• confocal Raman microscopy;
  • human skin;
  • depth profiling;
  • Raman spectral variability;
  • quantification

The opportunity of label-free and non-destructive detection of substances inside human skin by confocal Raman microscopy represents a novel approach for investigating drug penetration and permeation. Several studies already introduced confocal Raman microscopy for depth profiling in skin; however, the reported results show high deviations. Thus, analysis of the spectral variability of human skin itself is a necessary prerequisite for systematic quantitative investigations of drug penetration and permeation by confocal Raman microscopy.

In our work, we acquired Raman depth profiles from excised human skin samples after abdominal plastic surgery and investigated the absolute intensity fluctuation of four major skin derived Raman peaks. The results prove the expected high variability in spectral intensity, but we could not detect dissimilarities between different skin donors. A detailed analysis of the major endogenous skin components revealed differences in spatial distribution which consequently affects their individual applicability as reference peaks for relative depth profiling. Furthermore, we discovered an increase in signal variability in deeper stratum corneum layers, which has to be considered in future substance depth profiling investigations. In addition, we discovered an exponential decay of the Raman signal for all major skin components accounting for signal attenuation inside biological tissue. Based on this mathematical description, quantitative follow-up of substances in human skin can be realized. All in all, the results of this study elucidate the necessity of substantial understanding of endogenous spectral characteristics inside human skin as essential prerequisite for rational depth profiling of substances in human skin. Copyright © 2013 John Wiley & Sons, Ltd.