Fiber optic confocal reflectance microscopy: in vivo detection of pre-cancerous lesions in epithelial tissue

This dissertation describes the development of a fiber optic confocal reflectance microscope for imaging cell nuclei in cervical epithelium and oral mucosa in vivo. The ultimate goal of this research is to construct a non-invasive confocal imaging system to aid in the detection of pre-cancerous lesions in epithelial tissue. Confocal microscopy is a technique capable of imaging individual cells and cell nuclei within tissue by spatially isolating and detecting light from a small focal volume in the tissue. In vivo confocal images have only been obtained from skin and the lip due to the accessibility of these organs with conventional microscopes. Fiber optic confocal fluorescence microscopes with miniaturized objectives have been presented and used to obtain in vivo images from animal models. However, the choice of nontoxic fluorescent dyes suitable for clinical applications remains to be explored. A bench-top fiber optic confocal imaging system was designed and constructed to assess the feasibility of obtaining reflectance images of epithelial cells at 15 frames per second through a flexible fiber optic bundle. The performance of the bench-top system was tested by imaging standard samples and biological specimens. The spatial resolution and sensitivity were sufficient to permit imaging of sub-cellular structures in epithelial tissue. The in vivo imaging capability was achieved by incorporating a miniaturized objective lens and an axial scanning device into the system. Similar spatial resolution and sensitivity were obtained. The modified system was used in a pilot in vivo study: “Reflectance Confocal Imaging of Cervical Intraepithelial Neoplasia (CIN)”. The study protocol was approved by the Institutional Review Board at the University of Texas at Austin, and written informed consent was obtained from each of the participating patients before the experiments began. Images of cell nuclei were obtained from 15 cervical sites in 9 patients. Average nuclear size, nuclear-cytoplasmic ratio and scattering coefficient were measured from images of normal cervical epithelium and compared well with results from existing in vitro studies. The results described in this dissertation indicate that this technique can potentially improve early detection of precancers.