Subcellular Resolution Light Sheet Microscope with a Large Field of View

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PROJECT SUMMARY The ability to capture large regions of the neural network in living model organisms such as zebrafish and fruit flies at a subcellular scale will further advance neurological research. In this project, we aim to provide a microscopy platform that is able to capture images of a 286 to 300 micron area of the nervous system in living zebrafish and fruit files at subcellular resolution. We plan to combine super-resolution techniques with light sheet fluorescence microscopy to accomplish this goal. Aim 1 combines super-resolution structured illumination microscopy (SR-SIM) with multi-direction illumination light sheet fluorescence microscopy in a single objective configuration. We expect that the system should be able to achieve a resolution of 161nm in all lateral directions, and axial resolution of 458nm and 916nm in 3D SR-SIM mode and 2D SR-SIM mode, respectively. We expect that the proposed method will provide a highly detailed image of the entire midbrain structure and activity in 6 to 7 week post-fertilization zebrafish larvae. Aim 2 will achieve isotropic resolution at subcellular level (241nm lateral, 336nm axial) while maintaining a 286 to 300 micron field of view. We expect that the proposed method will result in the capability to image dynamics of postsynaptic filopodia across multiple muscle groups over a long period of time (longer than 1 hour). Aim 3 will develop a novel computer reconstruction algorithm to boost the effective frame rate and alleviate the artifacts in the resulting image. In the final result, we expect to see a 20% decrease in artifacts and an increase in speed by a factor of 3.