Retrofit implementation of Zernike phase plate imaging for cryo-TEM

Abstract

In-focus phase-plate imaging is particularly beneficial for cryo-TEM because it offers a substantial overall increase in image contrast, without an electron dose penalty, and it simplifies image interpretation. We show how phase-plate cryo-TEM can be implemented with an appropriate existing TEM, and provide a basic practical introduction to use of thin-film (carbon) phase plates. We point out potential pitfalls of phase-plate operation, and discuss solutions. We provide information on evaluating a particular TEM for its suitability.

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

“…Although both FEI and JEOL can now provide complete HFPP systems for their current-model TEMs, a PP retrofit could be adapted for any suitable TEM, as we have done for our JEOL JEM-4000FX (Marko et al, 2011), with the help of a competent machinist/engineer.…”

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

“…One of the key requirements for setting up a ZPC system on the JEM2200FS microscope is positioning the Zernike phase plate at the back focal plane of the objective lens 5,8,25 (Figs. 1, 2).…”

“…This setup is termed “on-plane conditions” (Fig. 2) 5,8,25 . Due to effects of the cut-on frequency, this requirement is much more stringent than the alignment requirement for the objective aperture, which can be fairly far from the back focal plane with no adverse effects (Fig.…”

“…The electron microscope used in the present protocol is equipped with electron optics designed to remedy this problem: the Zernike phase plate 4–8 , a thin carbon film with a small circular hole in the middle. When the electron beam passes through the phase plate, the scattered electrons undergo an approximately π/2 phase shift with respect to the unscattered central electron beam, so that the CTF switches from a sine to a cosine function.…”

“…That is, contrast remains strong at low resolutions (frequencies) close to the origin of the CTF, regardless of defocus. This effect, in principle, enables researchers to achieve nearly in-focus imaging without compromising low resolution contrast and eliminates the need to make corrections for the CTF 4,5,8–11 . As a test of the Zernike phase plate technology, the electron cryo-microscopy (cryoEM) structures of a 2D crystalline array of bacteriorhodopsin and of an icosahedral virus particle were obtained at ~10–12 Å without CTF correction 12 .…”