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.