Optical fluence-compensated functional optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) can image the blood oxygen saturation (sO₂) in vivo without labeling. OR-PAM assumes a linear relationship between the photoacoustic amplitude and the optical absorption coefficient and ignores the wavelength-dependent optical fluence attenuation in tissue. However, strong scattering in biological tissues may significantly change the optical energy deposition, leading to inaccurate sO₂ measurement. Here, we report fluence-compensated OR-PAM to correct the sO₂ imaging. In a narrow optical spectrum, we assume the scattered fluence is linearly related to the optical wavelength. Using three optical wavelengths, we can compensate for the scattering-induced photoacoustic signal change and thus improve the accuracy of sO₂ measurement. We use a Monta Carlo model to validate the linear assumption of the scattered fluence. In in vivo experiments, we demonstrate that the optical fluence compensation can effectively improve the sO₂ accuracy. The compensated arterial sO₂ values are in the range of 0.95 ∼ 0.99, which is consistent with normal physiological values. Compared with the uncompensated ones, the accuracy has been improved greatly. Enabled by the accurate sO₂ imaging tool, we can reliably observe the sO₂ gradient in the vascular network. We expect this new technique will further broaden the preclinical and clinical applications of functional OR-PAM.