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This study highlights the potential of scanning optoacoustic angiography (OA) in identifying alterations of superficial vasculature in patients with post-thrombotic syndrome (PTS) of the foot, a venous stress disorder associated with significant morbidity developing from long-term effects of deep venous thrombosis. The traditional angiography methods available in the clinics are not capable of reliably assessing the state of peripheral veins that provide blood outflow from the skin, a key hallmark of personalized risks of PTS formation after venous thrombosis. Our findings indicate that OA can detect an increase in blood volume, diameter, and tortuosity of superficial blood vessels. The inability to spatially separate vascular plexuses of the dermis and subcutaneous adipose tissue serves as a crucial criterion for distinguishing PTS from normal vasculature. Furthermore, our study demonstrates the ability of scanning optoacoustic angiography to detect blood filling decrease in an elevated limb position versus increase in a lowered position.

Abstract Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

Abstract Noninvasive monitoring of cardiac development can potentially prevent cardiac anomalies in adulthood. Mouse models provide unique opportunities to study cardiac development and disease in mammals. However, high‐resolution noninvasive functional analyses of murine embryonic cardiac models are challenging because of the small size and fast volumetric motion of the embryonic heart, which is deeply embedded inside the uterus. In this study, a real time volumetric optoacoustic spectroscopy (VOS) platform for whole‐heart visualization with high spatial (100 µm) and temporal (10 ms) resolutions is developed. Embryonic heart development on gestational days (GDs) 14.5–17.5 and quantify cardiac dynamics using time‐lapse‐4D image data of the heart is followed. Additionally, spectroscopic recordings enable the quantification of the blood oxygenation status in heart chambers in a label‐free and noninvasive manner. This technology introduces new possibilities for high‐resolution quantification of embryonic heart function at different gestational stages in mammalian models, offering an invaluable noninvasive method for developmental biology.

Abstract Tendon injuries resulting from accidents and aging are increasing globally. However, key tendon functional parameters such as microvascularity and oxygen perfusion remain inaccessible via the currently available clinical diagnostic tools, resulting in disagreements on optimal treatment options. Here, a new noninvasive method for anatomical and functional characterization of human tendons based on multispectral optoacoustic tomography (MSOT) is reported. Healthy subjects are investigated using a hand‐held scanner delivering real‐time volumetric images. Tendons in the wrist, ankle, and lower leg are imaged in the near‐infrared optical spectrum to utilize endogenous contrast from Type I collagen. Morphology of the flexor carpi ulnaris, carpi radialis, palmaris longus, and Achilles tendons are reconstructed in full. The functional roles of the flexor digitorium longus, hallicus longus, and the tibialis posterior tendons have been visualized by dynamic tracking during toe extension‐flexion motion. Furthermore, major vessels and microvasculature near the Achilles tendon are localized, and the global increase in oxygen saturation in response to targeted exercise is confirmed by perfusion studies. MSOT is shown to be a versatile tool capable of anatomical and functional tendon assessments. Future studies including abnormal subjects can validate the method as a viable noninvasive clinical tool for tendinopathy management and healing monitoring.

This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024). Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 µm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace. Infarct areas and volumes are assessed through MRI at two distinct time frames—three days (acute) and 28 days (chronic) following photothrombotic stroke induction. Subsequently, the brains are sectioned into 40 µm thick coronal slices, stained with NeuroTrace, and imaged as whole sections. The dataset holds considerable value for reuse, particularly for researchers focused on stroke volume estimation methods as well as those interested in comparing the efficacy of MRI and histological techniques.

Abstract Major biological discoveries are made by interrogating living organisms with light. However, the limited penetration of un‐scattered photons within biological tissues limits the depth range covered by optical methods. Deep‐tissue imaging is achieved by combining light and ultrasound. Optoacoustic imaging exploits the optical generation of ultrasound to render high‐resolution images at depths unattainable with optical microscopy. Recently, laser ultrasound has been suggested as a means of generating broadband acoustic waves for high‐resolution pulse‐echo ultrasound imaging. Herein, an approach is proposed to simultaneously interrogate biological tissues with light and ultrasound based on layer‐by‐layer coating of silica optical fibers with a controlled degree of transparency. The time separation between optoacoustic and ultrasound signals collected with a custom‐made spherical array transducer is exploited for simultaneous 3D optoacoustic and laser ultrasound (OPLUS) imaging with a single laser pulse. OPLUS is shown to enable large‐scale anatomical characterization of tissues along with functional multi‐spectral imaging of chromophores and assessment of cardiac dynamics at ultrafast rates only limited by the pulse repetition frequency of the laser. The suggested approach provides a flexible and scalable means for developing a new generation of systems synergistically combining the powerful capabilities of optoacoustics and ultrasound imaging in biology and medicine.

Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri‑infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Abstract Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (

The role of the transcription factor Fosl-2 in promoting myocardial fibrosis, arrhythmias and aberrant response to stress under immunofibrotic conditions is described using an in vivo mouse model and human cardiac fibroblasts.

Emerging evidence indicates crosstalk between the brain and hematopoietic system following cerebral ischemia. Here, we investigated metabolism and oxygenation in the spleen and spinal cord in a transient middle cerebral artery occlusion (tMCAO) model. Sham-operated and tMCAO mice underwent [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to assess glucose metabolism. Naïve, sham-operated and tMCAO mice underwent multispectral optoacoustic tomography (MSOT) assisted by quantitative model-based reconstruction and unmixing algorithms for accurate mapping of oxygenation patterns in peripheral tissues at 24 h after reperfusion. We found increased [18F]FDG uptake and reduced MSOT oxygen saturation, indicating hypoxia in the thoracic spinal cord of tMCAO mice compared with sham-operated mice but not in the spleen. Reduced spleen size was observed in tMCAO mice compared with sham-operated mice ex vivo. tMCAO led to an increase in the numbers of mature T cells in femoral bone marrow tissues, concomitant with a stark reduction in these cell subsets in the spleen and peripheral blood. The combination of quantitative PET and MSOT thus enabled observation of hypoxia and increased metabolic activity in the spinal cord of tMCAO mice at 24 h after occlusion compared to sham-operated mice.

A 3D wide-field fluorescence microscopy method is introduced based on optical astigmatism combined with fluorescence source localization. It enables transcranial cortical microcirculation mapping in murine brain with high spatiotemporal resolution.

We report on a fully hybridized magnetic resonance and optoacoustic tomography (MROT) system for concurrent noninvasive structural and functional imaging of the murine brain.

Optoacoustic tomography (OAT) provides a non-invasive means to characterize cerebral hemodynamics across an entire murine brain while attaining multi-parametric readouts not available with other modalities. This unique capability can massively impact our understanding of brain function. However, OAT largely lacks the soft tissue contrast required for unambiguous identification of brain regions. Hence, its accurate registration to a reference brain atlas is paramount for attaining meaningful functional readings. Herein, we capitalized on the simultaneously acquired bi-modal data from the recently-developed hybrid magnetic resonance optoacoustic tomography (MROT) scanner in order to devise an image coregistration paradigm that facilitates brain parcellation and anatomical referencing. We evaluated the performance of the proposed methodology by coregistering OAT data acquired with a standalone system using different registration methods. The enhanced performance is further demonstrated for functional OAT data analysis and characterization of stimulus-evoked brain responses. The suggested approach enables better consolidation of the research findings thus facilitating wider acceptance of OAT as a powerful neuroimaging tool to study brain functions and diseases.

Optoacoustic tomography is commonly performed with bulky and expensive short-pulsed solid-state lasers providing high per-pulse energies in the millijoule range. Light emitting diodes (LEDs) represent a cost-effective and portable alternative for optoacoustic signal excitation that can additionally provide excellent pulse-to-pulse stability. Herein, we introduce a full-view LED-based optoacoustic tomography (FLOAT) system for deep tissue in vivo imaging. It is based on a custom-made electronic unit driving a stacked array of LEDs, which attains 100 ns pulse width and highly stable (0.62 % standard deviation) total per-pulse energy of 0.48 mJ. The illumination source is integrated into a circular array of cylindrically-focused ultrasound detection elements to result in a full-view tomographic configuration, which plays a critical role in circumventing limited-view effects, enhancing the effective field-of-view and image quality for cross-sectional (2D) imaging. We characterized the FLOAT performance in terms of pulse width, power stability, excitation light distribution, signal-to-noise and penetration depth. FLOAT of the human finger revealed a comparable imaging performance to that achieved with the standard pulsed Nd:YAG laser. It is anticipated that this compact, affordable and versatile illumination technology will facilitate optoacoustic imaging developments in resource-limited settings for biological and clinical applications.

A number of optoacoustic (or photoacoustic) microscopy and mesoscopy techniques have successfully been employed for non-invasive tumor angiography. However, accurate rendering of tortuous and multidirectional neoplastic vessels is commonly hindered by the limited aperture size, narrow bandwidth and insufficient angular coverage of commercially available ultrasound transducers. We exploited the excellent flexibility and elasticity of a piezo polymer (PVDF) material to devise a fisheye-shape ultrasound detector with a high numerical aperture of 0.9, wide 1–30 MHz detection bandwidth and 27 mm diameter aperture suitable for imaging tumors of various size. We show theoretically and experimentally that the wide detector’s view-angle and bandwidth are paramount for achieving a detailed visualization of the intricate arbitrarily-oriented neovasculature in experimental tumors. The developed approach is shown to be well adapted to the tasks of experimental oncology thus allows to better exploit the angiographic potential of optoacoustics.

High-intensity focused ultrasound (HIFU) enables localized ablation of biological tissues by capitalizing on the synergistic effects of heating and cavitation. Monitoring of those effects is essential for improving the efficacy and safety of HIFU interventions. Herein, we suggest a hybrid optoacoustic-ultrasound (OPUS) approach for real-time assessment of heating and cavitation processes while providing an essential anatomical reference for accurate localization of the HIFU-induced lesion. Both effects could clearly be observed by exploiting the temperature dependence of optoacoustic (OA) signals and the strong contrast of gas bubbles in pulse-echo ultrasound (US) images. The differences in temperature increase and its rate, as recorded with a thermal camera for different HIFU pressures, evinced the onset of cavitation at the expected pressure threshold. The estimated temperatures based on OA signal variations were also within 10–20 % agreement with the camera readings for temperatures below the coagulation threshold (∼50 °C). Experiments performed in excised tissues as well as in a post-mortem mouse demonstrate that both heating and cavitation effects can be effectively visualized and tracked using the OPUS approach. The good sensitivity of the suggested method for HIFU monitoring purposes was manifested by a significant increase in contrast-to-noise ratio within the ablated region by > 10 dB and > 5 dB for the OA and US images, respectively. The hybrid OPUS-based monitoring approach offers the ease of handheld operation thus can readily be implemented in a bedside setting to benefit several types of HIFU treatments used in the clinics.

Optoacoustic tomography has been established as a powerful modality for preclinical imaging. However, efficient whole-body imaging coverage has not been achieved owing to the arduous requirement for continuous acoustic coupling around the animal. In this work, we introduce panoramic (3600) head-to-tail 3D imaging of mice with spiral volumetric optoacoustic tomography (SVOT). The system combines multi-beam illumination and a dedicated head holder enabling uninterrupted acoustic coupling for whole-body scans. Image fidelity is optimized with self-gated respiratory motion rejection and dual speed-of-sound reconstruction algorithms to attain spatial resolution down to 90 µm. The developed system is thus highly suitable for visualizing rapid biodynamics across scales, such as hemodynamic changes in individual organs, responses to treatments and stimuli, perfusion, total body accumulation, or clearance of molecular agents and drugs with unmatched contrast, spatial and temporal resolution.

Abstract Functional magnetic resonance imaging (fMRI) has massively contributed to the understanding of mammalian brain function. However, the origin and interpretation of the blood oxygen level‐dependent (BOLD) signals retrieved by fMRI remain highly disputed. This article reports on the development of a fully hybridized system enabling concurrent functional magnetic resonance optoacoustic tomography (MROT) measurements of stimulus‐evoked brain‐wide sensory responses in mice. The highly complementary angiographic and soft tissue contrasts of both modalities along with simultaneous multi‐parametric readings of stimulus‐evoked hemodynamic responses are leveraged in order to establish unequivocal links between the various counteracting physiological and metabolic processes in the brain. The results indicate that the BOLD signals are highly correlated, both spatially and temporally, with the total hemoglobin readings resolved with volumetric multi‐spectral optoacoustic tomography. Furthermore, the differential oxygenated and deoxygenated hemoglobin optoacoustic readings exhibit superior sensitivity as compared to the BOLD signals when detecting stimulus‐evoked hemodynamic responses. The fully hybridized MROT approach greatly expands the neuroimaging toolset to comprehensively study neurovascular and neurometabolic coupling mechanisms and related diseases.

Abstract Detailed characterization of microvascular alterations requires high‐resolution 3D imaging methods capable of providing both morphological and functional information. Existing optical microscopy tools are routinely used for microangiography, yet offer suboptimal trade‐offs between the achievable field of view and spatial resolution with the intense light scattering in biological tissues further limiting the achievable penetration depth. Herein, a new approach for volumetric deep‐tissue microangiography based on stereovision combined with super‐resolution localization imaging is introduced that overcomes the spatial resolution limits imposed by light diffusion and optical diffraction in wide‐field imaging configurations. The method capitalizes on localization and tracking of flowing fluorescent particles in the second near‐infrared window (NIR‐II, ≈1000–1700 nm), with the third (depth) dimension added by triangulation and stereo‐matching of images acquired with two short‐wave infrared cameras operating in a dual‐view mode. The 3D imaging capability enabled with the proposed method facilitates a detailed visualization of microvascular networks and an accurate blood flow quantification. Experiments performed in tissue‐mimicking phantoms demonstrate that high resolution is preserved up to a depth of 4 mm in a turbid medium. Transcranial microangiography of the entire murine cortex and penetrating vessels is further demonstrated at capillary level resolution.

Optoacoustic mesoscopy combines rich optical absorption contrast with high spatial resolution at tissue depths beyond reach for microscopic techniques employing focused light excitation. The mesoscopic imaging performance is commonly hindered by the use of inaccurate delay-and-sum reconstruction approaches and idealized modeling assumptions. In principle, image reconstruction performance could be enhanced by simulating the optoacoustic signal generation, propagation, and detection path. However, for most realistic experimental scenarios, the underlying total impulse response (TIR) cannot be accurately modelled. Here we propose to capture the TIR by scanning of a sub-resolution sized absorber. Significant improvement of spatial resolution and depth uniformity is demonstrated over 3 mm range, outperforming delay-and-sum and model-based reconstruction implementations. Reconstruction performance is validated by imaging subcutaneous murine vasculature and human skin in vivo. The proposed experimental calibration and reconstruction paradigm facilitates quantitative inversions while averting complex physics-based simulations. It can readily be applied to other imaging modalities employing TIR-based reconstructions.

Background: The glymphatic system is a brain waste clearance system mediated via cerebrospinal fluid (CSF) flow,1 with implications for influence on neurodegenerative diseases.2 Most preclinical glymphatic studies employ fluorescence imaging, which provides higher specificity, but a smaller field-of-view (FOV), or magnetic resonance imaging (MRI), which provides brain-wide FOV, but lower specificity. Functional optoacoustic neuro-tomography3 (FONT) offers a larger FOV compared to classical optical methods, and higher specificity compared to MRI. However, FONT has not yet been applied to probe the glymphatic system. In this study, we used fluorescence and optoacoustic imaging of a near-infrared dye, Janelia Fluor 669 (JF669), to track CSF and multimodal CSF-hemodynamic flows in mice. Methods: After observing strong fluorescence and optoacoustic signatures of JF669 in phantom experiments, we performed a series of in vivo experiments in isoflurane-anesthetized C57BL/6 mice (n=3 fluorescence and n=4 FONT experiments, respectively) (Figure 1A). The lumbar injection was applied to deliver JF669 at a rate of 2 µL/min for 30 minutes. A polyethylene tube was placed intrathecally at the lumbar region (L4-L5). The scalp was removed. Fluorescence or FONT images were obtained every 5 minutes after injection. Results: Fluorescence imaging and FONT probe CSF flow Images (Figure 1B) and time traces (Figure 1C) revealed time-dependent anatomical routes of paravascular influx, including the transport along the olfactory artery (OFA), superior cerebellar artery (SCA), and bilateral middle cerebral artery (MCA). For FONT imaging, since the OFA showed strong fluorescence (Figure 1B), we positioned the ultrasound transducer array at the anterior of the mouse brain with a FOV of 5x5 mm2 (Figure 1A). Standard filtered back-projection reconstruction was applied. Besides the OFA route of the paravascular influx, the dynamic images (Figure 1D) and time-traces (Figure 1E) also revealed time-dependent anatomical routes of CSF-interstitial fluid (ISF) exchange in the olfactory bulb (OFB) and paravascular efflux in the superior sagittal sinus (SSS) and the bilateral inferior cerebral vein (ICV). Next, we studied the aquaporin-4 (AQP4) dependence of glymphatic flow by subcutaneously injecting AQP4 inhibitor TGN020, in addition to the prior procedures. Under fluorescence imaging and FONT, we observed that TGN020 significantly decreased and spatially restricted the spread of JF669 in the brain. FONT spectral unmixing separates CSF and blood We swept the OPO through 680 nm to 750 nm, with 10 nm steps at 10 Hz in the phantom and in each animal. The multispectral reconstructions were unmixed using the known absorption spectra of hemoglobin and the JF669 OA spectrum obtained from the phantom.4 This enabled the separation of blood and JF669 signal (Figure 1D). Conclusions: We characterized anatomical routes of paravascular influx (OFA), CSF-ISF exchange (OFB) and paravascular efflux using FONT, while fluorescence imaging only enabled visualization of the paravascular influx. We thus demonstrated the feasibility and advantages of FONT for probing the glymphatic system. These findings present great opportunities for monitoring and understanding the glymphatic system during development, aging, disease, pharmacological interventions and genetic modifications.

Optoacoustic (OA, photoacoustic) imaging capitalizes on the low scattering of ultrasound within biological tissues to provide optical absorption-based contrast with high resolution at depths not reachable with optical microscopy. For deep tissue imaging applications, OA image formation commonly relies on acoustic inversion of time-resolved tomographic data. The excitation of OA responses and subsequent propagation of ultrasound waves can be mathematically described as a forward model enabling image reconstruction via algebraic inversion. These model-based reconstruction methods have been shown to outperform alternative inversion approaches and can further render OA images from incomplete datasets, strongly distorted signals or other suboptimally recorded data. Herein, we provide a general perspective on model-based OA reconstruction methods, review recent progress, and discuss the performance of the different algorithms under practical imaging scenarios.

Fast three-dimensional imaging of freely-swimming zebrafish is essential to understand the link between neuronal activity and behavioral changes during epileptic seizures. Studying the complex spatiotemporal patterns of neuronal activity at the whole-brain or -body level typically requires physical restraint, thus hindering the observation of unperturbed behavior. Here we report on real-time volumetric optoacoustic imaging of aberrant circular swimming activity and calcium transients in freely behaving zebrafish larvae, continuously covering their motion across an entire three-dimensional region. The high spatiotemporal resolution of the technique enables capturing ictal-like epileptic seizure events and quantifying their propagation speed, independently validated with simultaneous widefield fluorescence recordings. The work sets the stage for discerning functional interconnections between zebrafish behavior and neuronal activity for studying fundamental mechanisms of epilepsy and in vivo validation of treatment strategies.

Abstract Modern optical neuroimaging approaches are expanding the ability to elucidate complex brain function. Diverse imaging contrasts enable direct observation of neural activity with functional sensors along with the induced hemodynamic responses. To date, decoupling the complex interplay of neurovascular coupling and dynamical physiological states has remained challenging when employing single‐modality functional neuroimaging readings. A hybrid fluorescence optoacoustic tomography platform combined with a custom data processing pipeline based on statistical parametric mapping is devised, attaining the first noninvasive observation of simultaneous calcium and hemodynamic activation patterns using optical contrasts. Correlated changes in the oxy‐ and deoxygenated hemoglobin, total hemoglobin, oxygen saturation, and rapid GCaMP6f fluorescence signals are observed in response to peripheral sensory stimulation. While the concurrent epifluorescence serves to corroborate and complement the functional optoacoustic observations, the latter further aids in decoupling the rapid calcium responses from the slowly varying background in the fluorescence recordings mediated by hemodynamic changes. The hybrid imaging platform expands the capabilities of conventional neuroimaging methods to provide more comprehensive functional readings for studying neurovascular and neurometabolic coupling mechanisms and related diseases.

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.

Tumor microvascular responses may provide a sensitive readout indicative of radiation therapy efficacy, its time course and dose dependencies. However, direct high-resolution observation and longitudinal monitoring of large-scale microvascular remodeling in deep tissues remained challenging with the conventional microscopy approaches.We report on a non-invasive longitudinal study of morphological and functional neovascular responses by means of scanning optoacoustic (ОА) microangiography. In vivo imaging of CT26 tumor response to a single irradiation at varying dose (6, 12, and 18 Gy) has been performed over ten days following treatment. Tumor oxygenation levels were further estimated using diffuse optical spectroscopy (DOS) with a contact fiber probe.OA revealed the formation of extended vascular structures on the whole tumor scale during its proliferation, whereas only short fragmented vascular regions were identified following irradiation. On the first day post treatment, a decrease in the density of small (capillary-sized) and medium-sized vessels was revealed, accompanied by an increase in their fragmentation. Larger vessels exhibited an increase in their density accompanied by a decline in the number of vascular segments. Short-lasting response has been observed after 6 and 12 Gy irradiations, whereas 18 Gy treatment resulted in prolonged responses, up to the tenth day after irradiation. DOS measurements further revealed a delayed increase of tumor oxygenation levels for 18 Gy irradiations, commencing on the sixth day post treatment. The ameliorated oxygenation is attributed to diminished oxygen consumption by inhibited tumor cells but not to the elevation of oxygen supply.This work is the first to demonstrate the differential (size-dependent) nature of vascular responses to radiation treatments at varying doses in vivo. The OA approach thus facilitates the study of radiation-induced vascular changes in an unperturbed in vivo environment while enabling deep tissue high-resolution observations at the whole tumor scale.

Optoacoustic (OA) methods have become powerful tools in biomedical research capable of retrieving functional information from biological tissues in vivo. Acquisition of OA signals generally relies on direct physical contact of a transducer or an acoustic coupling medium with the tissue surface, which prevents applicability, e.g., in open surgeries or wounded tissues. Non-contact OA imaging has been achieved with air-coupled piezoelectric transducers, which provide a straightforward approach for remote sensing of ultrasound vibrations. However, sensitivity was hampered by a suboptimal alignment between the illumination and detection fields. Herein, we devised an air-coupled transducer featuring a central aperture for light delivery with coaxially aligned optical and acoustic foci, thus providing optimal sensitivity for OA signal detection. Imaging of phantoms and a mouse ear in vivo is showcased by raster-scanning the transducer with light being delivered through a multimode optical fiber.

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.

Summary: Functional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs. : Analytical Chemistry; Bioengineering; Biomaterials Subject Areas: Analytical Chemistry, Bioengineering, Biomaterials

The abnormal deposition of fibrillar beta-amyloid (Aβ) deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Here, we characterized curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of brain Aβ deposits in the arcAβ mouse model of AD cerebral amyloidosis. CRANAD-2 showed a specific and quantitative detection of Aβ fibrils in vitro, even in complex mixtures, and it is capable of distinguishing between monomeric and fibrillar forms of Aβ. In vivo epi-fluorescence microscopy and optoacoustic tomography after intravenous CRANAD-2 administration demonstrated higher cortical retention in arcAβ compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Aβ deposits in arcAβ mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for optical detection of Aβ deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ deposits.

Intense efforts are underway to develop functional imaging modalities for capturing brain activity at the whole organ scale with high spatial and temporal resolution. Functional optoacoustic (fOA) imaging is emerging as a new tool to monitor multiple hemodynamic parameters across the mouse brain, but its sound validation against other neuroimaging modalities is often lacking. Here we investigate mouse brain responses to peripheral sensory stimulation using both fOA and functional ultrasound (fUS) imaging. The two modalities operate under similar spatio-temporal resolution regime, with a potential to provide synergistic and complementary hemodynamic readouts. Specific contralateral activation was observed with sub-millimeter spatial resolution with both methods. Sensitivity to hemodynamic activity was found to be on comparable levels, with the strongest responses obtained in the oxygenated hemoglobin channel of fOA. While the techniques attained highly correlated hemodynamic responses, the differential fOA readings of oxygenated and deoxygenated haemoglobin provided complementary information to the blood flow contrast of fUS. The multi-modal approach may thus emerge as a powerful tool providing new insights into brain function, complementing our current knowledge generated with well-established neuroimaging methods.

Chronic, non-healing wounds constitute a major health problem, and the current therapeutic options are limited. Therefore, pharmaceuticals that can be locally applied to complicated wounds are urgently needed. Such treatments should directly target the underlying causes, which include diabetes mellitus, chronic local pressure and/or vascular insufficiency. A common consequence of these disorders is impaired wound angiogenesis. Here, we investigated the effect of topical application of a nitric oxide-releasing phosphodiesterase 5 inhibitor (TOP-N53)-containing liquid hydrogel on wound repair in mice. The drug-loaded hydrogel promoted re-epithelialization and angiogenesis in wounds of healthy and healing-impaired diabetic mice. Using a non-invasive label-free optoacoustic microscopy approach combined with automated vessel analysis, we show that the topical application of TOP-N53 formulation increases the microvascular network density and promotes the functionality of the newly formed blood vessels, resulting in enhanced blood perfusion of the wounds. These results demonstrate a remarkable healing-stimulating activity of topically applied TOP-N53 formulation, supporting its further development as a wound therapeutic.

Abstract Wound healing is a well‐coordinated process, necessitating efficient formation of new blood vessels. Vascularization defects are therefore a major risk factor for chronic, non‐healing wounds. The dynamics of mammalian tissue revascularization, vessel maturation, and remodeling remain poorly understood due to lack of suitable in vivo imaging tools. A label‐free large‐scale optoacoustic microscopy (LSOM) approach is developed for rapid, non‐invasive, volumetric imaging of tissue regeneration over large areas spanning up to 50 mm with a depth penetration of 1.5 mm. Vascular networks in dorsal mouse skin and full‐thickness excisional wounds are imaged with capillary resolution during the course of healing, revealing previously undocumented views of the angiogenesis process in an unperturbed wound environment. Development of an automatic analysis framework enables the identification of key features of wound angiogenesis, including vessel length, diameter, tortuosity, and angular alignment. The approach offers a versatile tool for preclinical research in tissue engineering and regenerative medicine, empowering label‐free, longitudinal, high‐throughput, and quantitative studies of the microcirculation in processes associated with normal and impaired vascular remodeling, and analysis of vascular responses to pharmacological interventions in vivo.

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.

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.

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.

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 (sO2) 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 sO2 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 sO2 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 sO2 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 sO2 of 42 ± 5% against 49 ± 4% (n = 3, P < .05) in the contralateral side, indicating their hypoxic status. Upon CA4P administration, sO2 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.

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. Keywords: Optoacoustic imaging, Ultrasonography, Multimodal imaging, Photoacoustics, Color Doppler

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.

Abstract 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 50mm3. 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.

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.

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.

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·HCl) and liposomal encapsulated doxorubicin hydrochloride (Dox·Lipo), two common drugs for anti-cancer 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.

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.

Volumetric optoacoustic tomography has been shown to provide unprecedented capabilities for ultrafast imaging of cardiovascular dynamics in mice. Three-dimensional imaging rates in the order of 100 Hz have been achieved, which enabled the visualization of transient cardiac events such as arrhythmias or contrast agent perfusion without the need for retrospective gating. The fast murine heart rates (400–600 beats per minute) yet impose limitations when it comes to compounding of multiple frames or accurate registration of multi-spectral data. Herein, we investigate on the capabilities of Fourier analysis of four-dimensional data for coregistration of independent volumetric optoacoustic image sequences of the heart. The fundamental frequencies and higher harmonics of respiratory and cardiac cycles could clearly be distinguished, which facilitated efficient retrospective gating without additional readings. The performance of the suggested methodology was successfully demonstrated by compounding cardiac images acquired by raster-scanning of a spherical transducer array as well as by unmixing of oxygenated and deoxygenated hemoglobin from multi-spectral optoacoustic data.

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.

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.

This paper comprehensively reviews the emerging topic of optoacoustic imaging from the image reconstruction and quantification perspective. Optoacoustic imaging combines highly attractive features, including rich contrast and high versatility in sensing diverse biological targets, excellent spatial resolution not compromised by light scattering, and relatively low cost of implementation. Yet, living objects present a complex target for optoacoustic imaging due to the presence of a highly heterogeneous tissue background in the form of strong spatial variations of scattering and absorption. Extracting quantified information on the actual distribution of tissue chromophores and other biomarkers constitutes therefore a challenging problem. Image quantification is further compromised by some frequently-used approximated inversion formulae. In this review, the currently available optoacoustic image reconstruction and quantification approaches are assessed, including back-projection and model-based inversion algorithms, sparse signal representation, wavelet-based approaches, methods for reduction of acoustic artifacts as well as multi-spectral methods for visualization of tissue bio-markers. Applicability of the different methodologies is further analyzed in the context of real-life performance in small animal and clinical in-vivo imaging scenarios.