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Optical Methods Fluorescence micrographs (top row) and brightfield images (bottom row) of short-term tissue cultures of normal (left) and dysplastic (right) cervical tissue. As dysplasia develops, the NADH fluorescence of the epithelium increases and the collagen fluorescence of the stroma decrease. We have developed fiber optic systems to measure tissue reflectance and fluorescence spectra over the UV-Vis-NIR spectral regions in near real time. In the UV and visible regions of the spectrum, tissue reflectance spectra provide information about the wavelength dependent scattering of tissue (related to changes in chromatin texture) as well as electronic absorption bands, primarily those of oxy- and deoxy hemoglobin. Similarly, fluorescence spectra provide information about electronic transitions. The most common naturally occurring biological fluorophores include the aromatic amino acids, the co-factors NAD(P)H and FAD, which describe the tissue metabolic rate, crosslinks associated with collagen and elastin, and porphyrins. We have developed novel tissue and organ culture models to explore the biological basis for differences in fluorescence and reflectance spectra of normal and precancerous tissue. Studies using these systems show that fluorescence and reflectance spectroscopy can provide information about the increase in metabolic activity associated with precancerous epithelial cells, the increase in stromal angiogenesis, increased nuclear size and texture, and the loss of collagen fluorescence that accompanies precancer.  Confocal Images of breast epithelial cells. An alternative approach to the detection and diagnosis of intra-epithelial neoplasia is to non-invasively image the cells within the epithelium using the light reflected from the tissue. In concept, this is very similar to the approach of histological analysis of biopsy specimens, except that 3D resolution is achieved without removing tissue and contrast is provided without histochemical stains. We are developing real time fiber optic imaging systems based on confocal microscopy to provide in vivo tissue images with histologic resolution which illustrate morphologic and biochemical changes. We have shown that these systems can achieve high sensitivity and specificity in vivo and have recently begun clinical trials examining cervical dysplasia and precancer in the oral cavity. 
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