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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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In vivo 3D multiphoton images of human skin – acquisition of combined 2PEF-FLIM (4 time channels)/SHG z-stacks compatible with Pseudo-FLIM melanin detection. 2PEF intensity is shown in cyan hot color, SHG in red and Pseudo-FLIM melanin mask pixels in purple. High 2PEF signal intensities appear in white color. Images are extracted from a z-stack of 70 en face images acquired with 2.346 µm z-step. (a) stratum corneum disjunctum; (b) stratum corneum disjunctum/compactum interface; (c, d) compactum / granulosum interface; (e, f) stratum granulosum; (g, h) stratum spinosum; (i) stratum basale; (j–o) stratum basale/dermis interface; (p) superficial dermis. Within the blood capillaries, Pseudo-FLIM detects cells with high slope values, fast decays, that most probably emit 2PEF signals from hemoglobin (see arrows). As the image acquisition time is slower than the blood flow, some blood cells appear with a deformed shape as indicated by the arrow in image m.