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.
MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as ...needles or plates measuring only a few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.
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.
•In-focus phase contrast is achieved with charging-free, single-sideband apertures.•Conformal coating with evaporated carbon avoids patch-potential effects.•Heating above 300°C avoids contamination effects.•Cryo-EM performance is evaluated with streptavidin monolayer crystals.
•Signal-loss associated with the VPP increases linearly as a function of spatial frequency.•This loss represents a decrease in the spectral detective quantum efficiency, DQE(s), which is attributable ...solely to the VPP.•At 2 Å resolution the DQE has been reduced to ~40% for both 200 kV and 300 kV beam energy.•Experiments have narrowed down the root cause of this reduction, pointing at a surface effect of the VPP material that influences both the unscattered and the scattered part of the electron beam.
The Volta Phase Plate (VPP) consists of a heated, thin film that is placed in the same plane as the focused diffraction pattern of an electron microscope. A change in surface potential develops at the point irradiated by the intense, unscattered electron beam, and this altered surface potential produces, in turn, a phase shift between the unscattered and scattered parts of the electron wave. While the VPP thus increases the image contrast for weak-phase objects at low spatial frequencies, we report here that it also leads to the loss of an increasing fraction of the signal at higher resolution. The approximately linear dependence (with increasing resolution) of this loss has been quantified at 200 kV and 300 kV, using evaporated-carbon films of different thicknesses as Volta phase plates. In all cases, the loss of signal remains almost independent of variation of the conditions and parameters that were tested. In spite of having done a number of additional, discovery-based experiments, the cause of this loss of signal remains unexplained at this point.
Abstract
We propose a scheme for constructing a phase plate for use in an ultrafast Zernike-type phase contrast electron microscope, based on the interaction of the electron beam with a strongly ...focused, high-power femtosecond laser pulse and a pulsed electron beam. Analytical expressions for the phase shift using the time-averaged ponderomotive potential and a paraxial approximation for the focused laser beam are presented, as well as more rigorous quasiclassical simulations based on the quantum phase integral along classical, relativistic electron trajectories in an accurate, non-paraxial description of the laser beam. The results are shown to agree well unless the laser beam is focused to a waist size below a wavelength. For realistic (off-the-shelf) laser parameters the optimum phase shift of -π/2 is shown to be achievable. When combined with RF-cavity based electron chopping and compression techniques to produce electron pulses, a femtosecond regime pulsed phase contrast microscope can be constructed. The feasibility and robustness of the scheme are further investigated using the simulations, leading to motivated choices for design parameters such as wavelength, focus size and polarization.
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.
•Investigation of optimum biprism position implementation into state-of-the-art TEM.•Reduction of artefacts, especially vignetting in double-biprism electron holography.•Biprism vibration and most stable imaging conditions in double-biprism holography.•Demonstration of the optimized double-biprism setup using a thin GaN-foil.
•Electrically conductive materials cause thermal magnetic field fluctuations.•Thermal magnetic field fluctuations from a laser phase plate causes resolution loss.•This loss is reduced by moving ...conductive materials farther from the electron beam.
We identify thermal magnetic field fluctuations, caused by thermal electron motion (“Johnson noise”) in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.
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