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. 2022 Jan 31;12:1642. doi: 10.1038/s41598-021-03114-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Global analysis process for in vivo 3D multiphoton images allowing 3D skin automatic layers segmentation and constituents quantification. (a) The first step of global 3D analysis of z-stacks of combined 2PEF-FLIM (4 time channels)/SHG images briefly consists in identifying the epidermal and dermal layers (3D automatic segmentation) and quantifying their morphology (thickness, DEJ 3D-shape) and the 3D dermal density of elastin and fibrillar collagens. (b) The z-stack of 2PEF-FLIM (4 time channels) images is further processed for melanin detection using Pseudo-FLIM method. The first 3-time channels are used for slope parametric image calculation and a melanin mask is obtained by applying a threshold to keep the high slopes values (above 70). (c) The 3D z-stack of melanin masks and the 3D automatic segmentation of the epidermis and its sub-layers are jointly used to estimate a 3D epidermal, SC or LED melanin density. By defining 12-thickness normalized epidermal layers, the epidermal melanin density z-distribution (z-profile of melanin density in the 12 thickness-normalized epidermal layers from 1—DEJ level to 12—SC level) can be assessed.