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Ultramicroscopy. 2018 Apr;187:26-33. doi: 10.1016/j.ultramic.2017.11.001. Epub 2018 Jan 12.

Ewald sphere correction using a single side-band image processing algorithm.

Author information

1
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: crusso@mrc-lmb.cam.ac.uk.
2
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

Abstract

Curvature of the Ewald sphere limits the resolution at which Fourier components in an image can be approximated as corresponding to a projection of the object. Since the radius of the Ewald sphere is inversely proportional to the wavelength of the imaging electrons, this normally imposes a limit on the thickness of specimen for which images can be easily interpreted to a particular resolution. Here we present a computational method for precisely correcting for the curvature of the Ewald sphere using defocused images that delocalise the high-resolution Fourier components from the primary image. By correcting for each approximately Friedel-symmetry-related sideband separately using two distinct complex transforms that effectively move the displaced Fourier components back to where they belong in the structure, we can determine the amplitude and phase of each of the Fourier components separately. This precisely accounts for the effect of Ewald sphere curvature over a bandwidth defined by the defocus and the size of the particle being imaged. We demonstrate this processing algorithm using: 1. simulated images of a particle with only a single, high-resolution Fourier component, and 2. experimental images of gold nanoparticles embedded in ice. Processing micrographs with this algorithm will allow higher resolution imaging of thicker specimens at lower energies without any image degradation or blurring due to errors made by the assumption of a flat Ewald sphere. Although the procedure will work best on images recorded with higher defocus settings than used normally, it should still improve 3D single-particle structure determination using images recorded at any defocus and any electron energy. Finally, since the Ewald sphere curvature is in a known direction in the third dimension which is parallel to the direction of view, this algorithm automatically determines the absolute hand of the specimen without the need for pairs of images with a known tilt angle difference.

KEYWORDS:

CTF; Depth of field; Ewald sphere; Single-particle reconstruction; Structure determination; cryoEM

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