Skip to main content Accessibility help
×
Home

An Improved Holey Carbon Film for Cryo-Electron Microscopy

  • Joel Quispe (a1), John Damiano (a2), Stephen E. Mick (a2), David P. Nackashi (a2), Denis Fellmann (a1), Teddy G. Ajero (a1), Bridget Carragher (a1) and Clinton S. Potter (a1)...

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.

Copyright

Corresponding author

Corresponding author. E-mail: joelq@scripps.edu

References

Hide All

REFERENCES

Downing, K.H. (2003). Support films with uniform hole size. Microsc Today 11, 54.
Dubochet, J., Adrian, M., Chang, J.J., Homo, J.C., Lepault, J., McDowall, A.W. & Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21, 129228.
Ermantraut, E., Wohlfart, K. & Tichelaar, W. (1998). Perforated support foils with re-defined hole size, shape and arrangement. Ultramicroscopy 74, 7581.
Henderson, R. (1995). The potential and limitations of neutrons, electrons, and X-rays for atomic resolution microscopy of unstained biological macromolecules. Q Rev Biophys 28, 171193.
Murray, J. (1987). Preparation of holey carbon films suitable for cryo-electron microscopy. J Electron Microsc Tech 5, 285290.
Quispe, J., Banez, R., Carragher, B. & Potter, C.S. (2004). Improving automation for cryo-EM specimen preparation. Microsc Microanal 10(Suppl. S02), 15081509.
Stagg, S.M., Lander, G., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D., Mallick, S.P., Avila, R.M., Carragher, B. & Potter, C.S. (2006). Automated cryoEM data acquisition and analysis of 284,742 particles of GroEL. J Struct Biol 155, 470481.
Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg, S., Potter, C.S. & Carragher, B. (2005). Automated molecular microscopy: The new Leginon system. J Struct Biol 151, 4160.
Taylor, K.A. & Glaeser, R.M. (1974). Electron diffraction of frozen, hydrated protein crystals. Science 186, 10361037.

Keywords

Metrics

Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed