Flash-and-freeze

A novel technique to capture membrane dynamics with electron microscopy

Research output: Contribution to journalArticle

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, "flashand-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.

Original languageEnglish (US)
Article numbere55664
JournalJournal of Visualized Experiments
Volume2017
Issue number123
DOIs
StatePublished - May 1 2017

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Electron microscopy
Electron Microscopy
Synaptic Vesicles
Membranes
Optogenetics
Freezing
Neurons
Fusion reactions
Light
Synaptic Membranes
Neurotransmitter Agents
Presynaptic Terminals
Cations
Visualization
Synaptic Transmission
Positive ions
Proteins
Membrane Proteins
Recovery
Electrons

Keywords

  • Cellular Biology
  • Channelrhodopsin
  • Electron microscopy
  • Endocytosis
  • Exocytosis
  • Flash-and-freeze
  • Freeze substitution
  • High-pressure freezing
  • Issue 123
  • Membrane dynamics
  • Optogenetics
  • Synapse
  • Time-resolved electron microscopy

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Flash-and-freeze : A novel technique to capture membrane dynamics with electron microscopy. / Li, Shuo; Raychaudhuri, Sumana; Watanabe, Shigeki.

In: Journal of Visualized Experiments, Vol. 2017, No. 123, e55664, 01.05.2017.

Research output: Contribution to journalArticle

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