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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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Comparison of FLIM bi-exponential fitting, Phasor and Pseudo-FLIM methods for melanin detection in vivo on human skin. Multiphoton 2D 2PEF FLIM images acquired at different depths from the skin surface to the dermis, within stratum cornuem (SC) disjunctum, corneum compactum, granulosum (SG), spinosum (SS), basale (SB) and superficial dermis were analyzed with the three methods. (top) FLIM bi-exponential fitting analysis images of the short τ₁ and long τ₂ fluorescence lifetime parameters. (middle) Phasor analysis images of g, s, phase lifetime and modulation lifetime parameters. The corresponding phasor plots (s versus g scatters) of the different skin layers are inserted in between the τ₁ and τ₂ images and color coded using the same color scale as for τ₁ and τ₂ parameters. An enlarge view is shown in Fig. S2. (bottom) Pseudo-FLIM analysis images of the slope parameter highlighting pixels with a fast decay. The arrows indicate the fast decay pixels within the blood capillary.