Time‐course quantitative mapping of caffeine within the epidermis, using high‐contrast pump–probe stimulated Raman scattering microscopy

BACKGROUND: An assessment of the drug penetration and distribution profiles within the skin is essential in dermatology and cosmetology. Recent advances in label‐free imaging technologies have facilitated the direct detection of unlabeled compounds in tissues, with high resolution. However, it remains challenging to provide quantitative time‐course distribution maps of drugs within the complex skin tissue. The present study aims at acquiring the real‐time quantitative skin penetration profiles of topically applied caffeine, by means of a combination of pump–probe phase‐modulated stimulated Raman scattering (PM‐SRS) and confocal reflection microscopy. The recently developed PM‐SRS microscopy is a unique imaging tool that can minimize strong background signals through a pulse‐shaping technique, while providing high‐contrast images of small molecules in tissues. MATERIALS AND METHODS: Reconstructed human skin epidermis models were used in order to analyze caffeine penetration in tissues. The penetration profiles of caffeine in an aqueous solution, an oil‐in‐water gel, and a water‐in‐oil gel were examined by combining PM‐SRS and confocal reflection microscopy. RESULTS: The characteristic Raman signal of caffeine was directly detected in the skin model using PM‐SRS. Integrating PM‐SRS and confocal reflection microscopy allowed real‐time concentration maps of caffeine to be obtained from formulation samples, within the skin model. Compared with the conventional Raman detection method, PM‐SRS lowered the background tissue‐oriented signals and supplied high‐contrast images of caffeine. CONCLUSION: We successfully established real‐time skin penetration profiles of caffeine from different formulations. PM‐SRS microscopy proved to be a powerful, non‐invasive, and real‐time depth‐profile imaging technique for use in quantitative studies of topically applied drugs.