Does contamination buildup limit throughput for automated cryoEM?

Journal of Structural Biology
Anchi ChengBridget Carragher

Abstract

The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film...Continue Reading

References

May 1, 1988·Quarterly Reviews of Biophysics·J DubochetP Schultz
Dec 21, 2000·Journal of Structural Biology·B CarragherA Reilein
May 14, 2005·Journal of Structural Biology·Christian SulowayBridget Carragher

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Citations

Dec 17, 2015·Microscopy·Yong Zi TanBridget Carragher
May 7, 2011·Nuclear Instruments & Methods in Physics Research. Section A, Accelerators, Spectrometers, Detectors and Associated Equipment·Xiaojing HuangChris Jacobsen
Oct 28, 2006·Journal of Structural Biology·Christopher R BoothWah Chiu
Jan 5, 2014·Journal of Structural Biology·Scott M StaggMichael S Chapman
Sep 8, 2010·Journal of Structural Biology·William V NicholsonJohn Trinick
Jun 28, 2011·Journal of Structural Biology·Andreas KorinekJürgen M Plitzko
Mar 6, 2009·Journal of Structural Biology·Gabriel C LanderBridget Carragher
Apr 7, 2010·Physical Review Letters·Xiaojing HuangChris Jacobsen

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