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Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

Published online by Cambridge University Press:  24 November 2016

Michael Marko ,
Chyongere Hsieh ,
Eric Leith ,
David Mastronarde  and
Sohei Motoki
Michael Marko*
Affiliation:
NY State Department of Health, Wadsworth Center, PO Box 509, Albany, NY 12201, USA College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY 12203, USA
Chyongere Hsieh
Affiliation:
NY State Department of Health, Wadsworth Center, PO Box 509, Albany, NY 12201, USA
Eric Leith
Affiliation:
Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
David Mastronarde
Affiliation:
Department of MCD Biology, University of Colorado Boulder, Boulder, CO 80309, USA
Sohei Motoki
Affiliation:
JEOL USA, 11 Dearborn Road, Peabody, MA 01960, USA
*
*Corresponding author. mike.marko.em@gmail.com
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Abstract

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

Keywords

phase platecontrast-transfer functioncryo electron tomography (cryo-ET)cryo electron microscopy (cryo-EM)SerialEM
Type
Instrumentation and Software Techniques
Information
Microscopy and Microanalysis , Volume 22 , Issue 6 , December 2016 , pp. 1316 - 1328
Copyright
© Microscopy Society of America 2016 

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References

Danev, R. & Baumeister, W. (2016). Cryo-EM single particle analysis with the Volta phase plate. Elife, 5. pii: e13046. doi: 10.7554/eLife.13046.Google Scholar
Danev, R., Buijsse, B., Khosouei, M., Plitzko, J.M. & Baumeister, W. (2014). Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc Nat Acad Sci U S A 111, 15635–15640.Google Scholar
Danev, R., Glaeser, R.M. & Nagayama, K. (2009). Practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy. Ultramicroscopy 109, 312–325.CrossRefGoogle ScholarPubMed
Glaeser, R.M. (2013). Methods for imaging weak-phase objects in electron microscopy. Rev Sci Instr 85, 111101–111117.Google Scholar
Hu, B., Margolin, W., Molineux, I.J. & Liu, J. (2015). Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc Nat Acad Sci U S A 112, E4919–E4928.Google Scholar
Mahamid, J., Pfeffer, S., Schaffer, M., Villa, E., Danev, R., Cuellar, L.K., Förster, F., Hyman, A.A., Plitzko, J.M. & Baumeister, W. (2016). Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972.Google Scholar
Malac, M., Beleggia, M., Kawasaki, M., Li, P. & Egerton, R.F. (2012). Convenient contrast enhancement by a hole-free phase plate. Ultramicroscopy 118, 77–89.Google Scholar
Marko, M., Leith, A., Hsieh, C. & Danev, R. (2011). Retrofit implementation of Zernike phase plate imaging for cryo-TEM. J Struct Biol 174, 400–412.Google Scholar
Marko, M., Meng, X., Hsieh, C., Roussie, J. & Striemer, C. (2013). Methods for testing Zernike phase plates and a report on silicon-based phase plates with reduced charging and improved ageing characteristics. J Struct Biol 184, 237–244.Google Scholar
Mastronarde, D.N. (1997). Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 120, 343–352.Google Scholar
Mastronarde, D.N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36–51.Google Scholar

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