In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate.

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C(s) corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.

Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate.

Imaging of weak amplitude and phase objects, such as unstained vitrified biological samples, by conventional transmission electron microscopy (TEM) suffers from poor object contrast since the amplitude and phase of the scattered electron wave change only very little. In phase contrast light microscopy the imaging of weak phase objects is greatly enhanced by the use of a quarter-wave phase plate, which produces high signal contrast by shifting the phase of the scattered light. An analogous quarter-wave plate for the electron microscope, designed as an electrostatic einzel lens, was proposed by Boersch in 1947 but the small dimensions of the device have impeded its realization up to now. We here present the first fabrication and application of a miniaturized electrostatic einzel lens driven as TEM quarter-wave phase plate. Phase modulation is generated by the electrostatic field confined to the inside of a microstructured ring electrode. This field affects the phase velocity of the unscattered part of the electron wave. By varying its strength the phase shift of the primary beam can be adjusted to pi/2, producing strong phase contrast independent of spatial frequency. The phase plate proves to be mechanically stable and does not impair image quality, in particular it does not reduce the high-resolution signal. The expected residual lens effect of the einzel lens is minimal. Our microlens is supported by conducting rods arranged in a threefold symmetry. This particular geometry provides optimized single-sideband signal transfer for spatial frequencies otherwise obstructed by the supporting rods.

Zero-loss image formation and modified contrast transfer theory in EFTEM.

For a weak phase/weak amplitude object the information transfer in the imaging process of TEM is described by the common formalism of the contrast transfer function (CTF). So far the effects of inelastic scattering were not accounted for in this formalism. In conventional imaging they were simply neglected. In energy filtering TEM (EFTEM), where removal of inelastic electrons leads to higher specimen contrast, they were modelled by a global increase of the elastic amplitude contrast. Thus, the description of inelastic and elastic scattering was mixed. Here a new ansatz is proposed which treats elastic and inelastic contrast transfer separately by adding an inelastic contribution to the scattering potentials. In EFTEM this has the effect of adding a filter contrast which depends on the characteristics of the inelastic scattering. For samples with dominant plasmon loss the additional filter contrast is restricted to low resolution. Because of its strong dependence on the nature of the inelastic scattering process, the filter contrast cannot in general be unified with the conventional elastic amplitude contrast. The modified CTF theory for EFTEM was tested experimentally on a variety of samples. Images of amorphous layers of copper, aluminium, and carbon films, as well as zero-loss images of proteins embedded in amorphous ice were evaluated. The values of the parameters of the additional filter contrast were determined for carbon film and proteins embedded in vitrified ice. Comparison of different CTF models used to reconstruct 3D volumes from zero-loss images confirmed that best agreement with the atomic model is attained with the new, modified CTF theory.