Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Journal of Visualized Experiments : JoVE
Shuo LiShigeki Watanabe

Abstract

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 microg...Continue Reading

Citations

May 23, 2019·Archives of Pharmacal Research·Yeonsun JinSung Hoon Lee

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Methods Mentioned

BETA
fluorescence microscopy
electron microscopy
correlative microscopy
light

Software Mentioned

ImageJ
CLEM
Matlab

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