Minimal-preparation Raman platform for tracking small-molecule penetration in ex vivo skin

Monitoring molecular penetration in skin is important for evaluating topical administration and transdermal delivery systems, but conventional methods provide limited spatiotemporal resolution and often require additional specialized instrumentation or extensive sample preparation. In this study, we present a conventional Raman spectroscopy-based analytical platform for monitoring the penetration dynamics of a target substance in ex vivo skin with minimal sample preparation and without cryo-sectioning. Raman signals were acquired using 785 nm excitation, and skin-induced signal changes were characterized and corrected to improve detection reliability. Using methylene blue (MB) as a model substance, the Raman band at 1619 cm –1 was used as the primary spectral marker to identify MB-positive regions and determine penetration depth, while its fluorescence background was retained as additional supporting evidence of MB presence rather than being removed as an unwanted signal. Penetration-depth analysis was performed from an application time of 5 min to 60 min, revealing the time-dependent evolution of molecular penetration in ex vivo skin. This minimal-preparation approach may serve as a practical tool for evaluating penetration dynamics in skin tissue.