Contrast analysis of cryo-images of n-paraffin recorded at 400 kV out to 2.1 A resolution.

n-Paraffin was used as a test specimen for evaluating the relative merits of 400-kV versus 100-kV electron microscopy in recording data for electron crystallographic analysis of beam-sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright-field images were recorded at 400 kV at a specimen temperature of -167 degrees C. Using the flood-beam imaging technique the best R-value is 0.08 for all reflections in the resolution zone from 4 to 3 A. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2A we have found R = 0.02. Using the spot-scan imaging technique, on the other hand, R was measured to be 0.42 for the reflections between 4- and 3-A resolution. This amount of relative contrast is 1.7 times that observed at 100 kV. Reflections at 3-2 A displayed an R-value of 0.05. Besides obtaining higher R-values when applying the spot-scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast-attenuating factors, including the modulation transfer function of the photographic film and the cryo-holder, envelope functions for spatial and temporal coherence and lens and high-tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot-scan does offer important improvements in contrast levels, which can be very useful in determining the three-dimensional structure from protein crystals.