Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

Tatiana B. Krasieva; Feng Liu; Chung-Ho Sun; Yu Kong; Mihaela Balu; Frank L. Meyskens; Bruce J. Tromberg

doi:10.1117/12.906490

9 February 2012 Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

Tatiana B. Krasieva, Feng Liu, Chung-Ho Sun, Yu Kong, Mihaela Balu, Frank L. Meyskens, Bruce J. Tromberg

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Proceedings Volume 8226, Multiphoton Microscopy in the Biomedical Sciences XII; 82262S (2012) https://doi.org/10.1117/12.906490
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λ_ex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6±0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55±0.05 and 0.17±0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

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Tatiana B. Krasieva, Feng Liu, Chung-Ho Sun, Yu Kong, Mihaela Balu, Frank L. Meyskens, and Bruce J. Tromberg "Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo", Proc. SPIE 8226, Multiphoton Microscopy in the Biomedical Sciences XII, 82262S (9 February 2012); https://doi.org/10.1117/12.906490

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