Multimodal photoacoustic-fluorescence microscopy for quantitative absorption coefficient mapping and precision lesion tracking in disease models

We present an integrated multimodal imaging system combining optical-resolution photoacoustic microscopy (OR-PAM) and fluorescence microscopy for quantitative mapping of optical absorption coefficients (${\mu }_a$) in biological tissues. Our platform synergistically merges the molecular specificity of fluorescence imaging with OR-PAM’s depth-resolved quantitative capabilities, enabling precise lesion localization and physiological monitoring. Our system features a novel reconstruction algorithm that integrates Monte Carlo light transport simulations with custom OR-PAM, enabling absolute ${\mu }_a$ determination − a capability unattainable with fluorescence microscopy and distinct from conventional OR-PAM approaches. The simulation experiment measured the optical absorption coefficient of blood vessels at 532 nm as 238 cm^–1, showing a 3.4 % deviation from the theoretical value of 230.5 cm^–1 (data sourced from the MCML software package). Experimental validation across three model systems demonstrated its diagnostic potential: (1) inflammatory monitoring revealed a 7.9 % ${\mu }_a$ increase at infection sites (258 vs. 239 cm^–1 in normal vasculature, p < 0.001) within 12 h; (2) tumor tracking showed 5.5 % higher ${\mu }_a$ in tumor vasculature (252 vs. 237 cm^–1); and (3) cerebral vascular imaging achieved ${\mu }_a$ quantification (235 cm^–1) following blood–brain barrier opening. These results establish that fluorescence microscopy provides exceptional molecular tracking capability, while OR-PAM delivers quantitative ${\mu }_a$ differences critical for distinguishing pathological tissues, offering a powerful tool for both fundamental research and clinical diagnostics.