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

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor control, motor learning and executive function. Central to BG function are striatal medium spiny neurons (MSNs) expressing D1 and D2 dopamine receptors. D1 and D2 MSNs are typically considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. While their opposite role is well documented for certain sensorimotor loops of the BG-thalamocortical network, it is unclear whether MSNs maintain a uniform functional role across the striatum and which influence they exert on brain areas outside the BG. Here, we addressed these questions by combining optogenetic activation of D1 and D2 MSNs in the mouse ventrolateral caudoputamen (vl CPu) with whole-brain functional MRI (fMRI) recordings. Neuronal excitation of either cell population in the vl CPu evoked distinct activity patterns in key regions of the BG-thalamocortical network including the pallidum, thalamus and motor cortex. Importantly, we report that striatal D1 and D2 MSN stimulation differentially engaged cerebellar and prefrontal regions. We characterised these long-range interactions by computational modelling of effective connectivity and confirmed that changes in D1 / D2 output drive functional relationships between regions within and beyond the BG. These results suggest a more complex functional organization of MSNs across the striatum than previously anticipated and provide evidence for the existence of an interconnected fronto - BG - cerebellar network modulated by striatal D1 and D2 MSNs.Graphical Abstract

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.

We present a fast, multispectral acoustic resolution optoacoustic microscope using a new burst-mode triggering scheme. Three pulsed laser sources are combined to retrieve spectral images across large field-of-views extending over 25mm by 25mm at 28μm lateral and 14μm axial resolution with an overfly scan of a few minutes. Highly sensitive PVDF transducer allows detection of structures 3.8mm below the human skin surface with per pulse energies of only 25μJ. The newly developed system overcomes limitations of previously reported scanning optoacoustic microscopy and mesoscopy implementations, offering a major leap forward in terms of clinical usability, laser safety, effective penetration depth and spectral unmixing capabilities.

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.

Acoustic-resolution optoacoustic microscopy (AR-OAM) visualizes internal tissue structures at millimeter to centimeter scale depths with high spatial resolution. The imaging performance mainly depends on the geometry and detection characteristics of the ultrasound transducer. Reconstruction methods incorporating transducer effects are essential to optimize achievable resolution, contrast and overall image quality. Model-based (MB) reconstruction has been shown to provide excellent imaging performance in several optoacoustic embodiments, due to its capacity to accurately model the transducer. However, the applicability of MB reconstruction methods in AR-OAM has been hampered by the high computational cost. Here, we propose an efficient MB reconstruction framework for largescale AR-OAM by considering scanning symmetries, which enabled capitalizing the computational power of a graphics processing unit. The suggested MB reconstruction method is shown to significantly improve the imaging performance of AR-OAM compared to synthetic aperture focusing technique, as validated in in vivo mouse skin experiment.

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

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

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

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.

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

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.

The highly complementary information provided by multispectral optoacoustics and pulse-echo ultrasound have recently prompted development of hybrid imaging instruments bringing together the unique contrast advantages of both modalities. In the hybrid optoacoustic ultrasound (OPUS) combination, images retrieved by one modality may further be used to improve the reconstruction accuracy of the other. In this regard, image segmentation plays a major role as it can aid improving the image quality and quantification abilities by facilitating modeling of light and sound propagation through the imaged tissues and surrounding coupling medium. Here, we propose an automated approach for surface segmentation in whole-body mouse OPUS imaging using a deep convolutional neural network (CNN). The method has shown robust performance, attaining accurate segmentation of the animal boundary in both optoacoustic and pulse-echo ultrasound images, as evinced by quantitative performance evaluation using Dice coefficient metrics.

Abnormal tau accumulation plays an important role in Alzheimer’s disease and frontotemporal dementia. There is a gap in preclinical high-resolution imaging of tau. Here we showed non-invasive whole-brain optoacoustic tau imaging in P301L tau mice.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor control, motor learning and executive function. Central to BG function are striatal medium spiny neurons (MSNs) expressing D1 and D2 dopamine receptors. D1 and D2 MSNs are typically considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. While their opposite role is well documented for certain sensorimotor loops of the BG-thalamocortical network, it is unclear whether MSNs maintain a uniform functional role across the striatum and which influence they exert on brain areas outside the BG. Here, we addressed these questions by combining optogenetic activation of D1 and D2 MSNs in the mouse ventrolateral caudoputamen (vl CPu) with whole-brain functional MRI (fMRI) recordings. Neuronal excitation of either cell population in the vl CPu evoked distinct activity patterns in key regions of the BG-thalamocortical network including the pallidum, thalamus and motor cortex. Importantly, we report that striatal D1 and D2 MSN stimulation differentially engaged cerebellar and prefrontal regions. We characterised these long-range interactions by computational modelling of effective connectivity and confirmed that changes in D1 / D2 output drive functional relationships between regions within and beyond the BG. These results suggest a more complex functional organization of MSNs across the striatum than previously anticipated and provide evidence for the existence of an interconnected fronto - BG - cerebellar network modulated by striatal D1 and D2 MSNs.

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., NeuroImage, 237, ISSN:1053-8119, ISSN:1095-9572

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.

Background The abnormal deposition of fibrillar beta‐amyloid deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease. Currently 3D imaging using positron emission tomography for plaque visualization is of a limited resolution (1 mm) in relation to the size of mouse brain in widely used AD models. Method We developed a novel high‐resolution non‐invasive volumetric multi‐spectral optoacoustic tomography (vMSOT) of 100 mm resolution using amyloid probe AOI987 to visualize amyloid‐beta distribution in arcAβ and APP/PS1 transgenic mouse models of cerebral amyloidosis. Immunohistochemical staining was performed with AOI987, anti‐Aβ antibody 6E10 and fibrillar amyloid conformation antibody OC on mouse brain sections. Result In vivo vMSOT detects higher Aβ load in the cortex, hippocampus and thalamus of arcAβ mice, and in the cortex of APP/PS1 mice compared to non‐transgenic littermates, corresponding with immunohistochemical staining results in mouse brain sections. Confocal microscopy showed co‐localization of AOI987, 6E10 and OC to parenchymal and cerebral amyloid angiopathy in brain tissue sections from arcAb and APP/PS1 mice, thus verifying the specificity of the vMSOT amyloid imaging approach. Conclusion We demonstrate a new high‐resolution in vivo 3D amyloid imaging platform across the murine brain in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ.

Background Tau abnormal aggregates have been visualized in‐vivo using intravital microscopy techniques with high resolution, but are limited to a small field‐of‐view and depth. Here, we report a new method for the transcranial detection of tau deposits accross the brain with 6 μm resolution. Method In vitro Thioflavin T fluorescence assay was performed to screen fluorescence imaging probes that bind to tau fibrils. P301L (Thy1.2) mouse models of frontal temporal lobe dementia with four repeat tau and non‐transgenic littermates were imaged in vivo using a novel large‐field multifocal illumination (LMI) fluorescence microscopy technique using luminescent conjugated oligothiophenes h‐FTAA. Immunohistochemical staining were performed on P301L mouse brain tissue sections using anti‐phosphorylated tau antibodies AT8 and AT100. Result In vitro Thioflavin T fluorescence assay and LMI imaging shows that h‐FTAA bind to recombinant full‐length tau fibrils. In vivo LMI imaging using h‐FTAA showed higher retention in the cortex of P301L mice at 10 month‐of‐age compared to age‐matched non‐transgenic littermates. Immunohistochemical staining on P301L mouse brain tissue sections showed co‐localization of h‐FTAA with anti‐phosphorylated tau antibodies AT8 and AT100, thus verifying the specificity of the probe to tauopathy in P301L mice. Conclusion We demonstrate a new in vivo high‐resolution imaging platform for detection of tauopathy in murine models of frontal temporal lobe tauopathy.

Background The abnormal deposition of beta‐amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimer’s disease. Currently available intravital microscopy techniques for high‐resolution plaque visualization commonly involve highly invasive procedures and are limited to small field‐of‐views within the rodent brain. Method We devised a large‐field multi‐focal illumination (LMI) fluorescence microscopy method that provides a 20 × 20 mm field‐of‐view and an axial resolution of ∼15 mm. In vivo and ex vivo transcranial LMI fluorescence microscopy and wide‐field fluorescence microscopy for amyloid deposits were performed in APP/PS1 and arcAb mouse models of Alzheimer’s disease amyloidosis using luminescent conjugated oligothiophene probe HS‐169. Immunohistochemical staining with HS‐169, anti‐Ab antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections Result LMI fluorescence microscopy detected amyloid‐beta deposits at a single plaque level in APP/PS1 and arcAb mice with high sensitivity and specificity. The contrast‐to‐noise ratio was approximately 24 times higher in LMI fluorescence microscopy of amyloid deposits compared to wide‐field fluorescence microscopy. Immunohistochemical staining showed HS‐169 co‐localized with 6E10 and OC stained compact parenchymal and vessel‐associated amyloid deposits. Conclusion The novel LMI in vivo amyloid imaging platform offers new prospects for studies into Alzheimer’s disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

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.

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

We demonstrate the feasibility of a compact real-time 3D optoacoustic (OA) imager employing a novel low-cost software-defined ultrasound digital acquisition platform. It supports simultaneous signal acquisition from up to 192 ultrasound channels and a direct optical link (2x 100G Ethernet) to the host-PC for high-frame-rate image acquisitions. Real-time 3D imaging experiments with light-absorbing phantoms and the wrist of a healthy volunteer are reported. These results pave the way toward a new generation of compact, affordable, and flexible hand-held OA scanners.

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 characterize curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography (MSOT) and fluorescence imaging of brain Aβ deposits in the arcAβ mouse model of AD cerebral amyloidosis. CRANAD-2 shows a specific and quantitative detection of A fibrilsin vitro,even in complex mixtures, and it is capable to distinguish between monomeric and fibrillar forms of A.In vivoepifluorescence and MSOT after intravenous CRANAD-2 administration demonstrated higher 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 fluorescence- and MSOT-based detection of Aβ deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ deposits.

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

Optoacoustic (OA) imaging has the capacity to effectively bridge the gap between macroscopic and microscopic realms in biological imaging. High-resolution OA microscopy has so far been performed via point-by-point scanning with a focused laser beam, thus greatly restricting the achievable imaging speed and/or field of view. Herein we introduce multifocal structured illumination OA microscopy (MSIOAM) that attains real-time 3D imaging speeds. For this purpose, the excitation laser beam is shaped to a grid of focused spots at the tissue surface by means of a beamsplitting diffraction grating and a condenser and is then scanned with an acousto-optic deflector operating at kHz rates. In both phantom and in vivo mouse experiments, a 10 mm wide volumetric field of view was imaged with 15 Hz frame rate at 28 μm spatial resolution. The proposed method is expected to greatly aid in biological investigations of dynamic functional, kinetic, and metabolic processes across multiple scales., Light: Science & Applications, 9 (1), ISSN:2047-7538

Imaging of cerebral vasculature is impeded with the existing fluorescence microscopy methods due to intense light scattering in living tissues and the need for highly invasive craniotomy procedures to resolve structures on a capillary scale. We propose a widefield fluorescence localization microscopy technique for high-resolution transcranial imaging and quantitative assessment of cortical perfusion in mice. The method is based on tracking single fluorescent microparticles sparsely distributed in the blood stream using a simple CMOS camera and a continuous-wave laser source. We demonstrate quantitative transcranial in vivo mapping of the blood flow velocity and direction at capillary level resolution (5 µm) across the entire cortex. The new technique opens a new high-resolution transcranial window into the brain function in health and disease.

The present chapter summarizes progress with optical methods that go beyond human vision. The focus is on two particular technologies: fluorescence molecular imaging and optoacoustic (photoacoustic) imaging. The rationale for the selection of these two methods is that in contrast to optical microscopy techniques, both fluorescence and optoacoustic imaging can achieve large fields of view, i.e., spanning several centimeters in two or three dimensions. Such fields of views relate better to human vision and can visualize large parts of tissue, a necessary premise for clinical detection. Conversely, optical microscopy methods only scan millimeter-sized dimensions or smaller. With such operational capacity, optical microscopy methods need to be guided by another visualization technique in order to scan a very specific area in tissue and typically only provide superficial measurements, i.e., information from depths that are of the order of 0.05-1 mm. This practice has generally limited their clinical applicability to some niche applications, such as optical coherence tomography of the retina. On the other hand, fluorescence molecular imaging and optoacoustic imaging emerge as more global optical imaging methods with wide applications in surgery, endoscopy, and non-invasive clinical imaging, as summarized in the following. The current progress in this field is based on a volume of recent review and other literature that highlights key advances achieved in technology and biomedical applications. Context and figures from references from the authors of this chapter have been used here, as it reflects our general view of the current status of the field.

Current intravital microscopy techniques visualize tauopathy with high-resolution, but have a small field-of-view and depth-of-focus. Herein, we report a transcranial detection of tauopathy over the entire cortex of P301L tauopathy mice using large-field multifocal illumination (LMI) fluorescence microscopy technique and luminescent conjugated oligothiophenes. In vitro assays revealed that fluorescent ligand h-FTAA is optimal for in vivo tau imaging, which was confirmed by observing elevated probe retention in the cortex of P301L mice compared to non-transgenic littermates. Immunohistochemical staining further verified the specificity of h-FTAA to detect tauopathy in P301L mice. The new imaging platform can be leveraged in pre-clinical mechanistic studies of tau spreading and clearance as well as longitudinal monitoring of tau targeting therapeutics.

Myocardial healing following myocardial infarction (MI) is a complex process that is yet to be fully understood. Clinical attempts in regeneration of the injured myocardium using cardiac stem cells faced major challenges, calling for a better understanding of the processes involved at a more basic level in order to foster translation.We examined the feasibility of volumetric optoacoustic tomography (VOT) in studying healing of the myocardium in different models of MI, including permanent occlusion (PO) of the left coronary artery, temporary occlusion (ischemia-reperfusion-I/R) and infarcted c-kit mutants, a genetic mouse model with impaired cardiac healing. Murine hearts were imaged at 100 Hz frame rate using 800 nm excitation wavelength, corresponding to the peak absorption of indocyanine green (ICG) in plasma and the isosbestic point of haemoglobin.The non-invasive real-time volumetric imaging capabilities of VOT have allowed the detection of significant variations in the pulmonary transit time (PTT), a parameter affected by MI, across different murine models. Upon intravenous injection of ICG, we were able to track alterations in cardiac perfusion in I/R models, which were absent in wild-type (wt) PO or kitClear differences in the PTT, ICG perfusion and cardiac cycle patterns were identified between the different models and days post MI. Overall, the results highlight the unique capacity of VOT for multi-parametric characterization of morphological and functional changes in murine models of MI.

Promising new developments include the introduction of near-infrared-shifted fluorescent molecules that can be used for in vivo labeling of deep tissue functional and molecular processes. Due to versatility and wide availability of optical molecular agents, functional and molecular imaging studies can be done using extrinsic contrast from, e.g. reporter genes or targeted biomarkers that can be genetically expressed or accumulated in a cell- or tissue-specific manner. Despite its great promise, similarly to ultrasonography, optoacoustic imaging possesses several limitations related to imaging through acoustically mismatched areas, such as lungs, skull, and bones. The initial goal of optoacoustic imaging and tomography is retrieval of the absorbed optical energy density inside the object. The highly heterogeneous nature of biological tissues may further lead to the appearance of significant image artifacts and compromise imaging performance and quantification. The true multi-scale imaging capabilities of optoacoustics can be better appreciated when comparing its dynamic imaging performance with other neuroimaging modalities.

We report on a new small animal imaging platform for concomitant noninvasive mapping of the absorbed optical energy, acoustic reflectivity, speed of sound and acoustic attenuation in whole mice with submillimeter resolution. In vivo mouse imaging experiments revealed fine details on the organ parenchyma, vascularization, tissue reflectivity, density and stiffness. The newly developed synergistic multimodal combination offers unmatched capabilities for imaging diverse tissue properties and biomarkers with high resolution, penetration and contrast.

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

Deposition of beta-amyloid (Aβ) deposits is one major histopathological hallmark of Alzheimer’s disease (AD). Here, we introduce volumetric multi-spectral optoacoustic tomography (vMSOT), which covers 10×10×10 mm3 field-of-view, capable of 3D whole mouse brain imaging. We show for the first time the optoacoustic properties of oxazine-derivative AOI987 probe, which binds to Aβ, and the application of vMSOT for the quantification of brain-wide Aβ deposition. Administration of AOI987 to two common transgenic mouse strains of AD amyloidosis led to a retention of the probe in Aβ-laden brain regions. Co-registered of vMSOT data to a brain atlas revealed strain-specific pattern of AOI987 uptake. A comparison with ex vivo light-sheet microscopy in cleared mouse brains showed a good correspondence in Aβ distribution. Lastly, we demonstrate the specificity of the AOI987 probe by immunohistochemistry. vMSOT with AOI987 facilitates preclinical brain region-specific studies of Aβ spread and accumulation, and the monitoring of putative treatments targeting Aβ.

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.

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

The abnormal deposition of beta-amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimer’s disease. Currently available intravital microscopy techniques for high-resolution plaque visualization commonly involve highly invasive procedures and are limited to a small field-of-view within the rodent brain. Here, we report the transcranial detection of amyloid-beta deposits at the whole brain scale with 20 μm resolution in APP/PS1 and arcAβ mouse models of Alzheimer’s disease amyloidosis using a large-field multifocal (LMI) fluorescence microscopy technique. Highly sensitive and specific detection of amyloid-beta deposits at a single plaque level in APP/PS1 and arcAβ mice was facilitated using luminescent conjugated oligothiophene HS-169. Immunohistochemical staining with HS-169, anti-Aβ antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections further showed that HS-169 resolved compact parenchymal and vessel-associated amyloid deposits. The novel imaging platform offers new prospects for in vivo studies into Alzheimer’s disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

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.

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

Scanning optical microscopy techniques are commonly restricted to a sub‐millimeter field‐of‐view (FOV) or otherwise employ slow mechanical translation, limiting their applicability for imaging fast biological dynamics occurring over large areas. A rapid scanning large‐field multifocal illumination (LMI) fluorescence microscopy technique is devised based on a beam‐splitting grating and an acousto‐optic deflector synchronized with a high‐speed camera to attain real‐time fluorescence microscopy over a centimeter‐scale FOV. Owing to its large depth of focus, the approach allows noninvasive visualization of perfusion across the entire mouse cerebral cortex, not achievable with conventional wide‐field fluorescence microscopy methods. The new concept can readily be incorporated into conventional wide‐field microscopes to mitigate image blur due to tissue scattering and attain optimal trade‐off between spatial resolution and FOV. It further establishes a bridge between conventional wide‐field macroscopy and laser scanning confocal microscopy, thus it is anticipated to find broad applicability in functional neuroimaging, in vivo cell tracking, and other applications looking at large‐scale fluorescent‐based biodynamics., Laser & Photonics Reviews, 14 (2), ISSN:1863-8880, ISSN:1863-8899

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

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., Neoplasia, 22 (12), ISSN:1522-8002, ISSN:1476-5586

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.