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

Light-sheet microscopy is ideal for imaging large and cleared tissues, but achieving a high isotropic resolution for a centimeter-sized sample is limited by slow and often aberrated, axially scanned light sheets. Here, we introduce a compact, high-speed light-sheet fluorescence microscope achieving 850 nm isotropic resolution across cleared samples up to 1 cm³ and refractive indices ranging from 1.33 to 1.56. Using off-the-shelf optics, we combine an air objective and a meniscus lens with an axially swept light sheet to achieve diffraction-limited resolution and aberration correction. The effective field of view is increased by twofold by correcting the field curvature of the light sheet using a concave mirror in the remote focusing unit. Adapting the light sheet’s motion with a closed-loop feedback enhances the imaging speed by tenfold, reaching 100 frames per second while maintaining resolution and field of view. We benchmark the system performance across scales, from subcellular structure up to centimeter scale, using various clearing methods.