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.
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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- Cell Biology
- Stem Cells and Regenerative Medicine
Conjugated linoleic acids (CLAs) can serve as a nutritional intervention to regulate quality, function, and fat infiltration in skeletal muscles, but the specific cytological mechanisms remain unknown. Here, we applied single-nucleus RNA-sequencing (snRNA-seq) to characterize the cytological mechanism of CLAs regulates fat infiltration in skeletal muscles based on pig models. We investigated the regulatory effects of CLAs on cell populations and molecular characteristics in pig muscles and found CLAs could promote the transformation of fast glycolytic myofibers into slow oxidative myofibers. We also observed three subpopulations including SCD+/DGAT2+, FABP5+/SIAH1+, and PDE4D+/PDE7B+ subclusters in adipocytes and CLAs could increase the percentage of SCD+/DGAT2+ adipocytes. RNA velocity analysis showed FABP5+/SIAH1+ and PDE4D+/PDE7B+ adipocytes could differentiate into SCD+/DGAT2+ adipocytes. We further verified the differentiated trajectory of mature adipocytes and identified PDE4D+/PDE7B+ adipocytes could differentiate into SCD+/DGAT2+ and FABP5+/SIAH1+ adipocytes by using high intramuscular fat (IMF) content Laiwu pig models. The cell-cell communication analysis identified the interaction network between adipocytes and other subclusters such as fibro/adipogenic progenitors (FAPs). Pseudotemporal trajectory analysis and RNA velocity analysis also showed FAPs could differentiate into PDE4D+/PDE7B+ preadipocytes and we discovered the differentiated trajectory of preadipocytes into mature adipocytes. Besides, we found CLAs could promote FAPs differentiate into SCD+/DGAT2+ adipocytes via inhibiting c-Jun N-terminal kinase (JNK) signaling pathway in vitro. This study provides a foundation for regulating fat infiltration in skeletal muscles by using nutritional strategies and provides potential opportunities to serve pig as an animal model to study human fat infiltrated diseases.
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- Cell Biology
- Medicine
Background:
It has been reported that loss of PCBP2 led to increased reactive oxygen species (ROS) production and accelerated cell aging. Knockdown of PCBP2 in HCT116 cells leads to significant downregulation of fibroblast growth factor 2 (FGF2). Here, we tried to elucidate the intrinsic factors and potential mechanisms of bone marrow mesenchymal stromal cells (BMSCs) aging from the interactions among PCBP2, ROS, and FGF2.
Methods:
Unlabeled quantitative proteomics were performed to show differentially expressed proteins in the replicative senescent human bone marrow mesenchymal stromal cells (RS-hBMSCs). ROS and FGF2 were detected in the loss-and-gain cell function experiments of PCBP2. The functional recovery experiments were performed to verify whether PCBP2 regulates cell function through ROS/FGF2-dependent ways.
Results:
PCBP2 expression was significantly lower in P10-hBMSCs. Knocking down the expression of PCBP2 inhibited the proliferation while accentuated the apoptosis and cell arrest of RS-hBMSCs. PCBP2 silence could increase the production of ROS. On the contrary, overexpression of PCBP2 increased the viability of both P3-hBMSCs and P10-hBMSCs significantly. Meanwhile, overexpression of PCBP2 led to significantly reduced expression of FGF2. Overexpression of FGF2 significantly offset the effect of PCBP2 overexpression in P10-hBMSCs, leading to decreased cell proliferation, increased apoptosis, and reduced G0/G1 phase ratio of the cells.
Conclusions:
This study initially elucidates that PCBP2 as an intrinsic aging factor regulates the replicative senescence of hBMSCs through the ROS-FGF2 signaling axis.
Funding:
This study was supported by the National Natural Science Foundation of China (82172474).
