The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0–2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm.

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