Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

Abstract

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

“…4, Supplementary Table 5). As we previously demonstrated 13 , reflective diSPIM enables the collection of additional specimen views ( Fig. 4a), increasing information content and boosting spatiotemporal resolution.…”

“…The instant structured illumination microscopy (iSIM) system has been previously described 13 . For all experiments, a 60X NA=1.42 oil immersion objective (Olympus PlanApo N 60x Oil) was used, resulting in an image pixel size of 55.5 nm and a lateral resolution of ~150 nm.…”

“…The geometry of the diSPIM (0.8/0.8 NA) used for conventional and reflective imaging has been previously described 13 . Glass coverslips (24 mm x 50 mm x 0.17 mm, VWR, Cat.…”

“…The exposure time for each plane was 8 ms, and the stage-scanning step size for the volumetric imaging was 0.4 m, corresponding to 209 nm along the optical axis after deskewing. When deconvolving the data with a spatially variant PSF for resolution recovery and removal of epifluorescence contamination 13 , the excitation pattern was based on the measured lattice light-sheet dimensions (propagation distance of ~26.6 µm FHWM along the optical axis and a waist of 0.99 µm FWHM), and the detection PSF was simulated as a widefield PSF with 1.1 NA using the PSF generator ImageJ plugin (http://bigwww.epfl.ch/algorithms/psfgenerator/). The light-sheet dimensions were measured by sweeping the sheet axially through a 0.1 µm diameter fluosphere (ThermoFisher) while stepping the bead along the propagation length of the sheet.…”

“…A related practical problem is the computational burden associated with iterative deconvolution, which scales with the number of iterations. While manageable for single-view microscopes, deconvolving large multiview datasets can take days 12,13 , in many cases drastically exceeding the time for data acquisition.…”