Multimodal optoacoustic imaging: methods and contrast materials.

Optoacoustic (OA) imaging offers powerful capabilities for interrogating biological tissues with rich optical absorption contrast while maintaining high spatial resolution for deep tissue observations. The spectrally distinct absorption of visible and near-infrared photons by endogenous tissue chromophores facilitates extraction of diverse anatomic, functional, molecular, and metabolic information from living tissues across various scales, from organelles and cells to whole organs and organisms. The primarily blood-related contrast and limited penetration depth of OA imaging have fostered the development of multimodal approaches to fully exploit the unique advantages and complementarity of the method. We review the recent hybridization efforts, including multimodal combinations of OA with ultrasound, fluorescence, optical coherence tomography, Raman scattering microscopy and magnetic resonance imaging as well as ionizing methods, such as X-ray computed tomography, single-photon-emission computed tomography and positron emission tomography. Considering that most molecules absorb light across a broad range of the electromagnetic spectrum, the OA interrogations can be extended to a large number of exogenously administered small molecules, particulate agents, and genetically encoded labels. This unique property further makes contrast moieties used in other imaging modalities amenable for OA sensing.

Nanoporous Submicron Gold Particles Enable Nanoparticle-Based Localization Optoacoustic Tomography (nanoLOT).

Localization optoacoustic tomography (LOT) has recently emerged as a transformative super-resolution technique breaking through the acoustic diffraction limit in deep-tissue optoacoustic (OA) imaging via individual localization and tracking of particles in the bloodstream. However, strong light absorption in red blood cells has previously restricted per-particle OA detection to relatively large microparticles, ≈5 µm in diameter. Herein, it is demonstrated that submicron-sized porous gold nanoparticles, ≈600 nm in diameter, can be individually detected for noninvasive super-resolution imaging with LOT. Ultra-high-speed bright-field microscopy revealed that these nanoparticles generate microscopic plasmonic vapor bubbles, significantly enhancing opto-acoustic energy conversion through a nano-to-micro size transformation. Comprehensive in vitro and in vivo tests further demonstrated the biocompatibility and biosafety of the particles. By reducing the detectable particle size by an order of magnitude, nanoLOT enables microangiographic imaging with a significantly reduced risk of embolisms from particle aggregation and opens new avenues to visualize how nanoparticles reach vascular and potentially extravascular targets. The performance of nanoLOT for non-invasive imaging of microvascular networks in the murine brain anticipates new insights into neurovascular coupling mechanisms and longitudinal microcirculatory changes associated with neurodegenerative diseases.

Hybrid spherical array for combined volumetric optoacoustic and B-mode ultrasound imaging.

Optoacoustic (OA) imaging has achieved tremendous progress with state-of-the-art systems providing excellent functional and molecular contrast, centimeter scale penetration into living tissues, and ultrafast imaging performance, making it highly suitable for handheld imaging in the clinics. OA can greatly benefit from efficient integration with ultrasound (US) imaging, which remains the routine method in bedside clinical diagnostics. However, such integration has not been straightforward since the two modalities typically involve different image acquisition strategies. Here, we present a new, to our knowledge, hybrid optoacoustic ultrasound (OPUS) imaging approach employing a spherical array with dedicated segments for each modality to enable volumetric OA imaging merged with conventional B-mode US. The system performance is subsequently showcased in healthy human subjects. The new OPUS approach hence represents an important step toward establishing OA in point-of-care diagnostic settings.

Spiral volumetric optoacoustic tomography of reduced oxygen saturation in the spinal cord of M83 mouse model of Parkinson's disease.

PURPOSE: Metabolism and bioenergetics in the central nervous system play important roles in the pathophysiology of Parkinson's disease (PD). Here, we employed a multimodal imaging approach to assess oxygenation changes in the spinal cord of the transgenic M83 murine model of PD overexpressing the mutated A53T alpha-synuclein form in comparison with non-transgenic littermates. METHODS: In vivo spiral volumetric optoacoustic tomography (SVOT) was performed to assess oxygen saturation (sO2) in the spinal cords of M83 mice and non-transgenic littermates. Ex vivo high-field T1-weighted (T1w) magnetic resonance imaging (MRI) at 9.4T was used to assess volumetric alterations in the spinal cord. 3D SVOT analysis and deep learning-based automatic segmentation of T1w MRI data for the mouse spinal cord were developed for quantification. Immunostaining for phosphorylated alpha-synuclein (pS129 α-syn), as well as vascular organization (CD31 and GLUT1), was performed after MRI scan. RESULTS: In vivo SVOT imaging revealed a lower sO2SVOT in the spinal cord of M83 mice compared to non-transgenic littermates at sub-100 µm spatial resolution. Ex vivo MRI-assisted by in-house developed deep learning-based automatic segmentation (validated by manual analysis) revealed no volumetric atrophy in the spinal cord of M83 mice compared to non-transgenic littermates at 50 µm spatial resolution. The vascular network was not impaired in the spinal cord of M83 mice in the presence of pS129 α-syn accumulation. CONCLUSION: We developed tools for deep-learning-based analysis for the segmentation of mouse spinal cord structural MRI data, and volumetric analysis of sO2SVOT data. We demonstrated non-invasive high-resolution imaging of reduced sO2SVOT in the absence of volumetric structural changes in the spinal cord of PD M83 mouse model.

Ultrafast four-dimensional imaging of cardiac mechanical wave propagation with sparse optoacoustic sensing.

Propagation of electromechanical waves in excitable heart muscles follows complex spatiotemporal patterns holding the key to understanding life-threatening arrhythmias and other cardiac conditions. Accurate volumetric mapping of cardiac wave propagation is currently hampered by fast heart motion, particularly in small model organisms. Here we demonstrate that ultrafast four-dimensional imaging of cardiac mechanical wave propagation in entire beating murine heart can be accomplished by sparse optoacoustic sensing with high contrast, ∼115-µm spatial and submillisecond temporal resolution. We extract accurate dispersion and phase velocity maps of the cardiac waves and reveal vortex-like patterns associated with mechanical phase singularities that occur during arrhythmic events induced via burst ventricular electric stimulation. The newly introduced cardiac mapping approach is a bold step toward deciphering the complex mechanisms underlying cardiac arrhythmias and enabling precise therapeutic interventions.

Biobased Agents for Single-Particle Detection with Optoacoustics.

Optoacoustic (OA, photoacoustic) imaging synergistically combines rich optical contrast with the resolution of ultrasound within light-scattering biological tissues. Contrast agents have become essential to boost deep-tissue OA sensitivity and fully exploit the capabilities of state-of-the-art OA imaging systems, thus facilitating the clinical translation of this modality. Inorganic particles with sizes of several microns can also be individually localized and tracked, thus enabling new applications in drug delivery, microrobotics, or super-resolution imaging. However, significant concerns have been raised regarding the low bio-degradability and potential toxic effects of inorganic particles. Bio-based, biodegradable nano- and microcapsules consisting of an aqueous core with clinically-approved indocyanine green (ICG) and a cross-linked casein shell obtained in an inverse emulsion approach are introduced. The feasibility to provide contrast-enhanced in vivo OA imaging with nanocapsules as well as localizing and tracking individual larger microcapsules of 4-5 µm is demonstrated. All components of the developed capsules are safe for human use and the inverse emulsion approach is known to be compatible with a variety of shell materials and payloads. Hence, the enhanced OA imaging performance can be exploited in multiple biomedical studies and can open a route to clinical approval of agents detectable at a single-particle level.

Coregistered transcranial optoacoustic and magnetic resonance angiography of the human brain.

Imaging modalities capable of visualizing the human brain have led to major advances in neurology and brain research. Multi-spectral optoacoustic tomography (MSOT) has gained importance for studying cerebral function in rodent models due to its unique capability to map changes in multiple hemodynamic parameters and to directly visualize neural activity within the brain. The technique further provides molecular imaging capabilities that can facilitate early disease diagnosis and treatment monitoring. However, transcranial imaging of the human brain is hampered by acoustic attenuation and other distortions introduced by the skull. Here, we demonstrate non-invasive transcranial MSOT angiography of pial veins through the temporal bone of an adult healthy volunteer. Time-of-flight (TOF) magnetic resonance angiography (MRA) and T1-weighted structural magnetic resonance imaging (MRI) were further acquired to facilitate anatomical registration and interpretation. The superior middle cerebral vein in the temporal cortex was identified in the MSOT images, matching its location observed in the TOF-MRA images. These initial results pave the way toward the application of MSOT in clinical brain imaging.

Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography.

PURPOSE: Abnormal tau accumulation within the brain plays an important role in tauopathies such as Alzheimer's disease and frontotemporal dementia. High-resolution imaging of tau deposits at the whole-brain scale in animal disease models is highly desired. METHODS: We approached this challenge by non-invasively imaging the brains of P301L mice of 4-repeat tau with concurrent volumetric multi-spectral optoacoustic tomography (vMSOT) at ~ 115 µm spatial resolution using the tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 (i.v.). In vitro probe characterization, concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice, followed by ex vivo validation using AT-8 antibody for phosphorylated tau. RESULTS: PBB5 showed specific binding to recombinant K18 tau fibrils by fluorescence assay, to post-mortem Alzheimer's disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brains. Dose-dependent optoacoustic and fluorescence signal intensities were observed in the mouse brains following i.v. administration of different concentrations of PBB5. In vivo vMSOT brain imaging of P301L mice showed higher retention of PBB5 in the tau-laden cortex and hippocampus compared to wild-type mice, as confirmed by ex vivo vMSOT, epi-fluorescence, multiphoton microscopy, and immunofluorescence staining. CONCLUSIONS: We demonstrated non-invasive whole-brain imaging of tau in P301L mice with vMSOT system using PBB5 at a previously unachieved ~ 115 µm spatial resolution. This platform provides a new tool to study tau spreading and clearance in a tauopathy mouse model, foreseeable in monitoring tau targeting putative therapeutics.

Hemodynamic response to sensory stimulation in mice: Comparison between functional ultrasound and optoacoustic imaging.

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.

Deep learning of image- and time-domain data enhances the visibility of structures in optoacoustic tomography.

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.

In situ characterization of microparticulate optoacoustic contrast agents in an intracardiac perfusion mouse model.

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.

Optoacoustic imaging of the skin.

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.

In vivo optoacoustic monitoring of percutaneous laser ablation of tumors in a murine breast cancer model.

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 (${\sim}{5}\;{\rm ns}$∼5ns) 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.

Real-time Volumetric Assessment of the Human Carotid Artery: Handheld Multispectral Optoacoustic Tomography.

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.

Endocardial irrigated catheter for volumetric optoacoustic mapping of radio-frequency ablation lesion progression.

Radiofrequency (RF) catheter ablation is widely employed for various minimally invasive procedures, including treatment of tumors, cardiac arrhythmias and varicose veins. Accurate real-time monitoring of the ablation treatments remains challenging with the existing clinical imaging modalities due to the lack of spatial or temporal resolution or insufficient tissue contrast for differentiating thermal lesions. Optoacoustic (OA) imaging has been recently suggested for monitoring temperature field and lesion progression during RF interventions. However, strong light absorption by standard metallic catheters hindered practical implementations of this approach. Herein, we introduce a new RF ablation catheter concept for combined RF ablation and OA lesion monitoring. The catheter tip encapsulates a multimode fiber bundle for OA excitation with near-infrared (NIR) light, whereas the electric current is conducted through the irrigation solution, thus avoiding direct exposure of the metallic parts to the excitation light. We optimized the catheter diameter and the saline flow rate in order to attain uniform and deep lesions. The newly introduced hybrid catheter design was successfully tested by real-time monitoring of the ablation process in smooth ventricle and rough atrium walls of a blood-filled ex vivo porcine heart, mimicking in vivo conditions in the clinical setting.

Integrated catheter for simultaneous radio frequency ablation and optoacoustic monitoring of lesion progression.

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

Hybrid system for in vivo epifluorescence and 4D optoacoustic imaging.

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

Noncontact monitoring of incision depth in laser surgery with air-coupled ultrasound transducers.

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

Simultaneous visualization of tumour oxygenation, neovascularization and contrast agent perfusion by real-time three-dimensional optoacoustic tomography.

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

High-contrast imaging of reversibly switchable fluorescent proteins via temporally unmixed multispectral optoacoustic tomography.

Photocontrol of reversibly switchable fluorescent proteins (RSFPs) was used to program optoacoustic signal time courses that were temporally unmixed to increase the proteins' contrast-to-noise-ratios (CNRs) in optoacoustic imaging. In this way, two variants of the RSFP Dronpa with very similar optoacoustic spectra could be readily discriminated in the presence of highly absorbing blood. Addition of temporal unmixing to multispectral optoacoustic tomography (tuMSOT) in conjunction with synthetic or genetically encoded photochromic contrast agents and customized photoswitching schedules can increase the performance of multiplexed and high-contrast molecular optoacoustic imaging.