A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics

Abstract

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Transcriptional profiling reveals potential involvement of microvillous TRPM5-expressing cells in viral infection of the olfactory epithelium

“…For three-dimensional imaging, a high numerical aperture (NA) oblique plane microscope was used [ 21 , 74 ]. Briefly, this variant on a light sheet microscope only uses one objective to interface with the sample.…”

“…Emitted fluorescence is detected through the same primary objective used for illumination. A secondary and tertiary objective optically resample the emitted fluorescence to image the fluorescence resulting from the obliquely launched light sheet onto a detector [ 21 , 74 ]. For the primary, secondary, and tertiary objectives we used a high-NA silicone immersion objective (Nikon × 100 NA 1.35, 0.28–0.31 mm working distance), a high-NA air immersion objective (Nikon × 40 NA 0.95, 0.25–0.16 mm working distance), and a bespoke glass-tipped objective (AMS-AGY v1.0, NA 1.0, 0 mm working distance), respectively.…”

“…For the primary, secondary, and tertiary objectives we used a high-NA silicone immersion objective (Nikon × 100 NA 1.35, 0.28–0.31 mm working distance), a high-NA air immersion objective (Nikon × 40 NA 0.95, 0.25–0.16 mm working distance), and a bespoke glass-tipped objective (AMS-AGY v1.0, NA 1.0, 0 mm working distance), respectively. Images were acquired by a high-speed scientific CMOS camera (Photometrics Prime BSI) using custom Python software ( [ 74 ], https://github.com/qi2lab/opm ).…”

A guidebook for DISCO tissue clearing

“…Alternatively, new LSFM utilizing axially swept light sheet such as MesoSPIM are promising for obtaining a more homogenous light penetration through the tissue, partially addressing this shortcoming. So far, LSFM is a growing field in microscopy and novel designs such as ctASLM (Chakraborty et al, 2019) or oblique plane microscopy (Sapoznik et al, 2020) substantially improve the resolution in xy and z, making the overall resolution better and more homogeneous. However, these new modalities are not commercially available, and it is therefore required that experts establish and maintain the microscope locally.…”

Absolute quantification and single-cell dose-response of cytosolic siRNA delivery

“…Other detection strategies are conceivable including modern large, high quantum-yield sCMOS-based, spinning-disk confocal systems with sparse pinhole patterns and high out-of-focus light suppression, but exploratory experiments indicated a higher detection limit for such a system. Various implementations of light sheet microscopy, for example, lattice light sheet 21 , HILO 22 , or other single-objective light sheet designs 23 would be attractive from a detection limit perspective but would generally restrict FOV and experimental throughput.…”