In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses

Abstract

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. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin. 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 1 year and with topical application of retinoic acid or retinol on human skin. Applications of Pseudo-FLIM based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, anti-photoaging, or photoprotection products evaluation.

Multiscale mapping of in vivo 3D epidermal melanin distribution of human skin using a fast large-area multiphoton exoscope (FLAME)

“…In this work, we utilized FLAME, a fast large-area multiphoton exoscope to evaluate the 3D melanin distribution over an imaging area of about two orders of magnitude (180×) larger than the 0.25×0.25 mm 2 FOV, commonly used for the melanin assessment in human skin using the commercial clinical multiphoton imaging technology. [16][17][18] Combined with selective detection of melanin by fluorescence temporal gating and binning, the data acquisition time for each subject was kept under ~7 minutes, which is at least an order of magnitude faster than the acquisition time that would be required for the current MPM clinical technology to scan over a similar area.…”

“…The rationale for emphasizing the skin heterogeneity when examining this area size is related to the fact that this is the skin area typically scanned with the current commercial clinical multiphoton tomograph employed in prior studies for in vivo melanin quantification in human skin. [16][17][18] The precision of global MVF measurements increases with an increase in the imaging area. The results described above show the heterogeneity in the 3D distribution of melanin in human skin for all skin types, which further suggests that a large sampling volume is critical for the reliability of the MVF measurements as they would capture this heterogeneity.…”

“…6,[16][17][18] TPEF laser-scanning microscopy technologies can generate in vivo 3D sub-micron resolution images of epidermal melanin distribution in human skin. 6,[16][17][18][19] The penetration depth of this imaging technique is limited to 150-200 m depending on the skin type and other imaging parameters. 20,21 Nonetheless, this depth is sufficient for capturing the entire thickness of the epidermis on most areas of the body.…”

“…18,19,23 A. M. Pena et al demonstrated the feasibility of this approach by studying the modulation of the 3D epidermal melanin following long-term topical treatments. 17,18 FLIM, among other methods such as pump-probe 24 and Coherent anti-Stokes Raman Scattering imaging, 25 has also been demonstrated as a potentially effective tool for selective detection of eu-and pheo-melanin in excised pigmented lesions from human skin and in vivo in murine model 24,25 and human skin. 23 To enhance the clinical imaging feasibility of this approach, our group and others have proposed and demonstrated significant enhancement in image acquisition time based on temporal binning 26 along with analysis of the fluorescence temporal decay slope.…”

“…23 To enhance the clinical imaging feasibility of this approach, our group and others have proposed and demonstrated significant enhancement in image acquisition time based on temporal binning 26 along with analysis of the fluorescence temporal decay slope. 17,18 While the potential of the two-photon FLIM approach for quantification of melanin and its depth-distribution in human skin has been demonstrated in the clinical setting, a key limitation remains. This limitation is related to the reduced field of view (0.25×0.25 mm 2 ) of the commercial clinical multiphoton tomograph 27,28 that has been used in prior studies for evaluating the potential of the TPEF intensity 6,16 and lifetime [17][18][19] detection in the assessment of melanin in human skin.…”