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J Struct Biol. 2014 Jan;185(1):42-7. doi: 10.1016/j.jsb.2013.11.002. Epub 2013 Nov 21.

Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device.

Author information

1
Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada.
2
Molecular Structure and Function Program, The Hospital for Sick Children Research Institute, Toronto, Canada; Department of Biochemistry, The University of Toronto, Canada; Department of Medical Biophysics, The University of Toronto, Canada. Electronic address: john.rubinstein@utoronto.ca.

Abstract

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 cryo-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 ∼500nm holes spaced 4μm 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.

KEYWORDS:

Contrast transfer function; Direct detector; Holey carbon; Nanofabrication; Specimen preparation; cryo-EM

PMID:
24269484
DOI:
10.1016/j.jsb.2013.11.002
[Indexed for MEDLINE]

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