Flash-and-freeze electron microscopy: Coupling optogenetics with high-pressure freezing

Shigeki Watanabe, M. Wayne Davis, Erik M. Jorgensen

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

A complete understanding of neuronal functions requires observation of the synapse with high spatial and temporal resolution. Recently, we developed a method to combine optogenetics with electron microscopy to capture dynamic changes in synaptic morphology during neurotransmission. First we generated transgenic C. elegans animals expressing channelrhodopsin in specific neurons. We stimulated these neurons in intact animals using a home-built light stimulation device and modified specimen holders for the Leica EM Pact2 high-pressure freezer. Samples were subsequently frozen 20 ms after the light stimulus. We demonstrated that synaptic vesicle fusion intermediates could be captured using this technique. This method can be readily applied to other light-activatable molecules, such as caged compounds, light-switchable ligands, and photoactivatable proteins, to study dynamic changes in cells.

Original languageEnglish (US)
Title of host publicationNanoscale Imaging of Synapses
Subtitle of host publicationNew Concepts and Opportunities
PublisherHumana Press Inc.
Pages43-57
Number of pages15
ISBN (Print)9781461491781
DOIs
StatePublished - 2014
Externally publishedYes

Publication series

NameNeuromethods
Volume84
ISSN (Print)0893-2336
ISSN (Electronic)1940-6045

Keywords

  • 4-D electron microscopy
  • High-pressure freezing
  • Neurotransmission
  • Optogenetics
  • Synaptic vesicle exocytosis
  • Time-resolved electron microscopy

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Psychiatry and Mental health

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