Tissue imaging based on two-photon autofluorescence and second harmonic generation

While autofluorescence is observed in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. Image guided spectral analysis is being developed to aid in extracting spectroscopic components from two-photon images. This methodology is being applied to the study of human skin. In ex vivo specimens, the overall bulk emission spectrum of the skin and the layer-resolved emission spectra of the stratum corneum, stratum spinosum, basal layer, and dermis have been measured. From the image guided spectral analysis, it was determined that there are approximately five factors that contribute to most of the luminescence signals from human skin. The autofluorescent species identified include tryptophan, NAD(P)H, melanin (or localizing species), and elastin. The collagen matrix produced a significant second harmonic signal. Because of the coherent nature of second harmonic emission, we are exploring the use of phase sensitive detection techniques to extend the imaging depth for collagen distribution.