Two-photon excited fluorescence lifetime imaging and spectroscopy of melaninsin vitroandin vivo

Abstract

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 ¼ 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 AE 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI ¼ 0.5 AE 0.05 and 0.17 AE 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.

In vivomeasurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy

“…19,20 The melanin appears in the MPM images as bright fluorescence spots in the cellular cytoplasm that represents aggregates of melanosomes. The excitation wavelength selected for this acquisition mode was 880 nm in order to maximize the melanin contrast against TPEF signals from other components of the epidermis (keratin, NADH/FAD).…”

“…19,20 Subjects ranged in pigmentation from very light skin to dark skin (Fitzpatrick types I-VI). All spatial frequency domain measurements are compared to data obtained from multiphoton microscopy using selective two-photon excited fluorescence (TPEF) of melanin.…”

Preclinical Advances with Multiphoton Microscopy in Live Imaging of Skin Cancers

“…There are endogenous fluorophores found in the skin, including keratin and melanin (Breunig et al, 2012; Krasieva et al, 2013). However, most of these give off fluorescence that is too weak to be detectable with typical imaging parameters used in timelapse intravital imaging.…”

Evidence for pheomelanin sheet structure

“…By contrast pheomelanin predominates in people with reddish hair and is thought to be im-plicated in producing cancer-inducing phototoxic species such as reactive oxygen species 2 and mutations. 3 The ratio of pheo-to eu-melanin has been demonstrated to be a potential cancer biomarker using two-photon excited fluorescence, [4][5][6] but the complexity of the approach makes it impractical and despite considerable research, optical biopsies have yet to replace excision in testing for melanoma. Nevertheless the urgent need for more practical and simpler alternatives to excision remains and optical approaches remain at the forefront of this area.…”