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

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

DOI:
10.3791/55664-v

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10:01 min

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May 01, 2017

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, ,
1Department of Cell Biology,Johns Hopkins School of Medicine, 2Department of Biochemistry and Molecular Biology,Johns Hopkins Bloomberg School of Public Health, 3Solomon H. Snyder Department of Neuroscience,Johns Hopkins School of Medicine

Chapters

  • 00:05Title
  • 00:52Flash-and-Freeze
  • 03:41Automated Freeze-substitution Unit-transfer
  • 06:40Sample Mounting and Imaging
  • 08:24Results: Representative Exocytosis and Endocytosis in Mouse Hippocampal Neurons
  • 08:56Conclusion

Summary

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We developed a novel technique in electron microscopy, "flash-and-freeze," that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

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Keywords: Electron MicroscopyMembrane DynamicsOptogeneticsSynaptic Vesicle RecyclingFlash-and-freezeHigh-pressure FreezingFree SubstitutionTime-resolved Imaging

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