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An Improved Holey Carbon Film for Cryo-Electron Microscopy

Published online by Cambridge University Press:  28 September 2007

Joel Quispe ,
John Damiano ,
Stephen E. Mick ,
David P. Nackashi ,
Denis Fellmann ,
Teddy G. Ajero ,
Bridget Carragher  and
Clinton S. Potter
Joel Quispe
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
John Damiano
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
Stephen E. Mick
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
David P. Nackashi
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
Denis Fellmann
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Teddy G. Ajero
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Bridget Carragher
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Clinton S. Potter
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
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Abstract

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

Keywords

TEMcryo-electron microscopyholey carbon filmsautomation
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 13 , Issue 5 , October 2007 , pp. 365 - 371
Copyright
© 2007 Microscopy Society of America

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References

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