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.

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.

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.

Which clinical features best predict occult scaphoid fractures? A systematic review of diagnostic test accuracy studies.

BACKGROUND: Plain radiographs cannot identify all scaphoid fractures; thus ED patients with a clinical suspicion of scaphoid injury often undergo immobilisation despite normal imaging. This study determined (1) the prevalence of scaphoid fracture among patients with a clinical suspicion of scaphoid injury with normal radiographs and (2) whether clinical features can identify patients that do not require immobilisation and further imaging. METHODS: This systematic review of diagnostic test accuracy studies included all study designs that evaluated predictors of scaphoid fracture among patients with normal initial radiographs. Quality assessment was undertaken using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Meta-analyses included all studies. RESULTS: Eight studies reported data on 1685 wrist injuries. The prevalence of scaphoid fracture despite normal radiographs was 9.0%. Most studies were at overall low risk of bias but two were at unclear risk; all eight were at low risk for applicability concerns. The most accurate clinical predictors of occult scaphoid fracture were pain when the examiner moved the wrist from a pronated to a supinated position against resistance (sensitivity 100%, specificity 97.9%, LR+ 45.0, 95% CI 6.5 to 312.5), supination strength <10% of contralateral side (sensitivity 84.6%, specificity 76.9%, LR+ 3.7, 95% CI 2.2 to 6.1), pain on ulnar deviation (sensitivity 55.2%, specificity 76.4%, LR+ 2.3, 95% CI 1.8 to 3.0) and pronation strength <10% of contralateral side (sensitivity 69.2%, specificity 64.6%, LR+ 2.0, 95% CI 1.2 to 3.2). Absence of anatomical snuffbox tenderness significantly reduced the likelihood of an occult scaphoid fracture (sensitivity 92.1%, specificity 48.4%, LR- 0.2, 95% CI 0.0 to 0.7). CONCLUSION: No single feature satisfactorily excludes an occult scaphoid fracture. Further work should explore whether a combination of clinical features, possibly in conjunction with injury characteristics (such as mechanism) and a normal initial radiograph might exclude fracture. Pain on supination against resistance would benefit from external validation. TRIAL REGISTRATION NUMBER: CRD42021290224.

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.

Shearwaters know the direction and distance home but fail to encode intervening obstacles after free-ranging foraging trips.

While displacement experiments have been powerful for determining the sensory basis of homing navigation in birds, they have left unresolved important cognitive aspects of navigation such as what birds know about their location relative to home and the anticipated route. Here, we analyze the free-ranging Global Positioning System (GPS) tracks of a large sample (n = 707) of Manx shearwater, Puffinus puffinus, foraging trips to investigate, from a cognitive perspective, what a wild, pelagic seabird knows as it begins to home naturally. By exploiting a kind of natural experimental contrast (journeys with or without intervening obstacles) we first show that, at the start of homing, sometimes hundreds of kilometers from the colony, shearwaters are well oriented in the homeward direction, but often fail to encode intervening barriers over which they will not fly (islands or peninsulas), constrained to flying farther as a result. Second, shearwaters time their homing journeys, leaving earlier in the day when they have farther to go, and this ability to judge distance home also apparently ignores intervening obstacles. Thus, at the start of homing, shearwaters appear to be making navigational decisions using both geographic direction and distance to the goal. Since we find no decrease in orientation accuracy with trip length, duration, or tortuosity, path integration mechanisms cannot account for these findings. Instead, our results imply that a navigational mechanism used to direct natural large-scale movements in wild pelagic seabirds has map-like properties and is probably based on large-scale gradients.

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.

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.

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.

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.

Acoustic Scattering Mediated Single Detector Optoacoustic Tomography.

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

Post-transplant malignancy in solid organ transplant recipients in Ireland, The Irish Transplant Cancer Group.

OBJECTIVE: Solid organ transplant recipients are at increased risk of cancer compared to the general population. To date, this risk in Ireland has not been investigated. We conducted a national registry study of cancer incidence following solid organ transplantation. METHODS: National centers for solid organ transplantation supplied their respective registry databases to cross-reference with episodes of malignancy from the National Cancer Registry Ireland (NCRI) between 1994 and 2014. Standardized incidence of cancer post-transplant was compared to the general population by means of standardized incidence ratios (SIRs), and between solid organ transplant types by incidence rate ratios. RESULTS: A total of 3346 solid organ transplant recipients were included in this study. Kidney transplant recipients constituted the majority of participants (71.2%), followed by liver (16.8%), heart (6.4%), and lung (5.6%) transplants. The most common cancers within the composite of all transplant recipients included the following (SIR [95% CI]): squamous and basal cell carcinoma (20.05 [17.97, 22.31] and 7.16 [6.43, 7.96], respectively), non-Hodgkin lymphoma (6.23 [4.26, 8.59]), and renal cell carcinoma (3.36 [1.96, 5.38]). CONCLUSIONS: This study reports the incidence of cancer following solid organ transplantation in Ireland. These results have significant national policy implications for surveillance, and early diagnosis in this patient group.

Calcium Sensor for Photoacoustic Imaging.

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

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.

Advanced optoacoustic methods for multiscale imaging of in vivo dynamics.

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