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Does contamination buildup limit throughput for automated cryoEM?

Anchi Cheng1, Denis Fellmann, James Pulokas

  • 1National Resource for Automated Molecular Microscopy and Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. acheng@scripps.edu

Journal of Structural Biology
|April 25, 2006
PubMed
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Automated cryo electron microscopy allows acquiring over 250,000 images in 24 hours. Contamination buildup is minimal (1 angstrom/h) without film, ensuring data quality for long-term cryo-electron microscopy (cryo-EM) experiments using CCD cameras.

Area of Science:

  • Cryo-electron microscopy
  • Structural biology
  • Materials science

Background:

  • Automated systems in cryo-electron microscopy (cryo-EM) enable acquisition of large image datasets from single specimen grids.
  • Experiments can yield over 250,000 single particle images within 24 hours.
  • Concerns exist regarding specimen contamination affecting data quality during extended cryo-EM data acquisition sessions.

Purpose of the Study:

  • To systematically characterize specimen contamination during long-term cryo-EM experiments.
  • To assess the feasibility of acquiring over 1,000,000 particles by extending data acquisition.
  • To evaluate the impact of contamination on data quality in cryo-EM.

Main Methods:

  • Development of a reliable method for estimating specimen ice thickness.

Related Experiment Videos

  • Calibration of image contrast against ice thickness under varying magnifications, aperture positions, and defoci.
  • Systematic characterization of contamination on specimens at liquid nitrogen temperatures using standard side entry cryo stages.

    • Main Results:

    • The anti-contamination aperture is crucial for preventing specimen contamination.
    • Loading film into the microscope significantly increases contamination rates, especially within the first 3 hours.
    • In the absence of film, contamination rates were reproducibly limited to approximately 1 angstrom/hour.

    Conclusions:

    • Specimen contamination is not a major limiting factor for long-term cryo-EM experiments when using a CCD camera.
    • Careful experimental setup, including the use of an anti-contamination aperture and avoiding film insertion, is essential.
    • Extended cryo-EM data acquisition sessions for over 1,000,000 particles are feasible without compromising data quality due to contamination.