Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation

Fluorescence of synthetic melanin in the solvents H2O, KOH, ethylene glycol monomethyl ether, and dimethyl sulfoxide has been excited by two-photon ab sorption at 800 nm using 120-fs pulses with photon flux densities of greate r than or equal to 10(27) cm(-2) s(-1). Compared to the one-photon (400-nm) -induced fluorescence of melanin, the overall spectral shape is red-shifted and shows a strong environment sensitivity. The decay of the two-photon-in duced fluorescence (TPF) of melanin is three-exponential, with a shortest m ain component of about 200 ps. The results of the TPF studies in line with the unique light absorption property of melanin of a monotonously decreasin g absorption spectrum between the near UV-region and the near infrared regi on indicate that the TPF is realized via stepwise absorption of two 800-nm photons. In comparison to the simultaneous absorption of two photons, the s tepwise process needs lower photon flux densities to get a sufficient popul ation of the fluorescent level. This stepwise process offers new possibilit ies of selective excitation of melanin in skin tissue in a spectral region when there is no overlap with any absorption of another fluorescent tissue component. The first results with different samples of excised human skin t issue (healthy, nevus cell nevi, malignant melanoma) suggest that fluoresce nce excited in this way yields information on malignant transformation.