Quantitative Energy-Filtered CRYO-EM of Hydrated Chromosome Fibers, Viruses, and Heavy Atoms

Abstract

We have calculated and experimentally measured the extent to which energy filtration improves the S/N in phase- and scattering-contrast images of frozen-hydrated molecules. Multiple scattering calculations based on PW (partial wave) complex scattering amplitudes agree quantitatively with 80 KV image intensities of 50-500nm-thick ice. For an objective aperture of 2.5 nm-1 the PW elastic mean free path is 280 nm. From the ratio of filtered to unfiltered image intensities from 50-500 nm of ice at 80 KV, we calculate an empirical inelastic mean free path of 180 nm. The PW calculations show that Beer's Law should be obeyed for thicknesses up to five mean free paths, whether or not energy filtration is used. Thus the bright-field signal can be used to determine the absolute mass distribution of molecules embedded in thick ice layers.Energy filtration removes background intensity due to inelastic scattering (Fig.1a), and increases the scattering signal from molecules (becoming the sum of both elastic electrons excluded by the objective aperture and inelastic electrons excluded by the energy filter). As the result, the calculated contrast is 2-15 times greater in the Zeiss EM902 than in a TEM without energy filtration (Fig.1b). 80 KeV energy-filtered images of frozen-hydrated molecules should have a higher S/N than unfiltered images taken at intermediate or high voltage (Fig. 1c,d). TMV contrast and S/N is improved by filtration and agrees with calculations (Fig.1b,2). The advantages of energy-filtration are important to the microscopy of chromatin fibers, which are excluded from ice layers ≤200 nm and viruses, which are usually excluded from layers ≤100 nm. Radial density reconstructions from near-focus energy-filtered images of frozen-hydrated chromatin from Thyone sperm show that the centers are not hollow, but very dense, probably containing DNA (Fig.3). Energy-filtered images of icosahedral viruses in even the thinnest possible ice have a much improved S/N ratio, decreasing the dose required for analysis (Fig.2a,b).