Novel Confocal Microscope for Molecular/Cellular Imaging

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DESCRIPTION (provided by applicant): We would like to develop a new method of in vivo imaging using a miniaturized confocal microscope to study the biological processes of tumor development on the molecular and cellular level. This method has the potential to revolutionize cancer research. The long term objective of this project is to develop a technique to observe how tumors originate, progress, and invade. The behavior of cells will be monitored from images of sub-cellular constituents tagged with green or yellow fluorescence protein (GFP, YFP). A miniaturized confocal prototype has already been developed and its performance has been demonstrated with ex vivo images. We plan to improve this microscope with a new design using a dual-angle-axis architecture. This design offers improved resolution and working distance, which will facilitate in vivo imaging. We then plan to study two tumor models, medulloblastoma in a mouse model that has been associated with decreased expression of the ptc1 gene, and dysplasia in Barrett's esophagus and colonic adenomas. We will use these models to develop the new microscope. First, the performance of the tabletop dual-angle-axis prototype will be characterized by reflectance and fluorescence images of cultured cells and tissues sections expressing GFP/YFP and by biopsy specimens of Barrett's esophagus and colonic adenomas. Then, images will be collected from post-natal ptc1 knock-out mice expressing GFP/YFP to monitor angiogenesis, tumor invasion, and response to therapy. Next, the results of these studies will be used to develop the redesigned miniaturized microscope using Micro-Electro-Mechanical Systems (MEMS) fabrication technology. The miniaturized microscope will be used to study medulloblastoma in ptc1 knock-out mice in vivo and dysplasia in Barrett's esophagus and colonic adenomas during routine endoscopy. The specific aims of this research are as follows: 1) Measure the transverse and axial resolution, signal-to-noise, and field of view of the tabletop dual-angle-axis confocal prototype. 2) Collect reflectance and fluorescence images from cultured cancer cells expressing GFP/YFP. 3) Confirm the performance of the tabletop prototype with reflectance and fluorescence images collected from biopsy specimens of Barrett's epithelium and colonic adenomas, as a model of dysplasia. 4) Monitor the temporal and spatial extent of proliferation from medulloblastoma in post-natal ptc1 mice using the tabletop microscope. 5) Design and fabricate the miniaturized microscope using MEMS technology. 6) Monitor the temporal and spatial extent of proliferation from medulloblastoma in post-natal ptc1 knock-out mice and transplanted tumor cells using the miniaturized microscope. 7) Collect images from Barrett's epithelium and colonic adenomas during routine endoscopy.