Pan-ASLM: a high-resolution and large field-of-view light sheet microscope for Expansion Microscopy

Expansion microscopy, a super-resolution fluorescence microscopy technique in which samples are expanded up to ∼8,000 times (after 20-fold expansion) their original volume, places high demands on the microscopes used to image the expanded samples. To reveal nanoscale cellular ultrastructure in meaningful sample volumes, the instruments need to feature a large field of view and working distance. Simultaneously, they need to offer a high three-dimensional resolution to avoid counteracting the resolution improvement achieved by the expansion process. Here, we present pan-ASLM, a high resolution, large field-of-view light-sheet microscope developed for expanded samples, based on the Axially Swept Light Sheet Microscopy (ASLM) technique. pan-ASLM allows imaging over a 640 µm x 640 µm field of view with lateral and axial resolutions of 566 nm and 457 nm, respectively, and features an image acquisition speed of up to 20 fps (183 Mvoxels/sec). It offers ∼1200x higher imaging speed, a ∼7x larger field of view, and ∼2x better axial resolution than the standard confocal microscopes typically used for expanded samples. We validate the new microscope design through imaging of pan-expanded HeLa cells as well as mouse kidney and brain tissue.