Deep tissue optoacoustic localization microangiography

Abstract 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 (<20 µm resolution) through the intact scalp and skull via optoacoustic localization of sparsely-distributed highly absorbing microparticles. This was enabled by devising 5 µm sized extremely absorbing dichloromethane microdroplets exhibiting four orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus facilitating 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.