Cortex-wide multiparametric photoacoustic microscopy based on real-time contour scanning

Large-scale, high-resolution imaging of cerebral hemodynamics is essential for brain research. Uniquely capable of comprehensive quantification of cerebral hemodynamics and oxygen metabolism in rodents based on the endogenous hemoglobin contrast, multiparametric photoacoustic microscopy (PAM) is ideally suited for this purpose. However, the out-of-focus issue due to the uneven surface of the rodent brain results in inaccurate PAM measurements and presents a significant challenge to cortex-wide multiparametric recording. We report a large-scale, high-resolution, multiparametric PAM system based on real-time surface contour extraction and scanning, which avoids the prescan and offline calculation of the contour map required by previously reported contour-scanning strategies. The performance of this system has been demonstrated in both phantoms and the live mouse brain through a thinned-skull window. Side-by-side comparison shows that the real-time contour scanning not only improves the quality of structural images by addressing the out-of-focus issue but also ensures accurate measurements of the concentration of hemoglobin (C^Hb), oxygen saturation of hemoglobin (sO²), and cerebral blood flow (CBF) over the entire mouse cortex. Furthermore, quantitative analysis reveals how the out-of-focus issue impairs the measurements of C^Hb, sO², and CBF.