Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin, NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.