Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.