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.
Large scale measurement and monitoring of neural activity at the subcellular level in the brains of model organisms such as zebrafish and fruit flies is of utmost importance for neuroscience. In this proposed project, we create a new approach to achieve subcellular resolution over a 286 to 300m field of view. The ability to observe how large areas of the neural network function in concert at the axonal and synaptic level is necessary to further advance our understanding of the brain, with direct impacts on human health.
