Abstract
The capability to image the 3D distribution of melanin in human skin in vivo with absolute quantities and microscopic details will not only enable noninvasive histopathological diagnosis of melanin-related cutaneous disorders, but also make long term treatment assessment possible. In this paper, we demonstrate clinical in vivo imaging of the melanin distribution in human skin with absolute quantities on mass density and with microscopic details by using label-free third-harmonic-generation (THG) enhancement-ratio microscopy. As the dominant absorber in skin, melanin provides the strongest THG nonlinearity in human skin due to resonance enhancement. We show that the THG-enhancement-ratio (erTHG) parameter can be calibrated in vivo and can indicate the melanin mass density. With an unprecedented clinical imaging resolution, our study revealed erTHG-microscopy's unique capability for long-term treatment assessment and direct clinical observation of melanin's micro-distribution to shed light into the unknown pathway and regulation mechanism of melanosome transfer and translocation.
Highlights
Melanin is the most important pigment in human and is the determinant of our skin color
As a dominant light absorber in human skin, melanin provides the strongest THG nonlinearity [22,23], through resonance enhancement with the absorption levels [23,31,32,33] those match the two-photon and three photon frequencies (Fig. 1(a)), while cytoplasm can be observed through melanin-enhanced THG in the basal layer of human skin [22]
These correspondences initially confirmed the dominance of melanin in the THG contrast in human skin
Summary
Melanin is the most important pigment in human and is the determinant of our skin color. The Fontana-Masson (FM) method [5] is a common technique to stain melanin, while humanmelanoma-black-45, Melan-A, or S-100 immunohistochemical stains [6] are exploited to image melanocytes [7], which synthesize melanin in melanosomes and transfer melanosomes to the neighboring keratinocytes through their dendrites. These exogenous stains for melanin or melanocytes are usually combined with the invasive and lengthy formalin-fixed paraffin-embedded (FFPE) immunohistochemistry processes [8], precluding their application for noninvasive onsite diagnosis with a histopathological accuracy. A noninvasive quantitative imaging tool, providing histopathological morphology information and a three-dimensional (3D) melanin density mapping, will be highly desirable for diagnosis and to serve as the critical part of a treatment evaluation program for melanin-related diseases
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