Abstract
Conventional microscopy views cells and tissue in culture using vital stains to enhance contrast. Recent advances in optics and molecular reagents allow for the development of intra-vital microscopy to view the complex, dynamic behavior of biology in remote areas. Furthermore, greater sensitivity and specificity can be achieved with small fluorescence-tagged peptides that preferentially bind to molecular markers. Here, the development of miniature confocal microscopes (diameter of 300 um to 3 mm) with single and dual axes configuration will be discussed.

The single axis is based on a tiny fiber optic bundle, and collects fluorescence to monitor lymphocyte trafficking in CXCR6-GFP mice and to detect pre-malignant colonic mucosa during endoscopy. Dual axes uses two low numerical aperture objectives oriented off-axis to significantly improve working distance, reduce axial resolution, and increase field of view. Scanning is performed with a post-objective MEMS (micro-electro-mechanical systems) mirror.

Furthermore, light scattered along the illumination path is rejected, resulting in improved image contrast. Examples from transgenic animals and normal and diseased colon, esophagus, and skin will be presented. In addition, phage display has been used to identify small peptides that preferentially bind to colonic dysplasia with a high target-to-background ratio. Upon conjugation to FITC, these reagents can enhance localization of pre-malignant gastrointestinal mucosa by confocal. The development of miniature confocal instruments and peptide reagents demonstrate an integrated optical approach to observe basic biology and pathology in vivo in the pre-clinical and clinical setting, thus opening a new frontier to be explored.

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