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Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy
Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze,...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565139/ https://www.ncbi.nlm.nih.gov/pubmed/28518090 http://dx.doi.org/10.3791/55664 |
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author | Li, Shuo Raychaudhuri, Sumana Watanabe, Shigeki |
author_facet | Li, Shuo Raychaudhuri, Sumana Watanabe, Shigeki |
author_sort | Li, Shuo |
collection | PubMed |
description | Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution. |
format | Online Article Text |
id | pubmed-5565139 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-55651392017-09-01 Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy Li, Shuo Raychaudhuri, Sumana Watanabe, Shigeki J Vis Exp Cellular Biology Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution. MyJove Corporation 2017-05-01 /pmc/articles/PMC5565139/ /pubmed/28518090 http://dx.doi.org/10.3791/55664 Text en Copyright © 2017, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Cellular Biology Li, Shuo Raychaudhuri, Sumana Watanabe, Shigeki Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title | Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title_full | Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title_fullStr | Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title_full_unstemmed | Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title_short | Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy |
title_sort | flash-and-freeze: a novel technique to capture membrane dynamics with electron microscopy |
topic | Cellular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565139/ https://www.ncbi.nlm.nih.gov/pubmed/28518090 http://dx.doi.org/10.3791/55664 |
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