Imaging of melanin distribution using multiphoton autofluorescence decay curves

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Abstract
Multiphoton fluorescence lifetime imaging (FLIM) is a technique that produces an image based on differences in the decay rate of fluorescence from a sample. Based on this method, the DermaInspect was developed to observe human skin components non-invasively. In this study, we used the DermaInspect to study melanin in skin. A human three-dimensional skin model containing melanocytes was embedded in an OCT compound, frozen and sectioned at 10 microm. The melanin distribution in each section was visualized by the DermaInspect using time-resolved single-photon counting and near-infrared femtosecond laser pulse excitation. The melanin distribution of the same sections was then visualized using the Fontana-Masson staining method. High-resolution images were generated from the ratio of a(1)/a(2) (a(1)e(-) (t/120)+a(2)e(-) (t/1100) was chosen to express the exponential fluorescent decay curve) obtained using the DermaInspect. Granules with a high a(1)/a(2) ratio, approximately 1 mum in diameter, were observed. Fontana-Masson staining identified these granules as melanin. This new technique was then applied for in vivo observation of melanin in human skin. 'Melanin caps' were visualized in the basal cell layer around the nuclei in images derived from the a(1)/a(2) ratio. Our study confirms that FLIM can non-invasively provide data of the melanin distribution with almost the same quality as the conventional Fontana-Masson staining method, and demonstrates that FLIM is useful for in vivo observation of melanin granules in human skin.