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Optoacoustic (OA, photoacoustic) imaging capitalizes on the synergistic combination of light excitation and ultrasound detection to empower biological and clinical investigations with rich optical contrast while effectively bridging the gap between micro and macroscopic imaging realms. State-of-the-art OA embodiments consistently provide images at micron-scale resolution through superficial tissue layers by means of focused illumination that can be smoothly exchanged for acoustic-resolution images at diffuse light depths of several millimetres to centimetres via ultrasound beamforming or tomographic reconstruction. Taken together, this unique multi-scale imaging capacity opens unprecedented capabilities for high-resolution in vivo interrogations of the skin at scalable depths. Moreover, diverse anatomical and functional information is retrieved via dynamic mapping of endogenous chromophores such as haemoglobin, melanin, lipids, collagen, water and others. This, along with the use of non-ionizing radiation, facilitates a clinical translation of the OA modalities. We review recent progress in OA imaging of the skin in preclinical and clinical studies exploiting the rich contrast provided by endogenous substances in tissues. The imaging capabilities of existing approaches are discussed in the context of initial translational studies on skin cancer, inflammatory skin diseases, wounds and other conditions.

Optoacoustic tomography (OAT) has recently been advanced toward ultrafast volumetric imaging frame rates in the kilohertz range. As a result, excessive data processing and storage capacity requirements are increasingly being imposed on the imaging systems. OAT data commonly exhibit significant sparsity across the spatial, temporal or spectral domains, which facilitated the development of compressed sensing algorithms exploiting various sparse acquisition and under-sampling schemes to reduce data rates. However, performance of compressed sensing critically depends on a priori knowledge on the type of acquired data and/or imaged object, commonly resulting in lack of general applicability and unpredictable image quality. In this work, we report on a fast adaptive OAT data compression framework operating on fully sampled tomographic data. It is based on a wavelet packet transform that maximizes the data compression ratio according to the desired signal energy loss. A dedicated reconstruction method was further developed that efficiently renders images directly from the compressed data. Up to 1000x compression ratios were achieved while providing efficient control over the resulting image quality from arbitrary datasets exhibiting diverse spatial, temporal and spectral characteristics. Our approach enables faster and longer acquisitions and facilitates long-term storage of large OAT datasets.

Reflection ultrasound computed tomography (RUCT) attains optimal image quality from objects that can be fully accessed from multiple directions, such as the human breast or small animals. Owing to the full-view tomography approach based on the compounding of images taken from multiple angles, RUCT effectively mitigates several deficiencies afflicting conventional pulse-echo ultrasound (US) systems, such as speckle patterns and interuser variability. On the other hand, the small interelement pitch required to fulfill the spatial sampling criterion in the circular transducer configuration used in RUCT typically implies the use of an excessive number of independent array elements. This increases the system's complexity and costs, and limits the achievable imaging speed. Here, we explore acquisition schemes that enable RUCT imaging with the reduced number of transmit/receive elements. We investigated the influence of the element size in transmission and reception in a ring array geometry. The performance of a sparse acquisition approach based on partial acquisition from a subset of the elements has been further assessed. A larger element size is shown to preserve contrast and resolution at the center of the field of view (FOV), while a reduced number of elements is shown to cause uniform loss of contrast and resolution across the entire FOV. The tradeoffs of achievable FOV, contrast-to-noise ratio, and temporal and spatial resolutions are assessed in phantoms and in vivo mouse experiments. The experimental analysis is expected to aid the development of optimized hardware and image acquisition strategies for RUCT and, thus, result in more affordable imaging systems facilitating wider adoption.

Spherical matrix arrays represent an advantageous tomographic detection geometry for non-invasive deep tissue mapping of vascular networks and oxygenation with volumetric photoacoustic tomography (VPT). Hybridization of VPT with ultrasound (US) imaging remains difficult with this configuration due to the relatively large inter-element pitch of spherical arrays. We suggest a new approach for combining VPT and US contrast-enhanced 3D imaging employing injection of clinically-approved microbubbles. Power Doppler (PD) and US localization imaging were enabled with a sparse US acquisition sequence and model-based inversion based on infimal convolution of total variation (ICTV) regularization. In vitro experiments in tissue-mimicking phantoms and in living mouse brain demonstrate the powerful capabilities of the new dual-mode imaging approach attaining 80 μm spatial resolution and a more than 10 dB signal to noise improvement with respect to a classical delay and sum beamformer. Microbubble localization and tracking allowed for flow velocity mapping up to 40 mm/s.

Extrinsically administered light-absorbing agents may greatly enhance the sensitivity and imaging performance of optoacoustic tomography (OAT). Beyond the use of targeted contrast agents in functional and molecular imaging applications, tracking of highly absorbing microparticles has recently been shown to facilitate super-resolution volumetric angiography and mapping of blood flow. However, in vivo characterization of new types of microparticulate absorbing agents is often hindered due to their potential toxicity, incompatible dimensions, or sub-optimal extinction spectrum shadowed by strong background absorption of hemoglobin. Herein, we used an intracardiac perfusion mouse model to individually track the perfusion of absorbing particles through the cerebral vasculature by acquiring a sequence of high-frame-rate 3D OAT images. The particles were injected in the left ventricle of the heart after substitution of blood by an artificial cerebrospinal fluid post mortem, which has further contributed to minimizing the background OAT signals induced by hemoglobin absorption. The presented approach can greatly aid the development of new microparticulate contrast agents with optimized performance for various OAT imaging applications.

Images rendered with common optoacoustic system implementations are often afflicted with distortions and poor visibility of structures, hindering reliable image interpretation and quantification of bio-chrome distribution. Among the practical limitations contributing to artifactual reconstructions are insufficient tomographic detection coverage and suboptimal illumination geometry, as well as inability to accurately account for acoustic reflections and speed of sound heterogeneities in the imaged tissues. Here we developed a convolutional neural network (CNN) approach for enhancement of optoacoustic image quality which combines training on both time-resolved signals and tomographic reconstructions. Reference human finger data for training the CNN were recorded using a full-ring array system that provides optimal tomographic coverage around the imaged object. The reconstructions were further refined with a dedicated algorithm that minimizes acoustic reflection artifacts induced by acoustically mismatch structures, such as bones. The combined methodology is shown to outperform other learning-based methods solely operating on image-domain data.

The rapidly evolving field of optoacoustic (photoacoustic) imaging and tomography is driven by a constant need for better imaging performance in terms of resolution, speed, sensitivity, depth and contrast. In practice, data acquisition strategies commonly involve sub-optimal sampling of the tomographic data, resulting in inevitable performance trade-offs and diminished image quality. We propose a new framework for efficient recovery of image quality from sparse optoacoustic data based on a deep convolutional neural network and demonstrate its performance with whole body mouse imaging in vivo. To generate accurate high-resolution reference images for optimal training, a full-view tomographic scanner capable of attaining superior cross-sectional image quality from living mice was devised. When provided with images reconstructed from substantially undersampled data or limited-view scans, the trained network was capable of enhancing the visibility of arbitrarily oriented structures and restoring the expected image quality. Notably, the network also eliminated some reconstruction artefacts present in reference images rendered from densely sampled data. No comparable gains were achieved when the training was performed with synthetic or phantom data, underlining the importance of training with high-quality in vivo images acquired by full-view scanners. The new method can benefit numerous optoacoustic imaging applications by mitigating common image artefacts, enhancing anatomical contrast and image quantification capacities, accelerating data acquisition and image reconstruction approaches, while also facilitating the development of practical and affordable imaging systems. The suggested approach operates solely on image-domain data and thus can be seamlessly applied to artefactual images reconstructed with other modalities. Optoacoustic imaging can achieve high spatial and temporal resolution but image quality is often compromised by suboptimal data acquisition. A new method employing deep learning to recover high-quality images from sparse or limited-view optoacoustic scans has been developed and demonstrated for whole-body mouse imaging in vivo.

The heart undergoes complex remodeling processes post myocardial infarction (MI), which are yet to be fully understood. Advanced preclinical in vivo imaging tools are key to a better understanding of the processes underlying ischemic damage and cardiac remodeling. Herein, volumetric optoacoustic tomography (VOT) was used to study remodeling patterns at different stages post MI by measuring the pulmonary transit time, ICG perfusion and cardiac cycle irregularities. The real-time, non-invasive, volumetric capabilities of VOT detected differences in remodeling patterns across different MI models, highlighting the feasibility of VOT as a preclinical cardiac imaging tool to study complex murine heart dynamics.

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.

Optoacoustic (OA) tomography has recently demonstrated ultrafast imaging rates, owing to its unique ability to capture volumetric image data with a single nanosecond-duration flash of light. The achievable spatial resolution of volumetric tomography is however limited by the narrow bandwidth and lack of scalability of the piezo-composite arrays currently employed for OA signal detection. Here we report on the first implementation of high-density spherical matrix array technology based on flexible polyvinylidene difluoride films featuring ultrawideband (38 MHz) sub-millimeter sized elements thus enabling volumetric imaging with ~35 μm resolution, superior image fidelity and excellent signal to noise performance permitting real-time imaging of deep tissues without employing signal averaging. We demonstrate real-time volumetric in vivo imaging capabilities of the new approach by performing cerebral angiography in mice. The new technology leverages the true potential of OA for high spatio-temporal resolution visualization of rapid biodynamics.

Abnormal microcirculatory changes in arterioles, venules and capillaries are key functional indicators of important diseases such as cancer, diabetes or cardiovascular disorders, as well as several neurodegenerative ailments. In vivo visualization of these structures has traditionally been restricted to the shallow depths

Optoacoustic tomography (OAT) and magnetic resonance imaging (MRI) are both endorsed with powerful imaging capabilities. Yet, their seamless integration into a hybrid system may additionally offer highly synergistic and complementary information for concurrent anatomical and functional imaging of living organisms. Herein, we investigate on the possibility of concurrent OAT-MRI image acquisition and provide experimental validation in phantom studies. Simultaneous recording was enabled by designing an MR-compatible copper-shielded spherical ultrasound array that provided real-time volumetric OAT imaging and could be safely introduced into a high-field 9.4 T MRI scanner. The interference between the two modalities was evaluated under commonly employed FLASH and EPI sequences. Phantom experiments revealed that the OAT probe caused minimal distortion in the MRI images, while synchronization of the laser and the MRI pulse sequences enabled defining artifact-free OAT acquisition windows. Good dynamic OAT contrast from superparamagnetic iron oxide nanoparticles, a widely used agent for MRI contrast enhancement, was also observed. The hybrid OAT-MRI system thus provides an excellent platform for cross-validating functional readings of both modalities. Overall, this initial study serves to establish the technical feasibility of developing hybrid OAT-MRI system for biomedical research.

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.

Understanding brain function mechanisms relies on tools capable of simultaneous structural and functional imaging at the whole brain scale, with high spatio-temporal resolution, often not attainable in a single imaging modality. Here, we employ spherical matrix array geometry to combine optoacoustic tomography (OAT) and ultrasound localisation microscopy (ULM), and obtain dual-mode volumetric images of the whole mouse brain. Together, OAT allows functional readouts of hemodynamics and oxygen metabolism, while ULM provides structural images of the brain vasculature with a resolution of 20 μm, and flow velocity information up to 10 mm/sec, for a comprehensive characterization of microvascular architecture and function.

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.

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., Laser and Photonics Reviews, 15 (3)

Optoacoustic tomography (OAT) and magnetic resonance imaging (MRI) provide highly complementary capabilities for anatomical and functional imaging of living organisms. Herein, we investigate on the feasibility of combining both modalities to render concurrent images. This was achieved by introducing a specifically-designed copper-shielded spherical ultrasound array into a preclinical MRI scanner. Phantom experiments revealed that the OAT probe caused minimal distortion in the MRI images, while synchronization of the laser and the MRI pulse sequence enabled defining artifact-free acquisition windows for OAT. Good dynamic OAT contrast from superparamagnetic iron oxide nanoparticles, a commonly used agent for MRI contrast enhancement, was also observed. The hybrid OAT-MRI system thus provides an excellent platform for cross-validating functional readings of both modalities. Overall, this initial study serves to establish the technical feasibility of developing a hybrid OAT-MRI system for biomedical research.

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., IEEE Transactions on Medical Imaging, 39 (10), ISSN:0278-0062, ISSN:1558-254X

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. © 2020 by the authors., Applied Sciences, 10 (18), ISSN:2076-3417

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.

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.

Multispectral optoacoustic tomography (MSOT) offers the unique capability to map the distribution of spectrally distinctive endogenous and exogenous substances in heterogeneous biological tissues by exciting the sample at various wavelengths and detecting the optoacoustically-induced ultrasound waves. This powerful functional and molecular imaging capability can greatly benefit from hybridization with pulse-echo ultrasound (US), which provides additional information on tissue anatomy and blood flow. However, speed of sound variations and acoustic mismatches in the imaged object generally lead to errors in the coregistration of compounded images and loss of spatial resolution in both imaging modalities. The spatially- and wavelength-dependent light fluence attenuation further limits the quantitative capabilities of MSOT. Proper segmentation of different regions and assignment of corresponding acoustic and optical properties turns then essential for maximizing the performance of hybrid optoacoustic and ultrasound (OPUS) imaging. Particularly, accurate segmentation of the boundary of the sample can significantly improve the images rendered. Herein, we propose an automatic segmentation method based on a convolutional neural network (CNN) for segmenting the mouse boundary in a pre-clinical OPUS system. The experimental performance of the method, as characterized with the Dice coefficient metric between the network output and the ground truth (manually segmented) images, is shown to be superior than that of a state-of-the-art active contour segmentation method in a series of two-dimensional (cross-sectional) OPUS images of the mouse brain, liver and kidney regions.

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.

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., Neoplasia, 22 (9), ISSN:1522-8002, ISSN:1476-5586

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.

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.

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

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.

Radio-frequency (RF) catheter ablation is routinely used in the clinics to treat arrhythmias, neoplastic lesions and other dysfunctional tissues. It is based on scarring the target tissue via localized heating induced by medium frequency alternating current in the proximity of a metallic ablation catheter. The outcome of RF interventions strongly relies on the temperature distribution within the treated tissue as well as on the exposure time, which cannot be efficiently monitored with existing clinical tools. Optoacoustic (OA) tomography has recently been shown to provide unique capabilities for RF ablation monitoring such as high spatio-temporal resolution, hand-held operation feasibility and high sensitivity to temperature changes and tissue coagulation. However, shallow light penetration in biological tissues strongly limits the accessible regions, particularly for cardiac procedures, while internal illumination through the catheter induces strong responses in the metallic parts that hamper soft tissue visualization. Herein, we present a new endocardial catheter based on saline irrigation that delivers electric current while being transparent for light. The new design averts OA image artifacts due to the presence of metallic electrodes while retaining the ablation efficiency.

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.

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.

Background Multispectral optical imaging has the capability of resolving 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 noninvasively. 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 µm) and real-time (10 volumes/sec) three-dimensional imaging, while further providing spectroscopic 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 assessment of carotid artery disease. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Mezrich in this issue.

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., Hybridized optoacoustic ultrasound computed tomography A three-in-one imaging platform combines the advantages of each individual technique to provide whole body tomographic imaging of small animals. Developed by the group of Daniel Razansky from the University of Zurich and ETH Zurich in Switzerland and collaborators in Germany and Spain, the hybrid platform combines optoacoustic tomography with reflection and transmission mode ultrasonography. By launching ultrasound and laser pulses into tissues, the technique allows the construction of cross-sectional tomographic images that reveal fine details on organ function, tissue vascularization, reflectivity, stiffness and density. As an added value of the hybrid combination, images retrieved by one modality are also used to enhance the reconstruction quality of the other two modalities. The platform could thus be used for probing and quantifying multiple anatomical, functional and molecular properties of tissues in health and disease.

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 pre-clinical 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.

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., Applied Sciences, 9 (13), ISSN:2076-3417

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 optimised 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. Raytracing 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. This article is protected by copyright.

Biomedical photoacoustic (or optoacoustic) tomography (PAT), or more generally, photoacoustic imaging (PAI), has been an active area of study and development in the last two decades [...]

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., Light: Science & Applications, 8 (1), ISSN:2047-7538

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.

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.

Speed of Sound (SoS) maps from ultrasound tomography (UST) provide valuable quantitative information for soft tissue characterization and identification of lesions, making this technique interesting for breast cancer detection. However, due to the complexity of the processes that characterize the interaction of ultrasonic waves with matter, classic and fast tomographic algorithms such as back-projection are not suitable. Consequently, the image reconstruction process in UST is generally slow compared to other more conventional medical tomography modalities. With the aim of facilitating the translation of this technique into real clinical practice, several reconstruction algorithms are being proposed to make image reconstruction in UST to be a fast and accurate process. The geometrical acoustic approximation is often used to reconstruct SoS with less computational burden in comparison with full-wave inversion methods. In this work, we propose a simple formulation to perform on-the-flight reconstruction for UST using geometrical acoustics with refraction correction based on quadratic Bézier polynomials. Here we demonstrate that the trajectories created with these polynomials are an accurate approximation to reproduce the refracted acoustic paths connecting the emitter and receiver transducers. The method is faster than typical acquisition times in UST. Thus, it can be considered a step towards real-time reconstructions, which may contribute to its future clinical translation.

Understanding brain functionality remains an arduous task despite decades of research employing a wide array of neural recording and imaging techniques. Genetically encoded calcium indicators (GECIs) are powerful and versatile tools for tagging fast neural activity in a large number of neurons with excellent spatial localization. Here we demonstrate that functional optoacoustic neuro-tomography (FONT) enables non-invasive imaging of sensory-evoked activity in GCaMP6-expressing mouse brain in vivo, thus holding promise for large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. The FONT imaging system has volumetric temporal resolution of 10msec and spatial resolution of 150µm across an effective field of view of 2cm3 covering an entire mouse brain. However, the effective depth in the current study was restricted to the cortical areas due to the limited penetration of light at the visible wavelengths used for the GCaMP6 excitation. The stimulation protocol involving somatosensory electrical hindpaw stimulation was specifically designed to minimize the influence of hemodynamic changes indirectly associated with neuronal activity, which are much slower than the GCaMP-related calcium transients. Rapid optoacoustic signal transients were observed in the activated brain regions but not inside major blood vessels, thus allowing for a clear differentiation of the calcium-related activity from the underlying hemodynamic responses. Our study is the first to examine fast volumetric optoacoustic signatures of GECIs non-invasively in living mice, further showing that the corresponding changes of their fluorescence are directly related to the optoacoustic responses.

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.

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.

Optoacoustic imaging provides high spatial resolution and the possibility to image specific functional parameters in real-time, therefore positioning itself as a promising modality for various applications. However, despite these advantages, the applicability of real-time optoacoustic imaging is generally limited due to a relatively small field of view. With this work, we aim at presenting a path towards panoramic optoacoustic tomographic imaging without requiring additional sensors or position trackers. We propose a two-step seamless stitching method for the compounding of multiple datasets acquired with a real-time 3D optoacoustic imaging system within a panoramic scan. The employed workflow is specifically tailored to the image properties and respective challenges. A comparison of the presented alignment on in-vivo data shows a mean error of $$628 \pm 512\,\upmu \hbox {m}$$ compared to ground truth tracking data. The presented compounding scheme integrates the physical resolution of optoacoustic data and hence can provide improved contrast in comparison with other compounding approaches based on addition or averaging. The proposed method can produce optoacoustic volumes with an enlarged field of view and improved quality compared to current methods in optoacoustic imaging. However, our study also shows challenges for panoramic scans. In this view, we discuss relevant properties, challenges, and opportunities and present an evaluation of the performance of the presented approach with different input data.

Multispectral optoacoustic tomography (MSOT) is a fast-developing imaging modality, combining the high contrast from optical tissue excitation with the high resolution and penetration depth of ultrasound detection. Since light is subject to absorption and scattering when travelling through tissue, adequate knowledge of the spatial fluence distribution is required in order to ensure quantification accuracy of MSOT. In order to reduce the systematic errors in spectral recovery due to fluence and to provide a visually more homogeneous image, correction for fluence is commonly performed on reconstructed images using one of the state-of-the-art methods. These require, as input, information on illumination geometry (a priori known from the system design) as well as spatial reference of an object in a form of either a binary map (assuming uniform optical properties), or a label map, in a more complex scenario of multiple regions with different optical properties. In order to generate such a map, manual segmentation is commonly used by delineating the outer border of the mouse body or major organs present in the slice, which is a time-consuming procedure, not efficient procedure, prone to operator errors. Here we evaluate methods for semi- and fully-automatic segmentation of hybrid optoacoustic and ultrasound images and characterize the performance of the methods using quantitative metrics for evaluating medical image segmentation against the ground truth obtained by manual segmentation.

We report on the study of multi-scale tumor dynamics in mice using spiral volumetric optoacoustic tomography. The approach enables, for the first time, non-invasive observation of tumor neovascularization, oxygenation and perfusion with high spatio-temporal resolution.

We report on optoacoustic micro-tomography (OMT), a novel approach for visualizing optical contrast with microscopic resolution at up to 5mm depths and volumetric imaging speed of 100 frames per second.

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. 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. 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. 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. • 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.