期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 185, 期 1, 页码 42-47出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2013.11.002
关键词
cryo-EM; Holey carbon; Specimen preparation; Direct detector; Contrast transfer function; Nanofabrication
资金
- SickKids Summer Research studentship
- Ontario Ministry of Research and Innovation
- Natural Science and Engineering Research Council Discovery Grant [401724-12]
Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient ciyo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with similar to 500 nm holes spaced 4 mu m apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently. (C) 2013 Elsevier Inc. All rights reserved.
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