High-Resolution, Large Imaging Volume, and Multi-View Single Objective Light-Sheet Microscopy

Abstract

Light-sheet microscopy has become the preferred method for long-term imaging of large living samples because of its low photo-invasiveness and good optical sectioning capabilities. Unfortunately, refraction and scattering often pose obstacles to light-sheet propagation and limit imaging depth. This is typically addressed by imaging multiple complementary views to obtain high and uniform image quality throughout the sample. However, multi-view imaging often requires complex multi-objective configurations that complicate sample mounting, or sample rotation that decreases imaging speed. Recent developments in single-objective light-sheet microscopy have shown that it is possible to achieve high spatio-temporal resolution with a single objective for both illumination and detection. Here we describe a single-objective light-sheet microscope that achieves: (i) high-resolution and large field-of-view imaging via a custom remote focusing objective; (ii) simpler design and ergonomics by remote placement of coverslips; (iii) fast volumetric imaging by means of light-sheet stabilised stage scanning – a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iv) multi-view imaging by means of dual orthogonal light-sheet illumination. Finally, we demonstrate the speed, field of view and resolution of our novel instrument by imaging zebrafish tail development.

Dual-view light-sheet imaging through a tilted glass interface using a deformable mirror

“…Another approach to the problem of light-sheet imaging through glass uses a single objective for both excitation and detection [ 7 – 10 ]. Recent implementations achieve spatial resolution that matches high-NA spinning disk confocal imaging [ 8 ], and multi-view imaging via beam inversion by two additional galvo mirrors [ 11 ]. The latter, however, has non-orthogonal PSFs from the two views, which is likely to reduce the improvement of z-resolution from multi-view fusion.…”

“…Glaser et al [ 6 ] used a solid-immersion lens (SIL) that minimized aberrations in both excitation and detection arms, also in a single-view arrangement. Alternatively, several other designs [ 7 – 11 ] used one high-NA objective for both excitation and detection, at the cost of adding secondary and tertiary objectives used to re-image the tilted detection PSF onto the sensor.…”

Reconstructing unobserved cellular states from paired single-cell lineage tracing and transcriptomics data

“…Utilizing time-series datasets from model organisms for which cell lineages are stereotypical, like that of C. elegans , could be of great use towards this aim (e.g., [22]). Beyond this, we envision that maturing light-sheet microscopy technologies [23, 24] could be paired with unbiased readout of cellular transcriptomic states to create comprehensive molecular atlases of dynamic processes.…”

Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

“…A third approach to the problem of light-sheet imaging through glass is by using a single objective for both excitation and detection (Dunsby, 2008;Sapoznik et al, 2020;Voleti et al, 2019;Yang et al, 2019). More recent implementations achieve spatial resolution that matches high-NA spinning disk confocal imaging (Sapoznik et al, 2020), and multi-view imaging via beam inversion by two additional galvo mirrors (Yang et al, 2020). These systems require samples which have refractive index close to the imaging medium.…”

“…Glaser et al (Glaser et al, 2019) used a solid-immersion lens (SIL) that minimized aberrations in both excitation and detection arms, also in single-view arrangement. Alternatively, several other SLFM designs (Dunsby, 2008;Sapoznik et al, 2020;Voleti et al, 2019;Yang et al, 2019Yang et al, , 2020 used one high-NA objective for both excitation and detection (single-view), at the cost of adding secondary and tertiary objectives used to re-image the tilted detection PSF onto the sensor.…”