Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of quantifying the cerebral hemodynamics and oxygen metabolism at the microscopic level. However, the limited depth of focus of conventional PAM is insufficient to encompass the depth variation of the mouse brain when imaging a large area. For instance, the surface contour of the mouse cortex is dome-shaped and spans several hundred microns along the depth direction. When out of focus, the resolution and sensitivity of PAM quickly degrades. Moreover, quantitative measurements (e.g., blood oxygenation and flow) are no longer accurate with the compromised resolution and sensitivity. Here, we report automated contour-scan multi-parametric PAM, which enables simultaneous imaging of blood perfusion, oxygenation and flow with high resolution and sensitivity over the entire mouse cortex. Different from the traditional contour-scan method that requires three steps (pre-scan, off-line calculation of the contour map, and contour scan), our technique can perform high-resolution wide-field contour scan without the first two steps, thereby significantly reducing the acquisition time. We first tested the feasibility of this technique by imaging a plastic ball coated with black ink. Then, we quantitatively analyzed the influence of out-of-focus on the measurement of blood flow in a vessel-mimicking phantom. Finally, we demonstrated cortex-wide multi-parametric PAM in the live mouse brain with high resolution and sensitivity.
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