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Title: Ace Day 1994: QUANTITATIVE ANALYSIS OF AUTOFLUORESCENT MICROSCOPE IMAGES OF COLON TISSUE FOR THE DIAGNOSIS OF PRE-CANCEROUS CHANGES

Written by: J.T. Arendt and R.M. Cothren, Ph.D.

Institutions: The Cleveland Clinic Foundation and The Ohio State University, Department of Biomedical Engineering

Date Written: April 7, 1994

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Clinical studies have demonstrated that in vivo fluorescence spectroscopy performed at endoscopy can distinguish between normal colonic mucosa and that with dysplasia. A fluorescence microscope with a backlit, thermoelectrically cooled CCD camera records images which reveal the fluorescence from the fluorophores within unstained frozen sections of colonic tissue taken from surgical resection. Normal tissue from 8 samples were imaged into 15 fields and 8 adenoma samples were imaged into 14 fields. By using barrier filters, the wavelengths emitted were determined. The following structures are fluorophores, organized by diminishing intensities: eosinophils, basement membranes, collagen fibers, and the cytoplasm of dysplastic crypt cells. Collagen fibers fluoresce strongly at blue wavelengths with a peak at 430 nm, whereas the dysplastic crypt cells in the adenoma fluoresce with some contribution to red wavelengths that are only weakly produced by the other fluorophores. Using a gray level image, the fluorophores were segmented by thresholding. A binary image was used as a mask, resulting in an image with only one type of fluorophore. The intensity contribution versus depth was computed by integrating parallel to the tissue surface. This produced a fluorescence density function for that particular fluorophore. In the mucosa of normal tissue, 92% of the fluorescence came from collagen and 8% from eosinophils. The eosinophils make up very little of the area in the image, so their contribution to the overall fluorescence is small. For adenoma, 56% came from collagen, 33% from crypt cells, and 11% from eosinophils. In this case, the very weak fluorescence of the crypt cells was significant due to the large percentage of the mucosa that consists of crypt cells in combination with the fluorescence of these crypt cells being stronger for adenoma than for normal tissue and to the crypt cells being larger and more numerous in adenoma than in normal tissue.

In related studies, the absorption and scattering properties of the tissue are being used to produce transfer functions and lineshapes of each fluorophore. These along with the fluorescence density functions are integrated through the depth of the tissue to find the predicted in vivo fluorescence. The lineshapes measured during in vivo spectroscopy and those calculated by the model match well. Both the measured and calculated lineshapes for adenoma have a weaker peak in the blue and have more contribution from the lower wavelengths than do the lineshapes for normal tissue.

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