Open-top axially swept light sheet microscopy

Open-top light sheet microscopy (OT-LSM) is a specialized microscopy for the high throughput cellular imaging of large tissues including optically cleared tissues by having all the optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with the improved axial resolution. OTAS-LSM swept the tightly focused excitation light sheet across the imaging FOV by using an electro tunable lens (ETL) and collected emission light at the focus of light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a water prism for simplicity and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment. The image resolutions were 1.5-1.6μm, and approximately 140% of the aberration-free theoretical values. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine and it could visualize neuronal networks in the single cell level. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues.