Isotropic, aberration-corrected light sheet microscopy for rapid high-resolution imaging of cleared tissue

Light sheet microscopy is the ideal technique for multiscale imaging of large and cleared tissues, and it is desirable to achieve the highest possible isotropic resolution across the entire sample. However, isotropic resolution for a centimeter-sized sample has only been achieved with slow and often aberrated, axially scanned light sheets, resulting in a low resolution of several micrometers. Here, we introduce a compact, high-speed light sheet fluorescence microscope with isotropic sub-micron resolution optimized for cleared tissue. We introduce three major opto-mechanical innovations using off-the-shelf optics to achieve an isotropic resolution of 850 nm across samples up to 1 cm ³ and refractive indices ranging from 1.33 to 1.56. We show that combining an air objective and a meniscus lens achieves an axially swept light sheet with sub-micron diffraction-limited resolution, free of aberrations. The effective field of view is increased 2-fold by correcting the field curvature of the light sheet with a concave mirror in the remote focusing unit. Furthermore, the imaging speed is enhanced 10- fold by adapting the light sheet’s motion with a closed-loop feedback, reaching 100 frames per second while maintaining isotropic resolution across the large field of view. Finally, we showcase the performance of our light sheet system for imaging from subcellular up to centimeter scale in cleared mouse cochlea and brain.