| Fluorescence diagnosis of epithelial precancer is based on the difference in spectral characteristics between normal and precancerous tissue. However, the bulk autofluorescence measured by conventional methods do not provide accurate diagnostic information such as structure and metabolism of tissue because the signal is a mixture of the fluorescence from different tissue layers. In this study, we demonstrated that depth-resolved fluorescence measurement can isolate the fluorescence signals from different tissue layers and provide more accurate information for tissue diagnosis.; We instrumented a confocal fluorescence spectroscopy system with single-photon excitation from 355--473 nm to investigate the layered structure and biochemistry of various epithelial tissues. It was found that strong keratin fluorescence from the keratinized epithelial layer creates severe interference in the assessment of NADH, FAD and collagen fluorescence. The depth-resolved fluorescence spectra excited at 355 nm produced sufficient contrast to resolve such fine structure, and the depth-resolved redox ratio from non-keratinized epithelium (the ratio of NADH fluorescence excited at 355nm over FAD fluorescence excited at 457nm) showed high correlation with tissue pathology. Furthermore, we demonstrated that confocal time-resolved measurement with single excitation from uv to violet can potentially provide accurate information for tissue diagnosis because different tissue layers exhibited different fluorescence time decays and the time decays of epithelial fluorescence were sensitive indicators of cellular metabolism.; The depth-resolved fluorescence measurement was also achieved using two-photon excitation from 710--810 nm. The two-photon excited fluorescence (TPEF) signals from the keratinized epithelial layer, non-keratinized epithelium and underlying stroma exhibited different spectral characteristics providing information on biomorphology and biochemistry of epithelial tissue. The second harmonic generation (SHG) signals served as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The results also demonstrated the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue. |
