Label-free human skin imaging with enhanced molecular contrast via time-resolved fluorescence and advanced phasor analysis.

Current state-of-the-art clinical skin imaging using label-free multiphoton microscopy (MPM) faces challenges due to limited molecular specificity, which hampers the accurate characterization of skin tissues because of overlapping fluorescence signals from multiple molecular components. In this study, we present a novel approach to enhance molecular contrast in MPM clinical skin imaging by leveraging advanced strategies to effectively unmix the various endogenous fluorophores present in the skin with the performance capabilities of a recently developed imaging platform for in vivo time-resolved fluorescence imaging of human skin. By identifying phasor positions of key endogenous skin fluorophores - such as keratin, melanin, free NADH, and protein-bound NADH - we effectively perform multicomponent unmixing in different skin types and conditions, including those with varying levels of pigmentation and metabolic states. The phasor analysis allows for the mapping and quantification of individual fluorescence species and provides comprehensive insight into the dynamic changes in molecular components associated with distinct clinical conditions. This study highlights the effective use of advanced imaging and phasor analysis to improve label-free molecular contrast in clinical skin imaging, leading to advancements in future research focused on precise and timely assessments of skin conditions and monitoring of therapeutic effects.