An Improved Holey Carbon Film for Cryo-Electron Microscopy

• Published in: Microscopy and microanalysis Vol. 13; no. 5; pp. 365 - 371
• Main Authors: Quispe, Joel, Damiano, John, Mick, Stephen E., Nackashi, David P., Fellmann, Denis, Ajero, Teddy G., Carragher, Bridget, Potter, Clinton S.
• Format: Journal Article
• Language: English
• Published: New York, USA Cambridge University Press 01.10.2007; Oxford University Press
• Subjects: Arrays; BIOLOGICAL APPLICATIONS; Carbon; Data collection; Electron microscopy; Ice thickness; Microscopy; Molecular weight; Silicon wafers; Specimen preparation; Thin films; automation; holey carbon films; TEM; cryo-electron microscopy
• ISSN: 1431-9276; 1435-8115
• DOI: 10.1017/S1431927607070791

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.