title: Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo.
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.
authors.affiliation: University of California, Beckman Laser Institute and Medical Clinic, Irvine, CA 92617-1475, USA.
authors.forename: Tatiana B
authors.initials: TB
authors.lastname: Krasieva
authors.forename: Chiara
authors.initials: C
authors.lastname: Stringari
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Chung-Ho
authors.initials: CH
authors.lastname: Sun
authors.forename: Yu
authors.initials: Y
authors.lastname: Kong
authors.forename: Mihaela
authors.initials: M
authors.lastname: Balu
authors.forename: Frank L
authors.initials: FL
authors.lastname: Meyskens
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Tromberg
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry; D006197:Hair / Q000737:chemistry; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry; D008543:Melanins / Q000032:analysis / Q000737:chemistry*; D008545:Melanoma / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D009243:NAD / Q000032:analysis / Q000737:chemistry; D061848:Optical Imaging / Q000379:methods*; D012867:Skin / Q000166:cytology
chemical_list: D008543:Melanins; D009243:NAD