Charging phenomena observed on biological specimens in a 400-kV electron cryo-microscope

Abstract

There is an ever present need in electron microscopy of biological specimens to improve the quality of the recorded data to approach the atomic resolution capability of the modem instruments. The method adopted which has yielded the only significant advance in this direction is to directly phase theelectron diffraction amplitudes of 2-dimensional crystals using the Fourier transforms of high resolution images. Cryo methods have improved the preservation and stability of the specimen to the relatively high electron dose irradiation which is required for imaging compared to electron diffraction. In the cases, however, where the actual power of the structure factors was compared with those that were transferred by the microscope’s imaging system, a large attenuation was measured. Spot-scan imaging has been shown to improve the image quality by reducing the beam-induced movement. Charging of the specimen appears to cause similar deleterious effects on image resolution, but it requires different methods aimed at resolution improvement. Proteins are insulators and because of secondary electron loss they will become positively charged during irradiation. Defocused diffraction brightfield imagescan display the charged nature of an irradiated specimen as an apparent increase in mass. A comparison wasmade of the degree of charging and discharging between using 100 and 400 keV electrons on streptavidin crystals in glucose and catalase crystals in amorphous ice. We show that when the region of the irradiated sample includes part of the supporting carbon film, the charge build-up is prevented.