Abstract
Here we have investigated the potential improvement in imaging vitrified biological specimens with the help of a chromatic aberration (Cc)-corrector. Using a newly developed chromatic aberration-corrected electron cryomicroscope (cryo-EM), the phase contrast micrographs comprising signals from both the zero loss and low energy loss (1-100 eV) channels were used to determine the structure of a pseudorabies virus (PRV). Using an energy selecting, electron energy loss spectrometer after the Cc corrector, datasets were collected separately yet sequentially on the same specimen to allow quantification of the signal in each of the respective channels. Both zero-loss first and low-loss first datasets were acquired. For further comparison, datasets from non-Cc-corrected cryo-EM were also collected. 3D reconstructions of the virus from all 4 above datasets are presented including two maps reconstructed only from electrons having lost 18-28 eV of energy whilst transiting the specimen. Although the amplitude contrast of the signals in the low-loss micrographs is opposite in sign to that of typical defocused images using only elastically scattered electrons, we show that the inelastic maps also contain detailed structural information which can be recovered using Cc correction. This can be verified by comparing the maps from each of the channels. Interestingly, the resolution of the reconstructed volume from the low-loss electrons decreases with defocus independently of the purely elastic electron images taken from the same specimen, which is consistent with previous theoretical predictions and experimental measurements of specimen induced decoherence using room temperature test specimens. Together, these results indicate that the inelastically scattered electrons do indeed contain useful phase contrast signals, particularly for thick specimens, but their recovery requires imaging as close to in-focus as possible. Combing the optical correction demonstrated here, with a lossless phase plate for in focus imaging, may offer the most straightforward way to achieve this in the future.