We propose to develop a miniature confocal theta fluorescence microscope for high resolution optical sectioning in biological tissue. This instrument will be developed for the in vivo study of molecular and cellular events in living animal models of human biology and disease. Standard confocal microscopes are awkward to use with small animals because the objective lenses are large and the working distances are short. In contrast, evaluating fluorescent markers in rodent models using a miniature confocal microscope with a long working distance that can be directed at an exposed tissue or implanted below the tissue surface offers several advantages. We plan to modify the collection optics of a previously developed miniature single axis confocal microscope fabricated with micro-electromechanical systems (MEMS) technology by using a dual axes architecture that uses two simple lenses oriented with the illumination and collection axes crossed at an angle theta. This design offers high axial resolution, long working distance, and reduced noise from scattered light. Moreover, we introduce the novel method of post-objective scanning so that the design can be scaled down to millimeter dimensions. We demonstrate 1 to 2 micron resolution at 488 nm with a tabletop prototype using readily available optics, and show fluorescence images with high SNR and contrast collected from specimens expressing GFP. In the R21 phase of this proposal, we will 1) modify the optics and detectors of the tabletop prototype to significantly improve the fluorescence throughput and achieve collection times compatible with in vivo imaging; 2) test this optimized tabletop prototype ex vivo, and 3) provide a detailed design of the MEMS confocal theta prototype. In the R33 phase, we will 1) fabricate, assemble, and package a 5 mm diameter confocal theta microscope, 2) characterize the imaging performance ex vivo, and 3) collect fluorescence images from transgenic mice in vivo. The 5 mm prototype is a demonstration of a new class of confocal microscopes whose ultimate size will approach a theoretical limit of 1 mm. Miniaturization to this scale will permit insertion through the uterine cervix or implantation into the cranium of small animal models for studying embryonic development, cell-cell interactions, cell migration, and tumor biology.
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