A versatile Oblique Plane Microscope for large-scale and high-resolution imaging of subcellular dynamics
- University of Texas Southwestern Medical Center, United States
- University of Queensland, Australia
- Michigan State University, United States
- Baylor College of Medicine and Texas Children's Hospital, United States
- Chan Zuckerberg Biohub, United States
- Duke University, United States
- University of Arizona, United States
- Calico Life Sciences LLC, United States
Abstract
We present an Oblique Plane Microscope 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.
Data availability
Manuscript data is available on Zenodo, under the doi:10.5281/zenodo.4266823.
Article and author information
Author details
Alexandre F Carisey
Yoshihiko Kobayashi
Purushothama Rao Tata
Funding
Cancer Prevention and Research Institute of Texas (RR160057)
- Reto P Fiolka
National Institutes of Health (5P30CA142543)
- Kevin M Dean
Damon Runyon Cancer Research Foundation (DFS-24-17)
- Jens C Schmidt
Chan Zuckerberg Initiative (HCA3-0000000196)
- Purushothama Rao Tata
Chan Zuckerberg Initiative (HCA3-0000000196)
- Doug P Shepherd
Chan Zuckerberg Initiative (HCA3-0000000196)
- Yoshihiko Kobayashi
ARC (FT190100516)
- Samantha J Stehbens
Rebecca Cooper Medical Foundation (PG2018168)
- Samantha J Stehbens
University of Queensland Early Career Award (RM2018002613)
- Samantha J Stehbens
Company of Biologists (JCSTF1903138)
- Robert J Ju
Robert A. Welch Foundation (I-1950-20180324)
- Konstantin Dubrovinski
National Institutes of Health (R00 GM120386)
- Jens C Schmidt
National Institutes of Health (R01GM110066)
- Konstantin Dubrovinski
National Institutes of Health (R01HL068702)
- Doug P Shepherd
National Institutes of Health (R33CA235254)
- Reto P Fiolka
National Institutes of Health (R35GM133522)
- Reto P Fiolka
National Institutes of Health (K25 CA204526)
- Erik S Welf
National Institutes of Health (P30 CA142543)
- Carlos L Arteaga
National Institutes of Health (1R01MH120131-01A1)
- Kevin M Dean
National Institutes of Health (1R34NS121873)
- Kevin M Dean
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Melike Lakadamyali, University of Pennsylvania, United States
Version history
- Received: April 8, 2020
- Accepted: November 9, 2020
- Accepted Manuscript published: November 12, 2020 (version 1)
- Accepted Manuscript updated: November 16, 2020 (version 2)
- Version of Record published: December 1, 2020 (version 3)
- Version of Record updated: December 7, 2020 (version 4)
- Version of Record updated: February 1, 2021 (version 5)
Copyright
© 2020, Sapoznik et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A versatile Oblique Plane Microscope for large-scale and high-resolution imaging of subcellular dynamics
eLife 9:e57681.
https://doi.org/10.7554/eLife.57681
Further reading
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While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.
