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We have fabricated and characterized a large-aperture electrooptic phase modulation device operating in the terahertz (THz) frequency range. The device consists of a 1.6 mm thick nematic liquid crystal placed between glass plates with a novel interdigitated electrode design. Using THz time-domain spectroscopy (THz-TDS) coupled with raster scanning imaging, we evaluated phase modulation across a 25 mm diameter LC device and mapped the spatial uniformity of phase shift. Our results confirm the functionality of the LC cell as a controllable quarter-wave plate at 0.26 THz and half-wave plate at 0.52 THz. This work contributes to the development of large-aperture and transmissive LC devices as low-cost phase plates for THz waves and paves the way for future applications in THz modulators. [ABSTRACT FROM AUTHOR]

The direct femtosecond laser writing of phase optical elements in glasses represents a promising technique for controlling light beams and developing new functional devices that operate on the birefringence effect. In this study, phase plates were written in the volume of silica and nanoporous glasses using a laser beam. For the first time, the chromatic dispersion curves of birefringence were measured for such plates using multiple methods. This allows the retardance of birefringent elements fabricated by direct laser writing to be estimated, even when using commercial birefringence analysis systems operating at a specific wavelength. [ABSTRACT FROM AUTHOR]

Stokes polarimetry was used to evaluate the birefringence of nanoporous alumina films. The transmittance of the film and the degree of polarization of the transmitted radiation were measured for angles of incidence at which the phase difference of the orthogonal polarized components of the transmitted radiation was λ/4 or λ/2. The porosity and film pore radii were estimated using the Maxwell–Garnet model. The possibility of creating achromatic phase plates based on nanoporous alumina film with a variable phase difference of the orthogonal polarized components of the transmitted radiation that can function as quarter-wave and half-wave plates is shown. [ABSTRACT FROM AUTHOR]

Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam‐scanning STED nanoscopy, require the co‐alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single‐mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off‐switching beam into a focal‐plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots. Here we describe novel designs of such so‐called 'easySTED phase plates' and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths. [ABSTRACT FROM AUTHOR]

Spatial resolution of the ground-based optical telescopes is limited by the Earth's atmospheric turbulence rather than the size of the telescope aperture. To restore the performance of a telescope to near diffraction limit, adaptive optics (AO) system should be designed and developed based on the turbulence characteristics at the telescope site. Crucial part in developing the AO system is, simulating the turbulence conditions of the atmosphere, estimation of its effect on wavefront and correction of the distorted wavefront in the laboratory. The essential components of laboratory AO system are phase plate and deformable mirrors (DMs). Phase plate simulates the turbulent conditions, and DM corrects the distorted wavefront. Performance of AO system depends on the characterization of these two devices. Fizeau interferometer and Shack–Hartmann wavefront sensor (SHWS) are used for the performance evaluation. Current work provides a detailed discussion about the tests carried out to characterize these two components and the results obtained. [ABSTRACT FROM AUTHOR]

In 1995, Hadley formulated an elegant effective-index model to describe the formation of transverse modes in optical cavities (Hadley 1995 Opt. Lett. 20 1483â€"5). We apply this model to Fabryâ€"Perot cavities and discuss its limitations, using the well-known paraxial solutions of these cavities as reference. We also introduce a new model, which we call the phase-plate model, that has less limitations and yields the correct first-order correction to the resonance frequencies for longer cavities. The analysis uses scalar optical fields in the paraxial limit. [ABSTRACT FROM AUTHOR]

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 $-\pi/2$ 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.

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. [ABSTRACT FROM AUTHOR]

Abstract Nowadays, the well-known cylindrical vector beams (CVBs) – the axially symmetric beam solution to the full-vector electromagnetic wave equation – are widely used for advanced laser material processing, optical manipulation and communication and have a great interest for data storage. Higher-order CVBs with polarisation order greater than one and superpositions of CVBs of various orders (hybrid CVBs) are especially of interest because of their great potential in contemporary optics. We performed a theoretical analysis of the transformation of first-order CVBs (radially and azimuthally polarised beams) into hybrid higher-order ones using phase elements with complex transmission functions in the form of the cosine or sine functions of the azimuthal angle. Binary multi-sector phase plates approximating such transmission functions were fabricated and experimentally investigated. The influence of the number of sectors and a height difference between neighbouring sectors, as well as the energy contribution of the different components in the generated hybrid higher-order CVBs were discussed in the context of polarisation transformation and vector optical field transformation in the focal region. The possibility of polarisation transformation, even in the case of weak focusing, is also demonstrated. The simple structure of the profile of such plates, their high diffraction efficiency and high damage threshold, as well as the easy-to-implement polarisation transformation principle provide advanced opportunities for high-efficient, quickly-switchable dynamic control of the generation of structured laser beams.

Modern subtractive and additive manufacturing techniques present new avenues for X‐ray optics with complex shapes and patterns. Refractive phase plates acting as glasses for X‐ray optics have been fabricated, and spherical aberration in refractive X‐ray lenses made from beryllium has been successfully corrected. A diamond phase plate made by femtosecond laser ablation was found to improve the Strehl ratio of a lens stack with a numerical aperture (NA) of 0.88 × 10−3 at 8.2 keV from 0.1 to 0.7. A polymer phase plate made by additive printing achieved an increase in the Strehl ratio of a lens stack at 35 keV with NA of 0.18 × 10−3 from 0.15 to 0.89, demonstrating diffraction‐limited nanofocusing at high X‐ray energies. [ABSTRACT FROM AUTHOR]

Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding and membrane insertion of C5b6, C7, C8, and C9. Using cryo-electron tomography with a Volta phase plate and subtomogram averaging, we imaged C5b-7, C5b-8, and C5b-9 complexes and determined the C5b-9 pore structure in lipid bilayers. The in situ C5b-9 pore structure at 2.3-nm resolution reveals a 10- to 11.5-nm cone-shaped pore starting with C5b678 and multiple copies of C9 that is poorly closed, yielding a seam between C9 and C6 substituting for the shorter β strands in C6 and C7. However, large variations of composite pore complexes are apparent in subtomograms. Oligomerized initiator complexes C5b-7 and C5b-8 show stages of membrane binding, deformation, and perforation that yield ∼3.5-nm-wide pores. These data indicate a dynamic process of pore formation that likely adapts to biological membranes under attack.

In a scanning transmission electron microscope (STEM), producing a high-resolution image generally requires an electron beam focused to the smallest point possible. However, the magnetic lenses used to focus the beam are unavoidably imperfect, introducing aberrations that limit resolution. Modern STEMs overcome this by using hardware aberration correctors comprised of many multipole elements, but these devices are complex, expensive, and can be difficult to tune. We demonstrate a design for an electrostatic phase plate that can act as an aberration corrector. The corrector is comprised of annular segments, each of which is an independent two-terminal device that can apply a constant or ramped phase shift to a portion of the electron beam. We show the improvement in image resolution using an electrostatic corrector. Engineering criteria impose that much of the beam within the probe-forming aperture be blocked by support bars, leading to large probe tails for the corrected probe that sample the specimen beyond the central lobe. We also show how this device can be used to create other STEM beam profiles such as vortex beams and probes with a high degree of phase diversity, which improve information transfer in ptychographic reconstructions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America.)

Highlights • Explanation of electron-beam induced charging of carbon thin film at LN2. • Observed negative charging is attributed to a local change in the work function. • Electron-stimulated desorption locally changes work function. • Scanning tunneling microscopy analysis of electron-beam induced conductivity. Abstract We studied the charging behavior of an amorphous carbon thin film kept at liquid-nitrogen temperature under focused electron-beam irradiation. Negative charging of the thin film is observed. The charging is attributed to a local change in the work function of the thin film induced by electron-stimulated desorption similar to the working principle of the hole free phase plate in its Volta potential implementation at elevated temperature. The negative bias of the irradiated film arises from the electron beam induced desorption of water molecules from the carbon film surface. The lack of positive charging, which is expected for non-conductive materials, is explained by a sufficient electrical conductivity of the carbon thin film even at liquid-nitrogen temperature as proven by multi-probe scanning tunneling microscopy and spectroscopy measurements. [ABSTRACT FROM AUTHOR]

Himalayan Chandra Telescope (HCT) is a 2-m class telescope located at Indian Astronomical observatory (IAO), Hanle, Jammu and Kashmir. It is one of the world’s highest observatories and is suitable for optical and infrared regions. Atmospheric turbulence plays a vital role deciding the seeing condition of an observatory. The atmospheric turbulance at IAO is simulated using a phase plate in an laboratory adaptive optical system. Wave-fronts are generated by the rotating phase plate and sensed using a Shack-Hartmann wavefront sensor (SHWS). Cross-correlation coefficients between the first wave-front and subsequent wave-fronts reduce as speed of phase plate increases. The range of phase plate speed where wave-front can be safely reconstructed are found with corresponding wind speed. Varying exposure times over which wind speed can be measured are also calculated. [ABSTRACT FROM AUTHOR]

First results on the experimental realisation of a 2 × 2 programmable phase plate for electrons are presented. The design consists of an array of electrostatic elements that influence the phase of electron waves passing through 4 separately controllable aperture holes. This functionality is demonstrated in a conventional transmission electron microscope operating at 300 kV and results are in very close agreement with theoretical predictions. The dynamic creation of a set of electron probes with different phase symmetry is demonstrated, thereby bringing adaptive optics in TEM one step closer to reality. The limitations of the current design and how to overcome these in the future are discussed. Simulations show how further evolved versions of the current proof of concept might open new and exciting application prospects for beam shaping and aberration correction. [ABSTRACT FROM AUTHOR]

Thin-film phase plates (PP) have become a valuable tool for the imaging of organic objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon (aC), which undergoes rapid aging under intense illumination with high-energy electrons. The limited lifetime of aC film PPs calls for alternative PP materials with improved material stability. This work presents thin-film PPs fabricated from the metallic glass alloy Zr 0.65 Al 0.075 Cu 0.275 (ZAC), which was identified as a promising PP material with beneficial properties, such as a large inelastic mean free path. An adverse effect of the ZAC alloy is the formation of a surface oxide layer in ambient air, which reduces the electrical conductivity and causes electrostatic charging in the electron beam. To avoid surface oxidation, the ZAC alloy is enclosed by thin aC layers. The resulting aC/ZAC/aC layer system is used to fabricate Zernike and Hilbert PPs. Phase-contrast TEM imaging is demonstrated for a sample of carbon nanotubes, which show strong contrast enhancement in PP TEM images. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware. These developments also greatly increased the usability and applicability of CET as a diagnostic and research tool, which is now enabling structural biologists to determine the structure of proteins in their native cellular environment to sub-nanometer resolution. These recent technical developments have stimulated us to update on our previous review (Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine. Ann Anat 191, 427–445) in which we described the fundamentals of CET. In this follow-up, we extend this basic description in order to explain the aforementioned recent advances, and describe related 3D techniques that can be applied to the anatomy of biological systems that are relevant for medicine. [ABSTRACT FROM AUTHOR]

We present the optical design and system characterization of an imaging microscope prototype at 121.6 nm. System engineering processes are demonstrated through the construction of a Schwarzschild microscope objective, including tolerance analysis, fabrication, alignment, and testing. Further improvements on the as-built system with a correction phase plate are proposed and analyzed. Finally, the microscope assembly and the imaging properties of the prototype are demonstrated. [ABSTRACT FROM AUTHOR]

Wavefront errors of rotationally parabolic refractive X-ray lenses made of beryllium (Be CRLs) have been recovered for various lens sets and X-ray beam configurations. Due to manufacturing via an embossing process, aberrations of individual lenses within the investigated ensemble are very similar. By deriving a mean single-lens deformation for the ensemble, aberrations of any arbitrary lens stack can be predicted from the ensemble with * = 0.034λ. Using these findings the expected focusing performance of current Be CRLs are modeled for relevant X-ray energies and bandwidths and it is shown that a correction of aberrations can be realised without prior lens characterization but simply based on the derived lens deformation. The performance of aberration-corrected Be CRLs is discussed and the applicability of aberration-correction demonstrated over wide X-ray energy ranges. [ABSTRACT FROM AUTHOR]

A systematic study on charging of carbon thin films under intense electron-beam irradiation was performed in a transmission electron microscope to identify the underlying physics for the functionality of hole-free phase plates. Thin amorphous carbon films fabricated by different deposition techniques and single-layer graphene were studied. Clean thin films at moderate temperatures show small negative charging while thin films kept at an elevated temperature are stable and not prone to beam-generated charging. The charging is attributed to electron-stimulated desorption (ESD) of chemisorbed water molecules from the thin-film surfaces and an accompanying change of work function. The ESD interpretation is supported by experimental results obtained by electron-energy loss spectroscopy, hole-free phase plate imaging, secondary electron detection and x-ray photoelectron spectroscopy as well as simulations of the electrostatic potential distribution. The described ESD-based model explains previous experimental findings and is of general interest to any phase-related technique in a transmission electron microscope. [ABSTRACT FROM AUTHOR]

Local spatial and temporal control of laser-induced microplasma (LIMP) allows to form a microrelief on fused silica plates for the fabrication of multilevel phase elements. In this work we suggested the optimal way for precision depth variation (up to 50 nm) of the microrelief (0.05 - 15 μm) on fused silica surface. New software and hardware were developed for the automation of optical phase elements calculation and its fabrication by LIMP. A few types of multilevel random phase plates were experimentally realized. The fabricated elements were successfully tested in the optical scheme for the correction of the highly coherent laser beam. The comparative analysis of binary and discrete phase plates was accomplished. [ABSTRACT FROM AUTHOR]

The image of a simple phase object produced by a round lens with a Foucault or Hilbert phase plate can be determined with Abbe imaging theory and a 2D transform expressed in cylindrical coordinates. The contributions to the image amplitude from a uniform disc object and an azimuthally varying plate can then be distinguished and their phases relative to the incident wave can be compared. It appears that the usual choice of added phase for a Hilbert plate causes the image of a weak disc object to vanish as the plate edge approaches the axis, but a different choice of plate thickness can enable a weak phase object to provide a linear contribution to image intensity. [ABSTRACT FROM AUTHOR]

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications. [ABSTRACT FROM AUTHOR]

Phase plates in cryo-electron tomography (cryoET) improve contrast, increasing the ability to discern separate molecules and molecular complexes in dense biomolecular environments. Here, we applied this new technology to the activation of the human complement system. Binding of C1 to antigen-antibody complexes initiates a cascade of proteolytic events that deposits molecules onto adjacent surfaces and terminates with the formation of membrane-attack-complex (MAC) pores in the targeted membranes. We imaged steps in this process using a Volta phase plate mounted on a Titan Krios equipped with a Falcon-II direct electron detector. The data show patches of single-layer antibodies on the surface and C1 bound to antibody platforms, with ca. ∼4% of instances where C1r and C1s proteases have dissociated from C1, and potentially instances of C1 transiently interacting with its substrate C4 or product C4b. Next, extensive deposition of C4b and C3b molecules is apparent, although individual molecules cannot always be properly distinguished with the current methods. Observations of MAC pores include formation of both single and composite pores, and instances of potential soluble-MAC dissociation upon failure of membrane insertion. Overall, application of the Volta phase plate cryoET markedly improved the contrast in the tomograms, which allowed for individual components to be more readily interpreted. However, variability in the phase shift induced by the phase-plate during the course of an experiment, together with incomplete sampling during tomogram acquisition, limited the interpretability of the resulting tomograms. Our studies exemplify the potential in studying molecular processes with complex spatial topologies by phase-plate cryoET. [ABSTRACT FROM AUTHOR]

Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.

A new advance in electron microscopy can reveal highly-detailed structures of protein complexes.

We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.

Material properties strongly depend on the nature and concentration of defects. Characterizing these features may require nano- to atomic-scale resolution to establish structure-property relationships. 4D-STEM, a technique where diffraction patterns are acquired at a grid of points on the sample, provides a versatile method for highlighting defects. Computational analysis of the diffraction patterns with virtual detectors produces images that can map material properties. Here, using multislice simulations, we explore different virtual detectors that can be applied to the diffraction patterns that go beyond the binary response functions that are possible using ordinary STEM detectors. Using graphene and lead titanate as model systems, we investigate the application of virtual detectors to study local order and in particular defects. We find that using a small convergence angle with a rotationally varying detector most efficiently highlights defect signals. With experimental graphene data, we demonstrate the effectiveness of these detectors in characterizing atomic features, including vacancies, as suggested in simulations. Phase and amplitude modification of the electron beam provides another process handle to change image contrast in a 4D-STEM experiment. We demonstrate how tailored electron beams can enhance signals from short-range order and how a vortex beam can be used to characterize local symmetry., Competing Interests: Conflict of Interest The authors declare that they have no competing interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ability to image light elements in both crystalline and noncrystalline materials at near atomic resolution with an enhanced contrast is highly advantageous to understand the structure and properties of a wide range of beam sensitive materials including biological specimens and molecular hetero-structures. This requires the imaging system to have an efficient phase contrast transfer at both low and high spatial frequencies. In this work we introduce a new phase contrast imaging method in a scanning transmission electron microscope (STEM) using a pre-specimen phase plate in the probe forming aperture, combined with a fast pixelated detector to record diffraction patterns at every probe position, and phase reconstruction using ptychography. The phase plate significantly enhances the contrast transfer of low spatial frequency information, and ptychography maximizes the extraction of the phase information at all spatial frequencies. In addition, the STEM probe with the presence of the phase plate retains its atomic resolution, allowing simultaneous incoherent Z-contrast imaging to be obtained along with the ptychographic phase image. An experimental image of Au nanoparticles on a carbon support shows high contrast for both materials. Multislice image simulations of a DNA molecule shows the capability of imaging soft matter at low dose conditions, which implies potential applications of low dose imaging of a wide range of beam sensitive materials. [ABSTRACT FROM AUTHOR]

The images produced from simple phase objects, lenses and Zernike phase plates when all have rotational symmetry can be calculated by 1D Fourier–Bessel transforms. For a simple disc object producing a uniform phase shift over its diameter, the resulting image can be defined for any size of object phase change. The monotonic range of intensity variation with object phase is found to depend strongly on the phase change introduced by the phase plate; this property of the system is not well predicted by the weak phase approximation. The effect of spreading the phase transition at the plate over a range of radius is beneficial if the plate phase change is sufficiently small. Weak-phase calculations for a phase distribution more typical of a spherical object are also shown. [ABSTRACT FROM AUTHOR]

Summary Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding and membrane insertion of C5b6, C7, C8, and C9. Using cryo-electron tomography with a Volta phase plate and subtomogram averaging, we imaged C5b-7, C5b-8, and C5b-9 complexes and determined the C5b-9 pore structure in lipid bilayers. The in situ C5b-9 pore structure at 2.3-nm resolution reveals a 10- to 11.5-nm cone-shaped pore starting with C5b678 and multiple copies of C9 that is poorly closed, yielding a seam between C9 and C6 substituting for the shorter β strands in C6 and C7. However, large variations of composite pore complexes are apparent in subtomograms. Oligomerized initiator complexes C5b-7 and C5b-8 show stages of membrane binding, deformation, and perforation that yield ∼3.5-nm-wide pores. These data indicate a dynamic process of pore formation that likely adapts to biological membranes under attack. [ABSTRACT FROM AUTHOR]

A variety of physical phase plate designs have been developed to maximize phase contrast for weak phase objects in the transmission electron microscope (TEM). Most progress towards application in structural biology has been made with Zernike PPs consisting of a ~30 nm film of amorphous carbon with a central hole. Although problems such as beam-induced deterioration of Zernike PPs remain unsolved, it is likely that thin film phase plates will be applied routinely in TEM of ice-embedded biological specimens in the near future. However, the thick carbon film of thin film PPs dampens high-resolution information, which precludes their use for single-particle electron cryo-microscopy at atomic resolution. In this work, an improved design for a thin film phase plate is proposed, combining the advantages of Zernike PPs and 2D materials, such as graphene. The improved design features a disc of phase-shifting material mounted on an ultrathin support film. The proposed device imparts a phase shift only to electrons scattered to low angles, whereas contrast at high resolution is generated by conventional defocusing. The device maximizes phase contrast at low spatial frequencies, where defocus contrast is limiting, while damping of information at high spatial frequencies is avoided. Experiments demonstrate that the fabrication of such a device is feasible. [ABSTRACT FROM AUTHOR]

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope’s contrast transfer function (CTF) to an image’s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled. [ABSTRACT FROM AUTHOR]

Because of the laser beam waist and diffraction effect of the lens, the focal spot light field in femtosecond laser microprocessing has an ellipsoidal spatial distribution. This leads to the gap between two processing layers increasing along the axial direction, and the distribution density of processing points decreasing along the horizontal direction. This directly reduces the resolution of the microprocessing, and badly affects the machining accuracy and surface quality. We established a mathematical model for three-dimensional (3-D) laser beam shaping based on the Fresnel diffraction theory and designed a kind of four-ring complex amplitude transmittance phase plate by using a global optimization algorithm and genetic algorithm to simultaneously realize transverse and axial 3-D shaping. We numerically showed that the transverse and axial gains of the focal facula after 3-D shaping are 0.77 and 0.68, respectively, where the corresponding peak energy ratio is 0.36, the transverse and axial sidelobe energies are 0.28 and 0.62, respectively, and the defocusing amount is -0.08. We also constructed a confocal/two-photon microscope system to experimentally achieve a better shaping effect in the case of femtosecond laser fabrication at a point on the thin film of a photochromic material. [ABSTRACT FROM AUTHOR]

Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for cryo-electron tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed cryo-electron tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in cryo-electron tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells. [ABSTRACT FROM AUTHOR]

The phase contrast of Au nanoparticles on amorphous-carbon films with different thicknesses is analyzed using an electrostatic Zach phase plate in a Zeiss 912 Ω transmission electron microscope with in-column energy filter. Specifically, unfiltered and plasmon-filtered phase-plate transmission electron microscopy (PP TEM) images are compared to gain insight in the role of coherence after inelastic scattering processes. A considerable phase-contrast contribution resulting from a combined elastic–inelastic scattering process is found in plasmon-filtered PP TEM images. The contrast reduction compared to unfiltered images mainly originates from zero-order beam broadening caused by the inelastic scattering process. The effect of the sequence of the elastic and inelastic scattering processes is studied by varying the position of the nanoparticles, which can be either located on top or at the bottom of the amorphous-carbon film with respect to the incident electron beam direction. [ABSTRACT FROM AUTHOR]

Charging of physical phase plates is a problem that has prevented their routine use in transmission electron microscopy of weak-phase objects. In theory, electrostatic phase plates are superior to thin-film phase plates since they do not attenuate the scattered electron beam and allow freely adjustable phase shifts. Electrostatic phase plates consist of multiple layers of conductive and insulating materials, and are thus more prone to charging than thin-film phase plates, which typically consist of only one single layer of amorphous material. We have addressed the origins of charging of Boersch phase plates and show how it can be reduced. In particular, we have performed simulations and experiments to analyze the influence of the insulating Si 3 N 4 layers and surface charges on electrostatic charging. To optimize the performance of electrostatic phase plates, it would be desirable to fabricate electrostatic phase plates, which (i) impart a homogeneous phase shift to the unscattered electrons, (ii) have a low cut-on frequency, (iii) expose as little material to the intense unscattered beam as possible, and (iv) can be additionally polished by a focused ion-beam instrument to eliminate carbon contamination accumulated during exposure to the unscattered electron beam (Walter et al., 2012, Ultramicroscopy , 116, 62–72). We propose a new type of electrostatic phase plate that meets the above requirements and would be superior to a Boersch phase plate. It consists of three free-standing coaxial rods converging in the center of an aperture (3-fold coaxial phase plate). Simulations and preliminary experiments with modified Boersch phase plates indicate that the fabrication of a 3-fold coaxial phase plate is feasible. [ABSTRACT FROM AUTHOR]