ИСТИНА

Melanin, the pigment mainly responsible for skin color, exhibits photoprotective, antioxidative, and photosensitizing properties and is directly involved in the life cycle of the healthy and pathological epidermis, including such a severe malignancy as melanoma [1]. Melanin localization can be assessed ex vivo and in vivo using its distinctive optical features, such as its characteristic Raman spectrum and discernible near-infrared excited fluorescence. Yet, a detailed analysis of the capabilities of depth-resolved confocal Raman and fluorescence microspectroscopy in the evaluation of melanin distribution in the skin is missing. Here we demonstrate how the melanin fraction at different depths in the human epidermis can be estimated in vivo from its Raman bands at 1380 and 1570 cm-1 utilizing multiple analysis techniques of Raman spectra, including simple ratiometric approach, spectral decomposition, and non-negative matrix factorization. We show that introduced approaches can be successfully applied to gain insights into the melanin distribution in the epidermis and might also provide information about the location of other skin constituents, such as collagen in the dermis and natural moisturizing factor in the stratum corneum. We also found that NIR excited fluorescence correlates well with melanin fraction, and its spectral band shape properties are correlated with the molecular organization of melanin. It was also found that the high NIR fluorescence is also observed in the dermis, suggesting that other molecular sources, such as oxidized proteins and lipids, might contribute to the red endogenous fluorescence. We believe that not only information about the distribution of melanin, but also insights into its molecular organization can be assessed by the combined Raman and NIR-fluorescence approach, which, in turn, can provide a new understanding of the behavior of melanin in healthy and pathological skin. [1] Brenner, M. & Hearing, V. J. The protective role of melanin against UV damage in human skin. Photochem. Photobiol. 84, 539–549.