An alternative to the defocus-based methods for improving image contrast is the use of phase plates. Three types have developed to improve image contrast.

1. The simplest is the Zernike phase plate which is a thin film of amorphous carbon with a small central hole that is placed at the back focal plane of the objective lens. This allows the non-scattered electrons to pass without phase shift creating a relative phase change between the transmitted wave and scattered waves from a specimen, leading to enhanced contrast in the image. Unfortunately, over time these easily degrade due to contamination, and so need to be changed regularly. They also produce fringe artefacts in the images and are difficult to use because of alignment issues.
2. The most commonly used is the Volta Phase Plate (VPP), is a hole-free thin film of amorphous carbon is also mounted at back focal plane. This thin film plate is electrically charged creating a change in electrostatic potential, causing a phase shift in the unscattered transmitted wave relative to the scattered waves. Being hole-free the Volta phase plate doesn’t suffer from the problems associated with the Zernike phase plates.
3. The laser-based phase plate involves use of mirrors to amplify a laser beam tens of thousands of times and to use this to cause the phase shift. As there are no physical materials in the electron beam path there will be no information loss. These are still in their development but show great promise for the future.

Comparison of the types of phase plates. A. Zernike phase plate which utilises a hole in a carbon film to produce the phase shift. B. The Volta phase plate where the phase shift is produced by electrical change in the film. C. laser-based phase plate. The intense laser beam causes the phase shift. After JEOL.

As it is impossible to achieve perfect focus at all regions, a small amount of defocus must still be used, which can be determined during data processing.

The limited defocus with phase plates makes contrast-transfer-function fitting and correction difficult. For the VPP the carbon film itself causes electron scattering and there is a loss of signal of around 20% across all spatial frequencies. However, the contrast enhancement is valuable and often the only way to visualise small features in the tomograms. Therefore, many cryo-ET studies that use sub-tomogram averaging use phase-plate imaging. While initially used for SPA of small particles and cryo-tomography to improve contrast, it has now been shown that the use of VPP does not help to achieve better resolution and their use is becoming less common, especially for high resolution studies. However, the new developments in this area are generating a lot of interest so they could be a more valuable tool in the future.

Carbon black observed A. in focus B. with -5µm defocus and C. in focus using a Volta phase plate. The CTF for each are simulated. The use of the phase plate increases the contrast in the image but the shape of the CTF for the in focus images makes image correction more difficult. Images courtesy of Georg Ramm, Monash, University.