A versatile Oblique Plane Microscope for large-scale and high-resolution imaging of subcellular dynamics
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
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.
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.
Metrics
-
- 31,502
- views
-
- 1,484
- downloads
-
- 137
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
Anionic lipid molecules, including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), are implicated in the regulation of epidermal growth factor receptor (EGFR). However, the role of the spatiotemporal dynamics of PI(4,5)P2 in the regulation of EGFR activity in living cells is not fully understood, as it is difficult to visualize the local lipid domains around EGFR. Here, we visualized both EGFR and PI(4,5)P2 nanodomains in the plasma membrane of HeLa cells using super-resolution single-molecule microscopy. The EGFR and PI(4,5)P2 nanodomains aggregated before stimulation with epidermal growth factor (EGF) through transient visits of EGFR to the PI(4,5)P2 nanodomains. The degree of coaggregation decreased after EGF stimulation and depended on phospholipase Cγ, the EGFR effector hydrolyzing PI(4,5)P2. Artificial reduction in the PI(4,5)P2 content of the plasma membrane reduced both the dimerization and autophosphorylation of EGFR after stimulation with EGF. Inhibition of PI(4,5)P2 hydrolysis after EGF stimulation decreased phosphorylation of EGFR-Thr654. Thus, EGFR kinase activity and the density of PI(4,5)P2 around EGFR molecules were found to be mutually regulated.
-
- Cell Biology
Cell survival in metazoans depends on cell attachment to the extracellular matrix (ECM) or to neighboring cells. Loss of such attachment triggers a type of programmed cell death known as anoikis, the acquisition of resistance to which is a key step in cancer development. The mechanisms underlying anoikis resistance remain unclear, however. The intracellular F-actin cytoskeleton plays a key role in sensing the loss of cell–ECM attachment, but how its disruption affects cell fate during such stress is not well understood. Here, we reveal a cell survival strategy characterized by the formation of a giant unilocular vacuole (GUVac) in the cytoplasm of the cells whose actin cytoskeleton is disrupted during loss of matrix attachment. Time-lapse imaging and electron microscopy showed that large vacuoles with a diameter of >500 nm accumulated early after inhibition of actin polymerization in cells in suspension culture, and that these vacuoles subsequently coalesced to form a GUVac. GUVac formation was found to result from a variation of a macropinocytosis-like process, characterized by the presence of inwardly curved membrane invaginations. This phenomenon relies on both F-actin depolymerization and the recruitment of septin proteins for micron-sized plasma membrane invagination. The vacuole fusion step during GUVac formation requires PI(3)P produced by VPS34 and PI3K-C2α on the surface of vacuoles. Furthermore, its induction after loss of matrix attachment conferred anoikis resistance. Our results thus show that the formation of a previously unrecognized organelle promotes cell survival in the face of altered actin and matrix environments.