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

Etai Sapoznik,Bin Yang,Purushothama Rao Tata,Arnaldo Marín,Jaewon Huh,Rory Kruithoff,Yoshihiko Kobayashi,Konstantin Doubrovinski,Bo-Jui Chang,David Broadbent,Theresa Pohlkamp,Alfred Millett-Sikking,Carlos L Arteaga ,Ariella B Hanker ,Erik S Welf ,Jens C Schmidt ,Andrew G York ,Kyung Min Lee ,Kevin M Dean ,Samantha J Stehbens ,Robert J Ju ,Douglas P Shepherd ,Alexandre F Carisey ,Evgenia V Azarova ,Reto Fiolka

doi:10.7554/elife.57681

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.

Highlights

Light-sheet fluorescence microscopy (LSFM) first generated significant interest in the biological community as a result of its ability to image developing embryos with single-cell resolution, inherent optical sectioning, low phototoxicity and high temporal resolution (Huisken et al, 2004)
High-resolution light-sheet microscopy has yet to be widely adopted in biological laboratories and core facilities owing to routine problems with sample drift, contamination, and lack of user friendliness
We show that an oblique plane microscope (OPM) with customized optics overcomes these challenges, and combines the ease of traditional sample mounting, environment maintenance, and multi-position stage control with the gentle, subcellular imaging afforded by selective plane illumination

Summary

Introduction

Light-sheet fluorescence microscopy (LSFM) first generated significant interest in the biological community as a result of its ability to image developing embryos with single-cell resolution, inherent optical sectioning, low phototoxicity and high temporal resolution (Huisken et al, 2004). Despite its advantages over other imaging modalities, its widespread adoption remains limited In part, this is due to the slow adoption of cutting-edge LSFM systems by commercial entities, and the requirement that each lab assemble, align, maintain, operate their own LSFM instruments. The highest resolution LSFM systems, the reliance on high-NA water-dipping objectives places the sample in direct contact with non-sterile optical surfaces, which compromises long-term imaging. These matters are made worse because modern LSFM systems often lack modalities that render microscopy routinely useful for non-experts, including sample environmental control (e.g. CO2, temperature, and humidity), oculars, and hardware-based autofocusing schemes

Methods

Results

Conclusion