Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

  • Sarah H. Shahmoradian, Mauricio R. Galiano, Chengbiao Wu, Shurui Chen, Matthew N. Rasband, William C. Mobley, Wah Chiu
  • Journal of Visualized Experiments, February 2014, MyJove Corporation
  • DOI: 10.3791/50783

Preparing neurons for nanoscale imaging without chemical fixation by cryo-electron microscopy

What is it about?

Neurons communicate through their long extensions (neurites/axons) to neighboring neurons. This article explains - both visually and textually - how to prepare rat neurons for seeing their neurites/axons at the nanoscale level in a close-to-native state, with no chemical fixation. They are preserved in such a state because the neurons are plunge-frozen in cryo temperatures instead of chemically fixed or treated, prior to being imaged by cryo-electron microscopy.

Why is it important?

In recent years, this technique has revealed several new features in neurites/neurons at the nanometer level. However, a detailed step-by-step guide has not been publicly available until now. Such a guide should help ensure reproducibility. Furthermore, the fact that the samples are preserved in vitreous ice (avoiding chemical fixation) is extremely appealing. This ensures the neurites/neurons are more "native." Thus, results obtained through this technique are more physiologically relevant.

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The following have contributed to this page: Sarah Shahmoradian