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.

Quantifying skin aging changes and characterizing its 3D structure and function in a non-invasive way is still a challenging area of research. In vivo multiphoton imaging offers means to assess skin constituents in 3D, however prior aging studies mostly focused on 2D analyses of dermal fibers through their signals’ intensities or densities.

We designed and implemented multiphoton multiparametric 3D quantification tools for in vivo human skin characterization. Despite the limited field of view of the technic, investigation of 2 regions of interest (ROIs) per zone per volunteer were found to be a good compromise in assessing 3D skin constituents in both epidermis and superficial dermis.

Skin aging was characterized on different UV exposed areas - ventral and dorsal forearms, face. Epidermal morphological changes occur late and were only objectified between extreme age groups. Melanin accumulated with age and chronic exposure on both forearms and appears earlier. Superficial dermal changes were mainly elastin density increase, also structurally modified from thin and straight fibers in young to dense and compact pattern in elder, and no obvious change in collagen density, both reflected by SHGto2PEF ratio and SAAID index decrease and ImbrN index increase. The second 20 μm-thickness normalized dermal sub-layer exhibited the highest aging differences. The 3D ImbrN index refines the share of photoaging in global aging on face and the 3D SAAID index on forearm.