methodological and systems level that aim at applying multispectral concepts to molecular imaging. Research was directed towards various application areas, from surface investigations of intrinsic tissue molecules to volumetric imaging of extrinsic molecular markers. The presented results show the capability of multispectral imaging to asses multiple molecules in-vivo, a property which is of tremendous value in systemic biomedical research. 

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.

The past decade marked an optical revolution in biology: an unprecedented number of optical techniques were developed and adopted for biological exploration, demonstrating increasing interest in optical imaging and in vivo interrogations. Optical methods have become faster and have reached nanoscale resolution, and are now complemented by optoacoustic (photoacoustic) methods capable of imaging whole specimens in vivo. Never before were so many optical imaging barriers broken in such a short time-frame: with new approaches to optical microscopy and mesoscopy came an increased ability to image biology at unprecedented speed, resolution, and depth. This review covers the most relevant techniques for imaging in developmental biology, and offers an outlook on the next steps for these technologies and their applications.

Optoacoustic (photoacoustic) imaging is often performed with one-dimensional transducer arrays, in analogy to ultrasound imaging. Optoacoustic imaging using linear arrays offers ease of implementation but comes with several performance drawbacks, in particular poor elevation resolution, i.e. the resolution along the axis perpendicular to