Multiscale mapping of in vivo 3D epidermal melanin distribution of human skin using a fast large-area multiphoton exoscope (FLAME)

Abstract

Melanin plays a significant role in the regulation of epidermal homeostasis and photoprotection of human skin. The assessment of its epidermal distribution and overall content is of great interest due to its involvement in a wide range of physiological and pathological skin processes. Among several spectroscopic and optical imaging methods that have been reported for non-invasive quantification of melanin in human skin, the approach based on the detection of two-photon excited fluorescence lifetime distinguishes itself by enabling selective detection of melanin with sub-cellular resolution, thus facilitating its quantification while also resolving its depth-profile. A key limitation of prior studies on the melanin assessment based on this approach is their inability to account for the skin heterogeneity due to the reduced field of view of the images, which results in high dispersion of the measurement values. Pigmentation in both normal and pathological human skin is highly heterogeneous and its macroscopic quantification is critical for reliable measurements of the epidermal melanin distribution and for capturing melanin-related sensitive dynamic changes as a response to treatment. In this work, we employ a fast large-area multiphoton exoscope (FLAME), recently developed by our group for clinical skin imaging, that has the ability to evaluate the 3D distribution of epidermal melanin content in vivo macroscopically (millimeter scale) with microscopic resolution (sub-micron) and rapid acquisition rates (minutes). We demonstrate significant enhancement in the reliability of the melanin density and distribution measurements across Fitzpatrick skin types I to V by capturing the intra-subject pigmentation heterogeneity enabled by the large volumetric sampling. We also demonstrate the potential of this approach to provide consistent measurement results when imaging the same skin area at different times. These advances are critical for clinical and research applications related to monitoring pigment modulation as a response to therapies against pigmentary skin disorders, skin aging, as well as skin cancers.

Significance of melanin distribution in the epidermis for the protective effect against UV light

“…For the subject with an ITA 52° (“light” skin), the average melanin area was 82.7 ± 3.0%, and for the subject with an ITA 39° (intermediate), it was 97.5 ± 0.8%. In individuals with lighter skin type, the area covered by melanin in the cells was not concentrated in a specific area, but the relative area size was distributed over a wider range, between 0 and 100%, as previously demonstrated by Vicente et al 11 . The mean melanin area per cell for the subject with an ITA 76° (“very light” skin) was 58.1 ± 4.4%, ITA 70° (“very light” skin) was 68.1 ± 3.9%.…”

“…The risk has often been attributed to differences in the content and composition of melanin in the skin 10 . Melanin, a complex biopolymer with condensed oxidized tyrosine as basis, is the most abundant skin chromophore, being presented as dark eumelanin and reddish pheomelanin 11 , 12 . Melanocytes synthesize and package melanin in the form of melanosomes and transfer it to adjacent keratinocytes, where the pigments arrange perinuclearly to protect the skin against UV-induced cell damage 11 , 13 .…”

“…Melanin, a complex biopolymer with condensed oxidized tyrosine as basis, is the most abundant skin chromophore, being presented as dark eumelanin and reddish pheomelanin 11 , 12 . Melanocytes synthesize and package melanin in the form of melanosomes and transfer it to adjacent keratinocytes, where the pigments arrange perinuclearly to protect the skin against UV-induced cell damage 11 , 13 . The distribution of melanin in a capsular structure over the keratinocyte nuclei is required to constitute a physical barrier that scatters, absorbs, and thus, reduces the penetration of UVR through the epidermis 14 , 15 .…”