Investigating melanin and its distribution in reconstructed pigmented epidermis model by fast 2D XZ multiphoton imaging
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Presented by: Shanshan ZANG
Investigating melanin’s density and z-epidermal distribution in skin is essential for medical and cosmetic applications. The in situ characterization of this skin pigment requires the use of label free non-invasive imaging methods such as multiphoton microscopy. Using endogenous autofluorescence signals from keratin, NAD(P)H and FAD metabolic coenzymes and melanin, multiphoton microscopy provides non-invasive 3D visualization of epidermis layers and melanin’s distribution in vivo in human skin 1,2 and in vitro in reconstructed pigmented epidermis (RPE) model 3. We previously reported on a multiphoton-based method for 3D quantitative assessment of melanin content in RPE model 3, but 3D imaging is very time consuming and greatly limits its routine use for efficacy evaluation of melanin modulators.
In this study, we report on a new faster method for melanin assessment in RPE model, based on the acquisition of several transversal 2D XZ multiphoton images, equivalent to the ones provided by histology Fontana-Masson (FM) staining. Nearly a hundred 2D XZ multiphoton images from stratum basal towards stratum corneum were acquired for every reconstructed skin sample, whereas only a few images are investigated in histology FM analysis. We also developed associated melanin quantification tools to extract quantitative parameters such as 2D melanin density and its z-distribution profile. For melanin distribution, an air/stratum basal boundary detection was implemented by determining the median position of the highest fluorescence intensity pixels in a surrounding interval in x dimension. The melanin containing pixels were then detected by applying Gaussian blur to further eliminate noise before detecting high fluorescence intensity melanin pixels using the same intensity-threshold as for 3D imaging 3. The average number of melanin containing pixels with respect to their distance from the basal epidermal boundary is used to estimate a melanin z-distribution profile from stratum basal up to 100 µm depth.
We applied melanin fast 2D XZ multiphoton imaging method to the study of melanin modulations in RPE model upon application of different associations of Ferulic Acid, Niacinamide and Phenylethyl Resorcinol, and compared the results to control samples. These three actives have strong whitening efficacy, especially Ferulic acid which is more easily absorbed into the body and stays in the blood longer than any other antioxidant, even longer than C vitamin. Since the importance of free radical affection of melanogenesis, evidence is showed that because of this anti-oxidation function, anti-pigmentation is found in vivo test compared to other anti-oxidation at lower concentration. Non-parametric test was used in analyzing the differences between control and treated groups. The results show a lowest melanin density for the association of Ferulic Acid, Niacinamide and Phenylethyl Resorcinol.
In conclusion, the association of fast 2D XZ multiphoton imaging with specific image processing tool allows the distribution and quantity of melanin to be visualized and quantified in RPE epidermis model with an operational time 5 times faster compared to 3D imaging, thus providing a powerful method for routine efficacy evaluation of melanin modulators in cosmetic and dermatological investigations.
References:
1 Ait El Madani, H. et al. In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation. J Biomed Opt 17, 026009, doi:https://doi.org/10.1117/1.JBO.17.2.026009 (2012).
2 Pena, A. M. et al. In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses. Scientific Reports 12, 1642, doi:https://doi.org/10.1038/s41598-021-03114-0 (2022).
3 Qiu, J. et al. The skin-depigmenting potential of Paeonia lactiflora root extract and paeoniflorin: In vitro evaluation using reconstructed pigmented human epidermis. Int J Cosmet Sci 38, 444-451, doi:http://onlinelibrary.wiley.com/doi/10.1111/ics.12309/epdf (2016).
In this study, we report on a new faster method for melanin assessment in RPE model, based on the acquisition of several transversal 2D XZ multiphoton images, equivalent to the ones provided by histology Fontana-Masson (FM) staining. Nearly a hundred 2D XZ multiphoton images from stratum basal towards stratum corneum were acquired for every reconstructed skin sample, whereas only a few images are investigated in histology FM analysis. We also developed associated melanin quantification tools to extract quantitative parameters such as 2D melanin density and its z-distribution profile. For melanin distribution, an air/stratum basal boundary detection was implemented by determining the median position of the highest fluorescence intensity pixels in a surrounding interval in x dimension. The melanin containing pixels were then detected by applying Gaussian blur to further eliminate noise before detecting high fluorescence intensity melanin pixels using the same intensity-threshold as for 3D imaging 3. The average number of melanin containing pixels with respect to their distance from the basal epidermal boundary is used to estimate a melanin z-distribution profile from stratum basal up to 100 µm depth.
We applied melanin fast 2D XZ multiphoton imaging method to the study of melanin modulations in RPE model upon application of different associations of Ferulic Acid, Niacinamide and Phenylethyl Resorcinol, and compared the results to control samples. These three actives have strong whitening efficacy, especially Ferulic acid which is more easily absorbed into the body and stays in the blood longer than any other antioxidant, even longer than C vitamin. Since the importance of free radical affection of melanogenesis, evidence is showed that because of this anti-oxidation function, anti-pigmentation is found in vivo test compared to other anti-oxidation at lower concentration. Non-parametric test was used in analyzing the differences between control and treated groups. The results show a lowest melanin density for the association of Ferulic Acid, Niacinamide and Phenylethyl Resorcinol.
In conclusion, the association of fast 2D XZ multiphoton imaging with specific image processing tool allows the distribution and quantity of melanin to be visualized and quantified in RPE epidermis model with an operational time 5 times faster compared to 3D imaging, thus providing a powerful method for routine efficacy evaluation of melanin modulators in cosmetic and dermatological investigations.
References:
1 Ait El Madani, H. et al. In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation. J Biomed Opt 17, 026009, doi:https://doi.org/10.1117/1.JBO.17.2.026009 (2012).
2 Pena, A. M. et al. In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses. Scientific Reports 12, 1642, doi:https://doi.org/10.1038/s41598-021-03114-0 (2022).
3 Qiu, J. et al. The skin-depigmenting potential of Paeonia lactiflora root extract and paeoniflorin: In vitro evaluation using reconstructed pigmented human epidermis. Int J Cosmet Sci 38, 444-451, doi:http://onlinelibrary.wiley.com/doi/10.1111/ics.12309/epdf (2016).