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Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological /pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic / pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining i) multiphoton FLIM, ii) fast image acquisition times, and iii) a melanin detection method, called pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. By comparison to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes / keratinocytes coculture and in vivo human skin, we demonstrate that the fast decay shapes of melanin correspond to high slope values, a characteristic one can use to identify melanin in vitro and in vivo. We show that pseudo-FLIM melanin detection is still feasible upon decreasing the image acquisition time, which opens the possibility to quantify melanin from 3D z-stacks. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over one year and with topical application of retinoic acid or retinol. Applications of pseudo-FLIM-based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, antiphotoaging, or photoprotection products evaluation. |
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