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Super-resolution methods have been developed to overcome physical diffraction limits of optical and ultrasound imaging techniques. Hybrid optoacoustic imaging approaches share the rich functional and molecular contrast advantages of optical methods while additionally attaining centimeter-scale penetration into living tissues due to reduced ultrasound scattering. The application of super-resolution principles for enhancing the optoacoustic imaging performance deep in mammalian tissues has so far been impeded by the high density of strongly absorbing red blood cells. Herein, we demonstrate three-dimensional microangiography of deep mouse brain beyond the acoustic diffraction limit (in vivo compatibility and single particle sensitivity in the presence of highly absorbing blood background. Accurate mapping of the blood flow velocity within microvascular structures was also accomplished with a dedicated high-frame-rate volumetric optoacoustic tomography system. We further show that the detected optoacoustic signal intensities from the localized particles may serve for estimating the light fluence distribution within optically heterogeneous tissues, a long-standing quantification challenge in biomedical optics. Given the intrinsic sensitivity of optoacoustics to various functional, metabolic and molecular events in living tissues, this new approach paves the way for non-invasive deep-tissue microscopic observations with unrivaled resolution, contrast and speed.

Optoacoustic microscopy (OAM) retrieves anatomical and functional contrast in vivo at depths not resolvable with optical microscopy. Recent progress on reconstruction algorithms have further advanced its imaging performance to provide high lateral resolution ultimately limited by acoustic diffraction. In this work, we suggest a new broadband model-based OAM (MB-OAM) framework efficiently exploiting scanning symmetries for an enhanced performance. By capitalizing on the large detection bandwidth of a spherical polyvinylidene difluoride (PVDF) film while accurately accounting for its spatial impulse response, the new approach significantly outperforms standard OAM implementations in terms of contrast and resolution, as validated by functional in vivo experiments in mice and human volunteers. Furthermore, L1-norm regularization enabled resolving structures separated by less than the theoretical diffraction-limited resolution. This unique label-free angiographic performance demonstrates the general applicability of MB-OAM as a super-resolution deep-tissue imaging method capable of breaking through the limits imposed by acoustic diffraction.

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

Individual light pulses with durations in the nanosecond range are most often employed in biomedical optoacoustic imaging. In this excitation regime, ultra-wideband acoustic responses are generated from tissues that cannot be efficiently captured with ultrasound transducers having a limited detection bandwidth. Here, we analyse a narrowband optoacoustic signal excitation mechanism consisting of a tone-burst of multiple equally-delayed pulses. The signal generation efficiency of single-pulse versus tone-burst excitation is compared for normal light exposure levels in the linear optoacoustic signal generation regime as well as when considering non-linearities associated with temperature increase or absorption saturation. The signal-to-noise ratio of the excited signals is also experimentally compared using a highly-absorbing ink phantom.

Fluorescence microscopy is a powerful enabling tool for biological discovery, albeit its effective penetration depth and resolving capacity are limited due to intense light scattering in living tissues. The recently introduced short-wave infrared cameras and contrast agents featuring fluorescence emission in the second near-infrared (NIR-II) window have extended the achievable penetration to about 2 mm. However, the effective spatial resolution progressively deteriorates with depth due to photon diffusion. Here we introduce diffuse optical localization imaging (DOLI) to enable super-resolution deep-tissue fluorescence microscopy beyond the limits imposed by light diffusion. The method is based on localization of flowing microdroplets encapsulating lead sulfide (PbS)-based quantum dots in a sequence of epi-fluorescence images acquired in the NIR-II spectral window. Experiments performed in tissue mimicking phantoms indicate that high-resolution detection of fluorescent particles can be preserved over 4 mm depth range, while in vivo microangiography of murine cerebral vasculature can be accomplished through intact scalp and skull. The method further enables retrieving depth information from planar fluorescence image recordings by exploiting the localized spot size. DOLI operates in a resolution-depth regime previously inaccessible with optical methods, thus massively enhancing the applicability of fluorescence-based imaging techniques.

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.

The speed of sound (SoS) in the imaged sample and in the coupling medium is an important parameter in optoacoustic tomography that must be specified in order to accurately restore maps of local optical absorbance. In this work, several hybrid focusing functions are described that successfully determine the most suitable SoS based on post-reconstruction images. The SoS in the coupling medium (water) can be determined from temperature readings. Thereby, this value is suggested to be used as an initial guess for faster SoS calibration in the reconstruction of tissues having a different SoS than water.

In this article, we present a novel scheme for segmenting the image boundary (with the background) in optoacoustic small animal in vivo imaging systems. The method utilizes a multiscale edge detection algorithm to generate a binary edge map. A scale dependent morphological operation is employed to clean spurious edges. Thereafter, an ellipse is fitted to the edge map through constrained parametric transformations and iterative goodness of fit calculations. The method delimits the tissue edges through the curve fitting model, which has shown high levels of accuracy. Thus, this method enables segmentation of optoacoutic images with minimal human intervention, by eliminating need of scale selection for multiscale processing and seed point determination for contour mapping., Comment: Engineering in Medicine and Biology Society (EMBC), 2015 37th Annual International Conference of the IEEE (Accepted version)