The fluorescence of melanin in human skin tissue reflects the structural changes in the process of skin cancer progression towards malignant melanoma. A selectively excitation of melanin in skin tissue, however, is only possible by stepwise two-photon absorption (TPA) in the NIR spectral range (λexc ≈ 800 nm). Due to the very short lifetime of the energy level populated by the absorption of the first photon (< 5 ps) an effective TPA process can be only achieved with laser pulses on the femtosecond time scale. To use the two-photon excited fluorescence (TPF) of melanin for early diagnosis of malignant melanoma a small, mobile TPF spectrometer was developed. It consists (i) of a fiber laser the radiation of which was amplified in a short pulse nitrogen laser pumped dye cell, (ii) an articulated arm with special mirrors for nearly lossless direction of the fs laser beam to the skin position of investigation, (iii) a camera objective which include visualization and excitation as well as acquisition of fluorescence and (iv) a monochromator with a multichannel photomultiplier and a PC with special software for spectrometer control and data processing. First investigations of the equipment concerning the early diagnosis of malignant melanoma were carried out.
Spectral and time-resolved fluorescence studies of different eumelanins (natural, synthetic, enzymatic) in solution have been carried out by two-photon excitation at 800 nm, using 80 fs pulses with photon flux densities ≤ 1027 cm-2.s-1. Whereas all samples show monotonously decreasing absorption between near UV and near IR, their fluorescence behavior indicates strong heterogeneity. With respect to the also measured one-photon excited fluorescence (OPF) of melanin at 400 nm, the overall spectral shape of the two-photon excited fluorescence (TPF) is red-shifted. Both OPF and TPF exhibit three-exponential decay with a shortest component £ 200 ps.
As is also shown, the fluorescence properties of melanin are dependent on the micro-environment. This allows the hypothesis, that the process of malignant transformation in skin tissue could be reflected in the fluorescence, provided the melanin in skin is selectively excited. The latter is realized by the described stepwise absorption of two 800 nm photons.
In this way, indeed characteristic differences between the TPF spectra of healthy tissue, nevus cell nevi and malignant melanoma have been found.
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