In-focus electrostatic Zach phase plate imaging for transmission electron microscopy with tunable phase contrast of frozen hydrated biological samples.

Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

Optimized Ar(+)-ion milling procedure for TEM cross-section sample preparation.

High-quality samples are indispensable for every reliable transmission electron microscopy (TEM) investigation. In order to predict optimized parameters for the final Ar(+)-ion milling preparation step, topographical changes of symmetrical cross-section samples by the sputtering process were modeled by two-dimensional Monte-Carlo simulations. Due to its well-known sputtering yield of Ar(+)-ions and its easiness in mechanical preparation Si was used as model system. The simulations are based on a modified parameterized description of the sputtering yield of Ar(+)-ions on Si summarized from literature. The formation of a wedge-shaped profile, as commonly observed during double-sector ion milling of cross-section samples, was reproduced by the simulations, independent of the sputtering angle. Moreover, the preparation of wide, plane parallel sample areas by alternating single-sector ion milling is predicted by the simulations. These findings were validated by a systematic ion-milling study (single-sector vs. double-sector milling at various sputtering angles) using Si cross-section samples as well as two other material-science examples. The presented systematic single-sector ion-milling procedure is applicable for most Ar(+)-ion mills, which allow simultaneous milling from both sides of a TEM sample (top and bottom) in an azimuthally restricted sector perpendicular to the central epoxy line of that cross-sectional TEM sample. The procedure is based on the alternating milling of the two halves of the TEM sample instead of double-sector milling of the whole sample. Furthermore, various other practical aspects are issued like the dependency of the topographical quality of the final sample on parameters like epoxy thickness and incident angle.