Microscopic charge fluctuations cause minimal contrast loss in cryoEM

The fluctuating granularity or "bee swarm" effect seen in highly defocussed transmission electron micrographs is caused by microscopic charge fluctuations in the specimen created by the illuminating beam. In the field of high-resolution single particle electron cryomicroscopy (cryoEM), there has been a concern that this fluctuating charge might cause defocus-dependent Thon ring fading which would degrade the final image. In this paper, we have analysed the 2.35 angstrom fringes from the (111) reflection in images of gold nanoparticles embedded in amorphous ice. We show that there is a small, yet detectable amount of defocus-dependent blurring of the lattice fringes when compared with those from a pure gold foil. The transverse electric field associated with the fluctuating charges on the insulating frozen water specimen deflects the electron beam locally and causes image blurring. The perturbation is small, decreasing the amplitude of the 2.35 angstrom reflection at 10 mu m defocus by about 7% (intensity by 14%). For smaller defocus values in the range 2-4 mu m and for resolutions that are typical in cryoEM, the effects of source incoherence and the bee swarm effect are negligible for all reasonable cryoEM imaging conditions, assuming that a field emission gun (FEG) is used for illumination. This leaves physical movement of the specimen due to radiation damage as the outstanding problem and the major source of contrast loss in cryoEM micrographs. (C) 2018 MRC Laboratory of Molecular Biology. Published by Elsevier B.V.