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Imaging Considerations for Cryo-Tomography of Organelles and Whole Cells at High Accelerating Voltage

Published online by Cambridge University Press:  02 July 2020

M. Marko ,
C.-E. Hsieh ,
C.A. Mannella  and
B.F. McEwen
M. Marko
Affiliation:
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
C.-E. Hsieh
Affiliation:
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
C.A. Mannella
Affiliation:
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
B.F. McEwen
Affiliation:
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
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Extract

Cryo-electron tomography can be used to obtain highly-detailed 3-D reconstructions of micrometer-sized biological structures which are unfixed, unstained and embedded in vitreous ice. Important practical considerations for acquiring the requisite tilt-series images are the choice of electron dose and defocus value, and the effects of contamination and ice thickness.

It is essential to reduce the total dose enough to prevent excessive ice radiolysis and damage to the specimen itself. To reduce the dose, a sensitive cooled CCD camera and microscope automation system are all but required. Because the specimens are thick, it is advantageous to use a high accelerating voltage, and/or energy filtering to reduce inelastic scattering. At high accelerating voltage, it is important to keep the dose high enough so that the signal is above the “scintillator noise” generated by the interaction of the high-energy electrons with the phosphor scintillator of the CCD camera. Initial test reconstructions indicate that scintillator noise, unlike electron shot noise, cannot be mitigated using dose fractionation. Fortunately, we find that large objects embedded in a thick (ca. 1 μm) vitrified ice layer are more resistant to beam damage (as well as charging) compared to smaller objects embedded in ice 100-200nm thick.

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy I. Molecular Approach
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 414 - 415
Copyright
Copyright © Microscopy Society of America

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References

1.McEwen, B.F., et al., these proceedings.Google Scholar
2.Mannella, C.A., et. al., these proceedings.Google Scholar
3.Grimm, R., et al., Biophys. J. 72(1997)482.CrossRefGoogle Scholar
4.Grimm, R., et al., Biophys. J. 74(1998)1031.CrossRefGoogle Scholar
5.Rath, B.K., et al., J. Struct. Biol. 120(1997)210.CrossRefGoogle Scholar
6.Downing, K. and Hendrickson, F.M., Ultramicroscopy 75(1999)215.CrossRefGoogle Scholar
7.McEwen, B.F., et al., Ultramicroscopy 60(1995)357.CrossRefGoogle Scholar
8.Murray, J.M., J. Ultrastr. Mol. Struct. Res. 95(1986)196CrossRefGoogle Scholar
9.Crowther, R.A., et al., Proc. R. Soc. London A 317(1970)319.Google Scholar
10. This research was supported by the Biological Microscopy and Image Reconstruction Resource which is funded by NIH grant RR01219.Google Scholar