Single-particle cryo-EM: alternative schemes to improve dose efficiency.

Imaging of biomolecules by ionizing radiation, such as electrons, causes radiation damage which introduces structural and compositional changes of the specimen. The total number of high-energy electrons per surface area that can be used for imaging in cryogenic electron microscopy (cryo-EM) is severely restricted due to radiation damage, resulting in low signal-to-noise ratios (SNR). High resolution details are dampened by the transfer function of the microscope and detector, and are the first to be lost as radiation damage alters the individual molecules which are presumed to be identical during averaging. As a consequence, radiation damage puts a limit on the particle size and sample heterogeneity with which electron microscopy (EM) can deal. Since a transmission EM (TEM) image is formed from the scattering process of the electron by the specimen interaction potential, radiation damage is inevitable. However, we can aim to maximize the information transfer for a given dose and increase the SNR by finding alternatives to the conventional phase-contrast cryo-EM techniques. Here some alternative transmission electron microscopy techniques are reviewed, including phase plate, multi-pass transmission electron microscopy, off-axis holography, ptychography and a quantum sorter. Their prospects for providing more or complementary structural information within the limited lifetime of the sample are discussed.

Experimental Demonstration of an Electrostatic Orbital Angular Momentum Sorter for Electron Beams.

The component of orbital angular momentum (OAM) in the propagation direction is one of the fundamental quantities of an electron wave function that describes its rotational symmetry and spatial chirality. Here, we demonstrate experimentally an electrostatic sorter that can be used to analyze the OAM states of electron beams in a transmission electron microscope. The device achieves postselection or sorting of OAM states after electron-material interactions, thereby allowing the study of new material properties such as the magnetic states of atoms. The required electron-optical configuration is achieved by using microelectromechanical systems technology and focused ion beam milling to control the electron phase electrostatically with a lateral resolution of 50 nm. An OAM resolution of 1.5ℏ is realized in tests on controlled electron vortex beams, with the perspective of reaching an optimal OAM resolution of 1ℏ in the near future.

Etching-Assisted Route to Heterophase Au Nanowires with Multiple Types of Active Surface Sites for Silane Oxidation.

The construction of multiple types of active sites on the surface of a metallic catalyst can markedly enhance its catalytic activity toward specific reactions. Here, we show that heterophase gold nanowires (Au NWs) with multiple types of active surface sites can be synthesized using an etching-assisted process, yielding the highest reported turnover frequency (TOF) for Au catalysts toward the silane oxidation reaction by far. We use synchrotron powder X-ray diffraction (PXRD) and aberration-corrected (scanning) transmission electron microscopy (TEM) to show that the Au NWs contain heterophase structures, planar defects, and surface steps. Moreover, the contribution to the catalytic performance from each type of active sites was clarified. Surface steps on the Au NW catalysts, which were identified using aberration-corrected (scanning) TEM, were shown to play the most important role in enhancing the catalytic performance. By using synchrotron PXRD, it was shown that a small ratio of metastable phases within Au NWs can enhance catalytic activity by a factor of 1.35, providing a further route to improve catalytic activity. Of the three types of surface active sites, surface terminations of planar defects such as twin boundaries (TB) and stacking faults (SF) are less active than metastable phases and surface steps for Au catalysts toward the silane oxidation reaction. Such an etching-assisted synthesis of heterophase Au NWs promises to open new possibilities for catalysis, plasmonic, optics, and electrical applications.

Manipulation of dipolar magnetism in low-dimensional iron oxide nanoparticle assemblies.

The manipulation of magnetic states in nanoparticle supercrystals promises new pathways to design nanocrystalline magnetic materials and devices. Trench-patterned silicon substrates were used as templates to guide the self-assembly of iron oxide nanoparticles. Grazing incidence small angle X-ray scattering shows that the nanoparticles form a long-range ordered structure along the trench direction while in the direction perpendicular to the trenches, no coherent structure is observable. Electron holography provides evidence of an ordered magnetic state of nanoparticle moments in the remanent state after the application of a saturation magnetic field parallel to the trenches. However, a disordered magnetic state was observed in a perpendicular geometry. Hysteresis loops indicate that the nanoparticle moments form a superferromagnetic state for the geometry parallel to the trenches. Memory effect investigations reveal that the disordered magnetic state corresponds to a collective superspin glass state in the perpendicular geometry, while the superferromagnetic state in the parallel geometry suppresses the superspin glass state.

Towards a holographic approach to spherical aberration correction in scanning transmission electron microscopy.

Recent progress in phase modulation using nanofabricated electron holograms has demonstrated how the phase of an electron beam can be controlled. In this paper, we apply this concept to the correction of spherical aberration in a scanning transmission electron microscope and demonstrate an improvement in spatial resolution. Such a holographic approach to spherical aberration correction is advantageous for its simplicity and cost-effectiveness.

TEM sample preparation of lithographically patterned permalloy nanostructures on silicon nitride membranes.

We have prepared ferromagnetic nanostructures intended for the investigation of high-frequency magnetization dynamics in permalloy (Py) nanodisks using Lorentz transmission electron microscopy (LTEM) and electron holography. Py nanodisks were fabricated on thin silicon nitride (SiN) membranes using three different fabrication methods: lift-off, ion beam etching (IBE), and stencil lithography. They were further analyzed using different instruments, including scanning electron microscopy, LTEM, and electron holography. A bilayer of positive PMMA resist was utilized in the first fabrication method to form an undercut structure that guarantees a clean lift-off procedure. The second approach used dry etching with an Ar beam to etch a thin Py film, while an electron-beam-patterned negative resist mask kept the desired structure. In the third process, nanostencils (shadow masks) with submicrometer apertures were milled on SiN membranes using a focused ion beam. Furthermore, we have developed a new TEM sample preparation method, where we fabricated Py nanostructures on a bulk substrate with a SiN buffer layer and etched the substrate to create a thin SiN membrane under the Py nanostructure. Finally, we observed the vortex dynamics of the Py nanodisk under magnetic fields using LTEM and off-axis electron holography. A correlation between preparation methods and the properties of the Py nanostructures was made.

Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene.

Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built de novo. However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphene-coated grids exhibit a higher b factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.

Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM.

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call "specimen induced decoherence", is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.

Shaping of Electron Beams Using Sculpted Thin Films.

Electron beam shaping by sculpted thin films relies on electron-matter interactions and the wave nature of electrons. It can be used to study physical phenomena of special electron beams and to develop technological applications in electron microscopy that offer new and improved measurement techniques and increased resolution in different imaging modes. In this Perspective, we review recent applications of sculpted thin films for electron orbital angular momentum sorting, improvements in phase contrast transmission electron microscopy, and aberration correction. For the latter, we also present new results of our work toward correction of the spherical aberration of Lorentz scanning transmission electron microscopes and suggest a method to correct chromatic aberration using thin films. This review provides practical insight for researchers in the field and motivates future progress in electron microscopy.

Efficient large field of view electron phase imaging using near-field electron ptychography with a diffuser.

Most implementations of ptychography on the electron microscope operate in scanning transmission (STEM) mode, where a small focussed probe beam is rapidly scanned across the sample. In this paper we introduce a different approach based on near-field ptychography, where the focussed beam is replaced by a wide-field, structured illumination, realised through a purpose-designed etched Silicon Nitride window. We show that fields of view as large as 100 µm2 can be imaged using the new approach, and that quantitative electron phase images can be reconstructed from as few as nine near-field diffraction pattern measurements.

Continuous illumination picosecond imaging using a delay line detector in a transmission electron microscope.

Progress towards analysing transitions between steady states demands improvements in time-resolved imaging, both for fundamental research and for applications in information technology. Transmission electron microscopy is a powerful technique for investigating the atomic structure, chemical composition and electromagnetic properties of materials with high spatial resolution and precision. However, the extraction of information about dynamic processes in the ps time regime is often not possible without extensive modification to the instrument while requiring careful control of the operation conditions to not compromise the beam quality. Here, we avoid these drawbacks by combining a delay line detector with continuous illumination in a transmission electron microscope. We visualize the gyration of a magnetic vortex core in real space and show that magnetization dynamics up to frequencies of 2.3 GHz can be resolved with down to ∼122ps temporal resolution by studying the interaction of an electron beam with a microwave magnetic field. In the future, this approach promises to provide access to resonant dynamics by combining high spatial resolution with sub-ns temporal resolution.

Design of electrostatic phase elements for sorting the orbital angular momentum of electrons.

The orbital angular momentum (OAM) sorter is a new electron optical device for measuring an electron's OAM. It is based on two phase elements, which are referred to as the "unwrapper" and "corrector" and are placed in Fourier-conjugate planes in an electron microscope. The most convenient implementation of this concept is based on the use of electrostatic phase elements, such as a charged needle as the unwrapper and a set of electrodes with alternating charges as the corrector. Here, we use simulations to assess the role of imperfections in such a device, in comparison to an ideal sorter. We show that the finite length of the needle and the boundary conditions introduce astigmatism, which leads to detrimental cross-talk in the OAM spectrum. We demonstrate that an improved setup comprising three charged needles can be used to compensate for this aberration, allowing measurements with a level of cross-talk in the OAM spectrum that is comparable to the ideal case.

Spherical aberration correction in a scanning transmission electron microscope using a sculpted thin film.

Nearly eighty years ago, Scherzer showed that rotationally symmetric, charge-free, static electron lenses are limited by an unavoidable, positive spherical aberration. Following a long struggle, a major breakthrough in the spatial resolution of electron microscopes was reached two decades ago by abandoning the first of these conditions, with the successful development of multipole aberration correctors. Here, we use a refractive silicon nitride thin film to tackle the second of Scherzer's constraints and demonstrate an alternative method for correcting spherical aberration in a scanning transmission electron microscope. We reveal features in Si and Cu samples that cannot be resolved in an uncorrected microscope. Our thin film corrector can be implemented as an immediate low cost upgrade to existing electron microscopes without re-engineering of the electron column or complicated operation protocols and can be extended to the correction of additional aberrations.

Generation of electron vortex beams using line charges via the electrostatic Aharonov-Bohm effect.

It has recently been shown that an electron vortex beam can be generated by the magnetic field surrounding the tip of a dipole-like magnet. This approach can be described using the magnetic Aharonov-Bohm effect and is associated with the fact that the end of a long magnetic rod can be treated approximately as a magnetic monopole. However, it is difficult to vary the magnetisation of the rod in such a setup and the electron beam vorticity is fixed for a given tip shape. Here, we show how a similar behaviour, which has the advantage of easy tuneability, can be achieved by making use of the electrostatic Aharonov-Bohm effect associated with an electrostatic dipole line. We highlight the analogies between the magnetic and electrostatic cases and use simulations of in-focus, Fresnel and Fraunhofer images to show that a device based on two parallel, oppositely charged lines that each have a constant charge density can be used to generate a tuneable electron vortex beam. We assess the effect of using a dipole line that has a finite length and show that if the charge densities on the two lines are different then an additional biprism-like effect is superimposed on the electron-optical phase.

Measuring the orbital angular momentum spectrum of an electron beam.

Electron waves that carry orbital angular momentum (OAM) are characterized by a quantized and unbounded magnetic dipole moment parallel to their propagation direction. When interacting with magnetic materials, the wavefunctions of such electrons are inherently modified. Such variations therefore motivate the need to analyse electron wavefunctions, especially their wavefronts, to obtain information regarding the material's structure. Here, we propose, design and demonstrate the performance of a device based on nanoscale holograms for measuring an electron's OAM components by spatially separating them. We sort pure and superposed OAM states of electrons with OAM values of between -10 and 10. We employ the device to analyse the OAM spectrum of electrons that have been affected by a micron-scale magnetic dipole, thus establishing that our sorter can be an instrument for nanoscale magnetic spectroscopy.