Maximum fluence for accurate functional photoacoustic microscopy

Photoacoustic microscopy (PAM) can map blood oxygen saturation (sO₂) in the brain and other organs at high spatial and temporal resolution, enabling many preclinical and clinical applications. In fast functional PAM, even when the laser power is within the safety limit, transient local thermal accumulation might adversely affect the accuracy of sO₂ measurement, which, however, has not been thoroughly studied. Here, we investigated the relationship between laser irradiation and sO₂ accuracy and established a numerical model to estimate the maximum laser fluence in typical fast functional PAM. In vivo experiments show that, when using 532-nm and 558-nm wavelengths for functional imaging, the maximum fluence ( $F_{\max }$ ) for accurate sO₂ measurement decreases with repetitive laser pulse number (N) on one spot as $F_{\max }=45N^{-0.41}$ mJ·cm^-2. We validated the model in a blood phantom and the mouse brain and tested its stability under long-term scanning. The maximum-fluence model provides practical guidance for selecting laser fluence in fast functional PAM.