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
- Arizona State University, 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
Human Frontiers Science Program Organization (LT000911/2018C)
- Jaewon Huh
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
Publication 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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Meiotic chromosome segregation relies on synapsis and crossover recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in 'homolog engagement' is sensed and transduced by meiotic cells is unknown. Here we report that in C. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break induction. Inactivation of CHK-2 terminates double-strand break formation and enables crossover designation and cell cycle progression. These findings illuminate how meiotic cells ensure crossover formation and accurate chromosome segregation.
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Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right nose, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.
