Isotropic imaging across spatial scales with axially swept light-sheet microscopy.

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems.

Imaging Subcellular Dynamics with Fast and Light-Efficient Volumetrically Parallelized Microscopy.

In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells.