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Laser scanning confocal autofluorescence microscopy (LSCAM) using 351- to 364-nm excitation light was used to quantitatively compare fluorescent spectral emission of unstained, frozen histological sections of normal, premalignant, and malignant colonic tissues. To identify the spatial origins of fluorescent signals accurately, the same frozen section slides used for microscopy were fixed and histochemically stained immediately following LSCAM imaging. Tissue fluorescence emission was quantified in terms of the intrinsic fluorescence coefficient β(λ), defined as the fluorescence power per unit tissue volume per unit wavelength (centered at λ) divided by the incident light irradiance. Over all emission wavelengths, colonic tissues emitted autofluorescence ranging from β(λ)∼10 -1.5 to 10 -3.0 cm -1. In the 530- to 610-spectral region, markedly increased autofluorescence (β up to 10 -2.5) was observed in the dysplastic cells of adenomatous polyps, as compared to normal epithelial cells. Compared to adenomatous polyps, decreased dysplastic cell autofluorescence was observed in adenocarcinoma. The brightest fluorescence in the lamina propria, which was attributed to eosinophils (β∼10 -2.5) in previous studies, was also observed in other granular structures (β up to 10 -1.4). LSCAM reveals quantitative significant differences in fluorescence emission between normal and diseased colonic tissues.