Non-Invasive Visualization of Melanin and Melanocytes by Reflectance-Mode Confocal Microscopy Toyonobu Yamashita, Tomohiro Kuwahara, Salvador González,

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Non-Invasive Visualization of Melanin and Melanocytes by Reflectance-Mode Confocal Microscopy Toyonobu Yamashita, Tomohiro Kuwahara, Salvador González, Motoji Takahashi Journal of Investigative Dermatology Volume 124, Issue 1, Pages 235-240 (January 2005) DOI: 10.1111/j.0022-202X.2004.23562.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reflectance-mode confocal microscopy (RCM) visualization of melanin in the human skin. (A) RCM images of the forearm at the dorsal side were taken at planes a and b shown in the scheme of the vertical section of epidermis. The back-scattered light from the supranuclear melanin caps (arrows) is strong. (B) RCM images of the forearm show less back-scattered light from the basal layer at the volar side (a) than at the dorsal side (b). The light scattering is attributed to melanin. (C) Photographs by a digital camera (a and b) and RCM images (c and d) of an age-dependent pigmentary lesion on the face are shown. The RCM images (c and d) clearly show supranuclear melanin caps (bright regions), but recognition of melanocytes is difficult. Journal of Investigative Dermatology 2005 124, 235-240DOI: (10.1111/j.0022-202X.2004.23562.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reflectance-mode confocal microscopy (RCM) visualization of melanosomes and melanocytes in pigmented animals' skin. (a, b) Comparison between the RCM image (a) and 3,4-dihydroxyphenylalanine (DOPA) staining (b) of the epidermal sheet of a (HR-1 × HR/De) F1 mouse: the RCM image (a) correlates well with the DOPA-stained epidermal sheet (b) and both show melanosome-like vesicles (arrows). (c, d) Comparison between the RCM image (c) with Fontana–Masson-stained horizontal section (d) of the dorsal skin of a Weiser–Maples guinea-pig: melanocytes (arrows) with high brightness are clearly observed with RCM (c), and histologically confirmed with Fontana–Masson staining (d). (e, f) Comparison between the RCM image (e) and Fontana–Masson-stained horizontal section (f) of the dorsal skin of a Yucatan micropig: melanocytes (arrows) are observed with both the methods (e and f). The epidermal layers were 31 μm [(HR-1 × HR/De) F1 mouse], 22 μm (Weiser–Maples guinea-pig), and 69 μm thick (Yucatan micropig), respectively, as measured on hematoxylin–eosin-stained transversal sections. Journal of Investigative Dermatology 2005 124, 235-240DOI: (10.1111/j.0022-202X.2004.23562.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Reflectance-mode confocal microscopy (RCM) visualization of melanocytes in the human volar forearm skin after ultraviolet (UV) exposure. (a) The changes after a single exposure UV (2.5 minimal erythema dose) were followed by RCM imaging. Before UV exposure, a slight reflectance depending on the melanin content is observed. (b) Three days after UV exposure, the images become dark. (c) On day 8, the intensity of reflectance is increased, suggesting that melanin is being produced by melanocytes. (d) After 18 d, the reflectance becomes stronger. (e, f) Twenty-nine and 70 d after UV exposure, the supranuclear melanin caps are clearly observed. (g, h) Expanded images of day 8 after UV exposure, except that images were taken with enhanced contrast. (h) UV-exposed area with clearly visible dendritic melanocytes (arrows), and (g) an area not exposed to UV, for comparison. Journal of Investigative Dermatology 2005 124, 235-240DOI: (10.1111/j.0022-202X.2004.23562.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions