Methods for testing Zernike phase plates and a report on silicon-based phase plates with reduced charging and improved ageing characteristics

Abstract

Imaging with Zernike phase plates is increasingly being used in cryo-TEM tomography and cryo-EM single-particle applications. However, rapid ageing of the phase plates, together with the cost and effort in producing them, present serious obstacles to widespread adoption. We are experimenting with phase plates based on silicon chips that have thin windows; such phase plates could be mass-produced and made available at moderate cost. The windows are coated with conductive layers to reduce charging, and this considerably extends the useful life of the phase plates compared to traditional pure-carbon phase plates. However, a compromise must be reached between robustness and transmission through the phase-plate film. Details are given on testing phase-plate performance by means of imaging an amorphous thin film and evaluating the power spectra of the images.

Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

“…Although good contrast could still be obtained, contrast reversal was often observed (Fig. 3), as was distortion of the expected power spectrum (Marko et al, 2013). Although contamination was suspected (which could lead to dark-field imaging or excessive phase shift of the unscattered beam), the cause was not determined.…”

Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages

“…Currently, charging of the phase plate is the biggest obstacle for getting high quality image data of the specimen. Each phase plate has a different usable lifespan due to various possible causes 8,27,28 . Some plates show signs of charging in the very first exposure to the electron beam, whereas others deteriorate only gradually after exposure 5,8 (Fig.…”

“…The manner and extent of changes in the FFT of charging phase plates vary depending on the initial quality of the phase plate and the cumulative electron exposure to the phase plate. Phase plate charging often manifests as an aberrant pattern or unusual Fourier oscillations at low spatial frequency that do not match the zeros of the CTF for any defocus value 27 (Fig. 5b–c).…”

On the role of inelastic scattering in phase-plate transmission electron microscopy

“…The electrostatic Zach PP with only one supporting rod provides reduced obstruction and a soft cut-on frequency [9]. The major limitation for the application of PPs is contamination and charging as well as degradation, which is addressed by varying the material of thin-film PPs [10,11] or implementing a heating device [12].…”