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. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument.

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. We demonstrate 3D imaging of intracellular processes, including cytoskeletal dynamics in single cell migration and collective wound healing for 1500 and 1000 time points, respectively. Further, we capture rapid biological processes, including trafficking of early endosomes with velocities exceeding 10 microns per second and calcium signaling in primary neurons.