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Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
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Biology
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JoVE Journal Biology
Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

DOI:
10.3791/62886-v

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08:20 min

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October 25, 2021

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, , , ,
1Max Planck Institute of Biochemistry, 2Max Planck Institute for Biophysics, 3Fondazione Human Technopole

Chapters

  • 00:04Introduction
  • 00:36Cryo-Fluorescence Light Microscopy (Cryo-FLM) of Plunge‐Frozen Grids with Cells
  • 01:51Focused Ion Beam (FIB) Milling
  • 05:47Correlative Transmission Electron Microscopy (TEM)
  • 07:02Results: Visualizing the Endocytic Protein Deposit in the Cell with Cryo‐ET
  • 07:38Conclusion

Summary

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Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Tags

Keywords: 3D-correlative FIB MillingCryo-electron TomographyCryo-fluorescence MicroscopyCryo-FIB-SEMSEM Grid OverviewFLM-SEM CorrelationIon Beam ImagingCellular Ultrastructure

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