Superresolution Light Sheet Microscopy for imaging model organisms with 200nm resolution

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Project Summary Light Sheet Microscopy has become an essential tool for investigating a wide variety of biological problems from developmental questions to mapping neural circuits. Light Sheet Microscopy is attractive due to its excellent optical sectioning in thick samples, low phototoxicity which can allow imaging over multiple days and high frame rates which make it possible to capture firing neurons. Many different types of Light Sheet Microscope have been designed and built, many of which have impressive capabilities. The high-speed Simultaneous Multi-View (hs- SiMView) system and the Swept Confocally Aligned Planar Excitation (SCAPE) microscope can perform volumetric imaging at 1 to 10 volumes per second with resolutions of 0.5 to 2 microns. While these systems produce wonderful results, they do not have the resolution to investigate sub-cellular details or trace axons and dendrites through dense neuropil. The Lattice Light Sheet Microscope (LLSM) can achieve an impressive sub- 200 nm resolution but the system is anisotropic and limited to smaller samples. Here we propose to develop a novel single-objective structured-illumination light-sheet microscope that will be capable of fast isotropic imaging at a resolution of 320 nm in all dimensions and resolutions down to 180 nm x 180 nm x 320 nm using a full structured illumination approach. This microscope will employ an easy upright sample mounting geometry and will further naturally incorporate a multi-direction light sheet which will help to eliminate shadowing artifacts. This system will have approximately 30 times the volumetric resolution of state- of-the-art Light Sheet Microscope systems for imaging zebrafish larvae or fruit flies. By allowing biologists to image model organisms with the resolution to investigate subcellular structures or trace neural processes, this project will help decode neural circuitry, decipher developmental pathways, and elucidate the mechanisms of disease.