Ultrasound-Aided Large-Scale Optoacoustic Microscopy for Volumetric Angiography and Oximetry

Given its direct relationship to tissue metabolism and various pathological processes, 3D mapping of blood oxygen saturation (sO₂) is essential for advancing our knowledge on oxygen delivery to tissues and evaluating therapeutic efficacy. Optoacoustic microscopy has enabled label-free estimation of sO₂ values by exploiting the spectrally distinctive absorption of hemoglobin in its oxygenated and deoxygenated forms. However, quantitative 3D mapping of sO₂ distribution over large heterogenous tissue regions is commonly hindered due to the strong spatial and spectral variability of the excitation light fluence. Herein, we capitalize on hybridization between pulse-echo ultrasound and large-scale spectroscopic optoacoustic microscopy readings to accurately delineate the tissue surface, achieve depth-resolved tissue layer segmentation, and comprehensively evaluate the main causes behind inaccurate sO₂ estimations with optoacoustic microscopy. Compensation for wavelength-dependent light fluence variations due to relative reflectance and attenuation through multiple tissue layers is further shown to remove spectral noise and restore physiologically relevant sO₂ values in the images recorded from the mouse ear and the dorsal murine skin. The ultrasound-aided large-scale optoacoustic microscopy (uLSOM) approach is thus expected to enhance applicability of optoacoustic microscopy for quantitative label-free imaging of tissue oxygenation and metabolism.