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. 2019 May;32(3):403-411.
doi: 10.1111/pcmr.12756. Epub 2018 Dec 21.

In vivo multiphoton microscopy of melasma

Griffin Lentsch  1 , Mihaela Balu  1 , Joshua Williams  1 , Sanghoon Lee  1   2 , Ronald M Harris  3 , Karsten König  4 , Anand Ganesan  3 , Bruce J Tromberg  1 , Nirmala Nair  5 , Uma Santhanam  6 , Manoj Misra  6
Affiliations

In vivo multiphoton microscopy of melasma

Griffin Lentsch et al. Pigment Cell Melanoma Res. 2019 May.

Abstract

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non-invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non-invasive imaging tool for characterizing melasma.

Keywords: in vivo imaging; melasma; microscopy.

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Conflict of interest statement

Conflicts of Interest: K. Koenig is the co-founder of Jenlab, GmbH. B.J. Tromberg and M. Balu report a pending patent, which is owned by the University of California, that is related to the technology described in this study. The Institutional Review Board and Conflict of Interest Office of the University of California, Irvine, have reviewed patent disclosures and did not find any concerns. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.. MPM image processing for estimating the melanin content.
Stacks of TPEF images acquired at different depths at (a) 790 nm and (b) 880 nm. (c) thresholded images corresponding to the stack shown in (b).
Figure 2.
Figure 2.. MPM imaging of dermal melanophages in melasma lesions–
MPM images showing: (A) clusters of small dermal melanophages (arrows), (B) clusters of large dermal melanophages (arrows), (C) dendritic melanophages (arrows), and (D) individual/scattered dermal melanophages (arrow). Images in A, B, C are from subjects with dermal/mixed melasma. Image D corresponds to epidermal melasma. Collagen and elastin fibers are visualized in dermis by their SHG (blue) and TPEF (green) signals, respectively. ‘z’ represents depth in all MPM images. Scale bar is 40 μm.
Figure 3.
Figure 3.. Distribution of the mean MVF values –
The distribution of the mean MVF values measured in all lesions diagnosed as epidermal melasma (*p=0.03) and dermal/mixed melasma (p=0.32) and in corresponding perilesional skin.
Figure 4.
Figure 4.. MPM imaging of epidermal melanin distribution in melasma lesion and perilesion–
MPM images showing: (a) melasma lesional skin, with pigment distribution localized mainly towards the cellular membrane, and (b) perilesional skin, with pigment distribution localized mainly towards the nuclear membrane (melanin caps). The insets in (a) and (b) show a close-up of the melanin distribution in the lesion and perilesion keratinocytes. These images were selected from the corresponding stacks acquired at different depths as shown in (c) and (d). MPM images are from a subject with dermal/mixed melasma. ‘z’ represents depth in all MPM images. Scale bar is 40 μm.
Figure 5.
Figure 5.. MPM imaging of elastosis in melasma lesion and perilesional skin –
MPM images of the dermis in the lesion (A1, B1, C1) showing severe elastosis with overproduction of normal elastin fibers (A1, C1), and abnormal elastin fibers (B1); MPM images of the dermis in the corresponding perilesion (A2, B2, C2) showing: moderate elastosis (A2), mild elastosis (B2), and moderate elastosis with overproduction of abnormal elastosis fibers (C2). Collagen and elastin fibers are visualized in dermis by their SHG (blue) and TPEF (green) signals, respectively. ‘z’ represents depth in all MPM images. Scale bar is 40 μm.
Figure 6.
Figure 6.. Representative MPM images of epidermal pigment distribution in the basal layer vs. elastosis severity in melasma lesion and perilesional skin –
MPM image of the epidermis in the lesion (A) showing an epidermal pigment distribution localized towards the cellular membrane, and perilesion (B) showing an epidermal pigment distribution localized towards the nuclear membrane (melanin caps); MPM images of the dermis in the lesion (C) showing severe elastosis with overproduction of abnormal elastosis fibers, and perilesion (D) showing mild elastosis. Collagen and elastin fibers are visualized in dermis by their SHG (blue) and TPEF (green) signals, respectively. ‘z’ represents depth in all MPM images. Scale bar is 40 μm.
Figure 7.
Figure 7.. Representative MPM images of epidermal pigment distribution in the basal layer vs. presence of dermal melanophages in melasma lesion and perilesional skin –
MPM image of the epidermis in the lesion (A), showing an epidermal pigment distribution localized towards the cellular membrane, and perilesion (B), showing an epidermal pigment distribution localized towards the nuclear membrane (melanin caps); MPM images of the dermis in the lesion (C), showing clusters of dermal melanophages, and perilesion (D), showing no dermal melanophages. Collagen and elastin fibers are visualized in dermis by their SHG (blue) and TPEF (green) signals, respectively. ‘z’ represents depth in all MPM images. Scale bar is 40 μm.
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