Self-fluence-compensated functional photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) can image blood oxygen saturation (sO2) in vivo with high resolution and excellent sensitivity and offers a great tool for neurovascular study and early cancer diagnosis. OR-PAM ignores the wavelength-dependent optical attenuation in superficial tissue, which cause errors in sO2 imaging. Monte Carlo simulation shows that variations in imaging depth, vessel diameter, and focal position can cause up to ~60% decrease in sO2 imaging. Here, we develop a self-fluence-compensated OR-PAM to compensate for the wavelength-dependent fluence attenuation. We propose a linearized model to estimate the fluence attenuations and use three optical wavelengths to compensate for them in sO2 calculation. We validate the model in both numerical and physical phantoms and show that the compensation method can effectively reduce the sO2 errors. In functional brain imaging, we demonstrate that the compensation method can effectively improve sO2 accuracy, especially in small vessels. Compared with uncompensated ones, the sO2 values are improved by 10~30% in the brain. We monitor ischemic-stroke-induced brain injury which demonstrates great potential for the preclinical study of vascular diseases.