Bart Buijsse

Volta potential phase plate for in-focus phase contrast transmission electron microscopy

Significance Biological electron cryomicroscopy is limited by the radiation sensitivity of the samples and the consequent need to minimize exposure to the beam. This, in turn, results in low-contrast images with a poor signal-to-noise ratio. The current practice to improve phase contrast by defocusing results in contrast transfer functions necessitating image restoration to provide interpretable data. Phase plates enable in-focus phase contrast, but the existing ones, including the thin film Zernike-type phase plate, suffer from severe limitations, such as a short usable life span, fringing artifacts, and problems in using them in automated data acquisition procedures. The Volta phase plate presented here solves those problems and has the potential to become a practical solution for in-focus phase contrast in transmission electron microscopy. We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to ∼200 °C to prevent contamination and enable the Volta potential effect. The phase shift is created “on the fly” by the central diffraction beam eliminating the need for precise phase plate alignment. Images acquired with the Volta phase plate (VPP) show higher contrast and unlike Zernike phase plate images no fringing artifacts. Following installation into the microscope, the VPP has an initial settling time of about a week after which the phase shift behavior becomes stable. The VPP has a long service life and has been used for more than 6 mo without noticeable degradation in performance. The mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the VPP it is used in a constructive way. The exact physics and/or chemistry behind the process causing the Volta potential are not fully understood, but experimental evidence suggests that radiation-induced surface modification combined with a chemical equilibrium between the surface and residual gases in the vacuum play an important role.

Design of a hybrid double-sideband/single-sideband (schlieren) objective aperture suitable for electron microscopy.

A novel design is described for an aperture that blocks a half-plane of the electron diffraction pattern out to a desired scattering angle, and then--except for a narrow support beam--transmits all of the scattered electrons beyond that angle. Our proposed tulip-shaped design is thus a hybrid between the single-sideband (ssb) aperture, which blocks a full half-plane of the diffraction pattern, and the conventional (i.e. fully open) double-sideband (dsb) aperture. The benefits of this hybrid design include the fact that such an aperture allows one to obtain high-contrast images of weak-phase objects with the objective lens set to Scherzer defocus. We further demonstrate that such apertures can be fabricated from thin-foil materials by milling with a focused ion beam (FIB), and that such apertures are fully compatible with the requirements of imaging out to a resolution of at least 0.34nm. As is known from earlier work with single-sideband apertures, however, the edge of such an aperture can introduce unwanted, electrostatic phase shifts due to charging. The principal requirement for using such an aperture in a routine data-collection mode is thus to discover appropriate materials, protocols for fabrication and processing and conditions of use such that the hybrid aperture remains free of charging over long periods of time.

Minimizing electrostatic charging of an aperture used to produce in-focus phase contrast in the TEM

Microfabricated devices designed to provide phase contrast in the transmission electron microscope must be free of phase distortions caused by unexpected electrostatic effects. We find that such phase distortions occur even when a device is heated to 300 °C during use in order to avoid the formation of polymerized, carbonaceous contamination. Remaining factors that could cause unwanted phase distortions include patchy variations in the work function of a clean metal surface, radiation-induced formation of a localized oxide layer, and creation of a contact potential between an irradiated area and the surround due to radiation-induced structural changes. We show that coating a microfabricated device with evaporated carbon apparently eliminates the problem of patchy variation in the work function. Furthermore, we show that a carbon-coated titanium device is superior to a carbon-coated gold device, with respect to radiation-induced electrostatic effects. A carbon-coated, hybrid double-sideband/single-sideband aperture is used to record in-focus, cryo-EM images of monolayer crystals of streptavidin. Images showing no systematic phase error due to charging are achievable under conditions of low-dose data collection. The contrast in such in-focus images is sufficient that one can readily see individual streptavidin tetramer molecules. Nevertheless, these carbon-coated devices perform well for only a limited length of time, and the cause of failure is not yet understood.

Advanced double-biprism holography with atomic resolution.

The optimum biprism position as suggested by Lichte (Ultramicroscopy 64 (1996) 79 [10]) was implemented into a state-of-the-art transmission electron microscope. For a setup optimized for atomic resolution holograms with a width of 30nm and a fringe spacing of 30pm, we investigated the practical improvements on hologram quality. The setup is additionally supplemented by a second biprism as suggested by Harada et al. (Applied Physics Letters 84 (2004) 3229 [12]). In order to estimate the possibilities and limitations of the double biprism setup, geometric optics arguments lead to calculation of the exploitable shadow width, necessary for strong reduction of biprism-induced artefacts. Additionally, we used the double biprism setup to estimate the biprism vibration, yielding the most stable imaging conditions with lowest overall fringe contrast damping. Electron holograms of GaN demonstrate the good match between experiment and simulation, also as a consequence of the improved stability.

Challenges in Phase Plate Product Development

The use of a phase plate in electron microscopy has shown renewed interest, triggered by a publication on this topic in 2001 by Danev and Nagayama [1]. This interest can be understood from the fact that many samples that are studied in TEM are weak phase objects. The use of a phase plate is the obvious method of choice to convert otherwise invisible phase modulations into visible amplitude modulations in the detected intensity profile. A phase plate can provide in-focus phase contrast, unlike the conventional method where a strong defocus is needed to generate contrast at low spatial resolutions, with the added consequence of introducing contrast reversals as a function of frequency.

Optimizing the FEI Volta Phase Plate for Efficient and Artefact-free Data Acquisition

FEI introduced the Volta Phase Plate (VPP) as a new product in 2014. It has been known for a long time that a phase plate should facilitate the observation of weak phase objects such as cryo-EM samples, but only with the introduction of the VPP have we started to exploit this advantage in practical applications. VPP-based tomography has revealed otherwise invisible particles, which could successfully be used in subtomogram averaging techniques [1]. VPP-based single particle work has enabled a near atomic reconstruction of a protein imaged close to focus [2]. The current insight is that VPP technology may provide answers in situations where conventional cryo-EM is reaching a limit, e.g. due to small particle size, or conformational heterogeneity of the particle.

Applications and New Investigations of the Volta Phase Plate

The use of the Volta Phase Plate for cryoTEM has increased significantly in the last year. Recently two important papers described the novelty of the technique [1] and highlighted the importance of the technique in making new biological discoveries [2]. Here we present our work on both new applications of the Volta Phase Plate to improve imaging of small protein complexes, and new explorations to enhance the information transfer of low coherency sources through the ability to work in focus.

Practical Aspects and Usage Tips for the Volta Phase Plate

Compared to other technical advances in transmission electron microscopy (TEM), such as brighter electron sources, energy filters, better optics and direct detection cameras, phase plates have lagged behind in both development and applications. The main reason for that is the difficulty in solving practical issues, such as beam-induced electrostatic charging, which cannot be reliably predicted or avoided based on theoretical research. Another reason is that phase plates add more complexity to the operation of the microscope and until not so long ago were not usable for automated data acquisition.

Automated Cryo-tomography and Single Particle Analysis with a New Type of Phase Plate

Recent years have shown an increased interest in the development and use of phase plates in cryo-EM. The oldest and the most productive type of phase plate is the carbon film Zernike phase plate [1]. It has been successfully used in cryo-tomography [2] and single particle analysis applications [3]. Despite its good performance the Zernike phase plate has a few pitfalls. One major practical hindrance is its short lifetime [4]. Typically within 10 days after being installed into the microscope its performance deteriorates to the point where it has to be exchanged. Another disadvantage of the Zernike phase plate is that it produces fringes around high-contrast features in the image, such as lipid membranes, support film edges etc [5]. Despite its shortcomings the Zernike phase plate has been the main motivation and experience generator in the last years.

A ‘tulip aperture’ providing in-focus phase-contrast

As is well known, a phase plate is aimed at providing optimal contrast of phase objects under infocus imaging conditions. Without using a phase plate, in TEM one has to rely on defocused-based imaging conditions, which can only provide contrast at the cost of resolution loss, contrast inversions (CTF oscillations) and associated delocalization. In principle, three types of phase plate schemes can be devised that provide in-focus contrast [1]: Zernike phase contrast (ZPC), Schlieren contrast (SC) and Hilbert differential contrast (HDC). All these schemes turn phase modulations of the exit wave of the specimen into intensity modulations at the detector. They all use a physical device in the back focal plane of the microscope. In the case of ZPC this can be a thin-film or electrostatic phase plate. For SC it is a knife edge that blocks half of the diffraction pattern (therefore the technique is sometimes called single-sideband imaging). For HDC the knife edge consists of a thin film that provides an appropriate phase shift to half the diffracted beam. SC is an attractive scheme in the sense that it does not depend on providing a well-defined phase shift to (part of) the diffraction pattern, it only requires a blocking device. However, there are disadvantages as well. Fifty percent of the diffracted beam is not used in the image formation, and the phases of the remaining Fourier component that build up the image are shifted by (π/2-γ(s)), with γ(s) the aberration function. A practical disadvantage is the extreme sensitivity of this technique to charging or patch field effects that occur at the edge of the device, causing unpredictable shifts in the phases of the Fourier components of the image. Here we report on a device that is a variation on the SC method [2]. It is an open aperture with a central tulip-shaped feature, as shown in Figure 1A. The tulip provides a knife edge only for the electrons that are scattered under low angles, corresponding with low spatial frequencies. Using this method, images can be recorded at Scherzer defocus. The usual Scherzer band of optimal contrast transfer is provided by the open area of the aperture, outside the central semi-disk, while the central area provides Schlieren contrast with constant contrast transfer of 0.5. Figure 1B shows the theoretical contrast transfer function (CTF). We demonstrate that tulip apertures can be fabricated from thin-foil materials by milling with a focused ion beam (FIB). Various materials have been tested, in search for a material that did not generate any potential fields beyond the device borders due to charging or change of the intrinsic material properties after e-beam exposure. One such a material, molybdenum, showed particularly good properties and showed no distortion of the Thon ring pattern, distortions which are indicative of unwanted phase changes of the diffracted beam. Figure 2 shows these Thon ring patterns in the presence of the device for various values of defocus. In-focus images were acquired of graphitized carbon particles. Imaging results show the improved contrast at low spatial frequencies while retaining the high-resolution information. As an example, the 0.34 nm lattice planes of graphite were visible in all directions. Computational image restoration is required when applying a tulip aperture. One must correct the systematic phase shifts of π/2-γ(s) that occur for the lower spatial frequencies of the image. This image restoration operation is nevertheless arguably less likely to cause errors than is the currently required CTF correction. For example, there are no zeros in the CTF that one must deal with, and, in addition, the images can be recorded with little or no defocus.