Label-free skin penetration analysis using time-resolved, phase-modulated stimulated Raman scattering microscopy

Skin penetration analysis of topically applied drugs or active compounds is essential in biomedical applications. Stimulated Raman scattering (SRS) microscopy is a promising label-free skin penetration analysis tool. However, conventional SRS microcopy suffers from limited signal contrast owing to strong background signals, which prevents its use in low-concentration drug imaging. Here, we present a skin penetration analysis method of topical agents using recently developed phase-modulated SRS (PM-SRS) microscopy. PM-SRS uses phase modulation and time-resolved signal detection to suppress both nonlinear background signals and Raman background signals from a tissue. A proof-of-concept experiment with a topically applied skin moisturizing agent (ectoine) in an in vitro skin tissue model revealed that PM-SRS with 1.7-ps probe delay yields a signal contrast 40 times higher than that of conventional amplitude-modulated SRS (AM-SRS). Skin penetration measurement of a topical therapeutic drug (loxoprofen sodium) showed that the mean drug concentration at the tissue surface layer after 240 min was 47.3 ± 4.8 mM. The proposed PM-SRS microscopy can be employed to monitor the spatial and temporal pharmacokinetics of small molecules in the millimolar concentration regime.