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Electron cryotomography sample preparation using the Vitrobot.

Cristina V Iancu1, William F Tivol, Jordan B Schooler

  • 1Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.

Nature Protocols
|April 5, 2007
PubMed
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This study details protocols for electron cryotomography sample preparation, focusing on vitrification using an automated plunge-freezing device. These methods enable high-resolution structural analysis of biological specimens in a near-native state.

Area of Science:

  • Structural biology
  • Biophysics
  • Cryo-electron microscopy

Background:

  • Electron cryotomography (ECT) offers the highest resolution for studying complex biological structures.
  • Specimen preservation in a near-native state is crucial for accurate structural determination.
  • Optimizing sample preparation, including ice thickness and fiducial markers, is essential for successful ECT.

Purpose of the Study:

  • To provide detailed protocols for electron cryotomography sample preparation.
  • To describe methods for introducing fiducial markers and achieving optimal sample vitrification.
  • To detail the operation and parameters of an automated plunge-freezing device for sample vitrification.

Main Methods:

  • Detailed protocols for sample preparation in electron cryotomography.

Related Experiment Videos

  • Introduction of fiducial markers (gold nanoparticles) for image alignment.
  • Vitrification of samples using an automated, climate-controlled plunge-freezing device (FEI Vitrobot).

    • Main Results:

    • Established protocols for sample preparation, including fiducialization and vitrification.
    • Demonstrated the effectiveness of an automated plunge-freezing device for reproducible sample vitrification.
    • Optimized parameters for the FEI Vitrobot, enabling efficient sample preparation (8 grids in 1.5-2 hours).

    Conclusions:

    • The described protocols facilitate high-resolution structural analysis using electron cryotomography.
    • Automated plunge-freezing significantly improves the efficiency and reproducibility of sample vitrification.
    • These detailed methods are valuable for researchers studying the structure of large protein complexes, viruses, organelles, and cells.