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

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

doi:10.1117/1.JBO.18.3.031107

12 December 2012 Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

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

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Journal of Biomedical Optics, Vol. 18, Issue 3, 031107 (December 2012). https://doi.org/10.1117/1.JBO.18.3.031107

Abstract

Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λ_ex = 1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6 ± 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI = 0.5 ± 0.05 and 0.17 ± 0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

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Tatiana B. Krasieva, Chiara Stringari, Feng Liu, Chung-Ho Sun, Yu Kong, Mihaela Balu, Frank L. Meyskens, Enrico Gratton, and Bruce J. Tromberg "Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo," Journal of Biomedical Optics 18(3), 031107 (12 December 2012). https://doi.org/10.1117/1.JBO.18.3.031107

Published: 12 December 2012

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