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

Abstract

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

Charging of carbon thin films in scanning and phase-plate transmission electron microscopy

“…Both groups used an aC thin film very similar to the EB aC analyzed in this work and worked at a thin film temperature of 60 • C and 225 • C [14] and 260 • C [31]. The beam diameter on the thin film was 1 µm and 25 nm for the series acquired at 225 • C (red line in Figure 7a) and 60…”

“…• C (blue line in Figure 7a), respectively. The beam diameter is specified to be between 20 and 100 nm for the HFPP image series from [31] whose analysis is depicted in Figure 7b. frames followed by an almost linear increase (blue line in Figure 7a).…”

“…If the beam is switched off, water molecules will start to re-adsorb on the illuminated area and possibly diffuse along the sample surface. However, both processes are assumed to happen in a time scale of minutes and hours which can explain the fact that HFPP patches remain charged for a longer period of time [14,31].…”

“…The described charging phenomenon is a plausible explanation for the functionality of the HFPP [8,31] and its implementations including the Volta PP [14]. The aC thin film used as Volta PP in [14] and as HFPP in [31] was fabricated using the identical device type as the EB aC thin film analyzed in this work. The Volta PP is used at a temperature of 225 • C which is slightly lower than the EB aC thin-film and HFPP temperature of 260 • C and the beam diameters were 1 µm, 20 nm and 100 nm, respectively.…”

Investigation of hole-free phase plate performance in transmission electron microscopy under different operation conditions by experiments and simulations

“…Particular attention has to be paid to the vertical position of the HFPP film which must coincide with the BFP of the objective lens. The position of the BFP is aligned by adjusting the C2 lens current [22]. If the HFPP is not in the BFP, the granularity of the HFPP film becomes visible in the image at intermediate magnification.…”