Showing papers on "Resolution (electron density) published in 2017"

MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy

Abstract: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.

Addressing Preferred Specimen Orientation in Single-Particle Cryo-EM through Tilting

Abstract: We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

Abstract: Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 A resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 A using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 A from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 A between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously

Diffraction and microscopy with attosecond electron pulse trains

Abstract: Attosecond spectroscopy 1–7 can resolve electronic processes directly in time, but a movie-like space–time recording is impeded by the too long wavelength (~100 times larger than atomic distances) or the source–sample entanglement in re-collision techniques 8–11 . Here we advance attosecond metrology to picometre wavelength and sub-atomic resolution by using free-space electrons instead of higher-harmonic photons 1–7 or re-colliding wavepackets 8–11 . A beam of 70-keV electrons at 4.5-pm de Broglie wavelength is modulated by the electric field of laser cycles into a sequence of electron pulses with sub-optical-cycle duration. Time-resolved diffraction from crystalline silicon reveals a < 10-as delay of Bragg emission and demonstrates the possibility of analytic attosecond–angstrom diffraction. Real-space electron microscopy visualizes with sub-light-cycle resolution how an optical wave propagates in space and time. This unification of attosecond science with electron microscopy and diffraction enables space–time imaging of light-driven processes in the entire range of sample morphologies that electron microscopy can access. Attosecond light pulses are used to probe ultrafast processes. The experimental observation of attosecond electron pulses now promises the marriage of these techniques with electron microscopy and diffraction.

Probing the electronic and catalytic properties of a bimetallic surface with 3 nm resolution

Abstract: Tip-enhanced Raman spectroscopy in conjunction with scanning tunnelling microscopy can be used to correlate chemical properties and surface topography of bimetallic catalysts with high spatial resolution. An atomic- and molecular-level understanding of heterogeneous catalysis is required to characterize the nature of active sites and improve the rational design of catalysts1,2,3. Achieving this level of characterization requires techniques that can correlate catalytic performances to specific surface structures, so as to avoid averaging effects1. Tip-enhanced Raman spectroscopy4,5,6,7 combines scanning probe microscopy with plasmon-enhanced Raman scattering and provides simultaneous topographical and chemical information at the nano/atomic scale from ambient8,9,10 to ultrahigh-vacuum11,12 and electrochemical environments13,14. Therefore, it has been used to monitor catalytic reactions15,16,17,18 and is proposed to correlate the local structure and function of heterogeneous catalysts19. Bimetallic catalysts, such as Pd–Au, show superior performance in various catalytic reactions20,21, but it has remained challenging to correlate structure and reactivity because of their structural complexity. Here, we show that TERS can chemically and spatially probe the site-specific chemical (electronic and catalytic) and physical (plasmonic) properties of an atomically well-defined Pd(sub-monolayer)/Au(111) bimetallic model catalyst at 3 nm resolution in real space using phenyl isocyanide as a probe molecule ( Fig. 1a ). We observe a weakened N≡C bond and enhanced reactivity of phenyl isocyanide adsorbed at the Pd step edge compared with that at the Pd terrace. Density functional theory corroborates these observations by revealing a higher d-band electronic profile for the low-coordinated Pd step edge atoms. The 3 nm spatial resolution we demonstrate here is the result of an enhanced electric field and distinct electronic properties at the step edges.

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

Abstract: Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons. Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE.

Abstract: SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 A structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

Abstract: The orientation distribution of a single-particle electron cryomicroscopy specimen limits the resolution of the reconstructed density map. Here we define a statistical quantity, the efficiency, E od, which characterises the orientation distribution via its corresponding point spread function. The efficiency measures the ability of the distribution to provide uniform information and resolution in all directions of the reconstruction, independent of other factors. This metric allows rapid and rigorous evaluation of specimen preparation methods, assisting structure determination to high resolution with minimal data.A number of parameters influence the resolution of a cryo-EM structure. Here the authors investigate the effects of specimen orientation in single particle cryo-EM and present open-source software for rapidly assessing orientation distributions to improve data collection.

Light-Field Image Super-Resolution Using Convolutional Neural Network

Abstract: Commercial light field cameras provide spatial and angular information, but their limited resolution becomes an important problem in practical use. In this letter, we present a novel method for light field image super-resolution (SR) to simultaneously up-sample both the spatial and angular resolutions of a light field image via a deep convolutional neural network. We first augment the spatial resolution of each subaperture image by a spatial SR network, then novel views between super-resolved subaperture images are generated by three different angular SR networks according to the novel view locations. We improve both the efficiency of training and the quality of angular SR results by using weight sharing . In addition, we provide a new light field image dataset for training and validating the network. We train our whole network end-to-end, and show state-of-the-art performances on quantitative and qualitative evaluations.

Vibronic Spectroscopy with Submolecular Resolution from STM-Induced Electroluminescence

Abstract: A scanning tunneling microscope is used to excite the fluorescence of single molecules, leading to the observation of well resolved vibronic features. The work opens the way to vibronic spectroscopy with atomic-scale resolution.

A classical description of subnanometer resolution by atomic features in metallic structures

Abstract: Recent experiments have evidenced sub-nanometer resolution in plasmonic-enhanced probe spectroscopy. Such a high resolution cannot be simply explained using the commonly considered radii of metallic nanoparticles on plasmonic probes. In this contribution the effects of defects as small as a single atom found on spherical plasmonic particles acting as probing tips are investigated in connection with the spatial resolution provided. The presence of abundant edge and corner sites with atomic scale dimensions in crystalline metallic nanoparticles is evident from transmission electron microscopy (TEM) images. Electrodynamic calculations based on the Finite Element Method (FEM) are implemented to reveal the impact of the presence of such atomic features in probing tips on the lateral spatial resolution and field localization. Our analysis is developed for three different configurations, and under resonant and non-resonant illumination conditions, respectively. Based on this analysis, the limits of field enhancement, lateral resolution and field confinement in plasmon-enhanced spectroscopy and microscopy are inferred, reaching values below 1 nanometer for reasonable atomic sizes.

Achieving better-than-3-A resolution by single-particle cryo-EM at 200 keV

Abstract: Nearly all single-particle cryo-EM structures resolved to better than 4-A resolution have been determined using 300-keV transmission electron microscopes (TEMs) We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-A resolution using a 200-keV TEM These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules

Resolution-enhanced Fourier ptychographic microscopy based on high-numerical-aperture illuminations.

Abstract: High-resolution and wide field-of-view (FOV) microscopic imaging plays a central role in diverse applications such as high-throughput screening and digital pathology. However, conventional microscopes face inherent trade-offs between the spatial resolution and FOV, which are fundamental limited by the space-bandwidth product (SBP) of the optical system. The resolution-FOV tradeoff can be effectively decoupled in Fourier ptychography microscopy (FPM), however, to date, the effective imaging NA achievable with a typical FPM system is still limited to the range of 0.4–0.7. Herein, we report, for the first time, a high-NA illumination based resolution-enhanced FPM (REFPM) platform, in which a LED-array-based digital oil-immersion condenser is used to create high-angle programmable plane-wave illuminations, endowing a 10×, 0.4 NA objective lens with final effective imaging performance of 1.6 NA. With REFPM, we present the highest-resolution results with a unprecedented half-pitch resolution of 154 nm at a wavelength of 435 nm across a wide FOV of 2.34 mm2, corresponding to an SBP of 98.5 megapixels (~50 times higher than that of the conventional incoherent microscope with the same resolution). Our work provides an important step of FPM towards high-resolution large-NA imaging applications, generating comparable resolution performance but significantly broadening the FOV of conventional oil-immersion microscopes.

Advances in Atomic Resolution In Situ Environmental Transmission Electron Microscopy and 1 Angstrom Aberration Corrected In Situ Electron Microscopy

Abstract: Advances in atomic resolution in situ environmental transmission electron microscopy for direct probing of gas-solid reactions, including at very high temperatures are described. In addition, recent developments of dynamic real time in situ studies at the Angstrom level using a hot stage in an aberration corrected environment are presented. In situ data from Pt and Pd nanoparticles on carbon with the corresponding FFT (optical diffractogram) illustrate an achieved resolution of 0.11 nm at 500 C and higher in a double aberration corrected TEM and STEM instrument employing a wider gap objective pole piece. The new results open up opportunities for dynamic studies of materials in an aberration corrected environment.

Image Super Resolution Using Generative Adversarial Networks and Local Saliency Maps for Retinal Image Analysis

Abstract: We propose an image super resolution (ISR) method using generative adversarial networks (GANs) that takes a low resolution input fundus image and generates a high resolution super resolved (SR) image upto scaling factor of 16. This facilitates more accurate automated image analysis, especially for small or blurred landmarks and pathologies. Local saliency maps, which define each pixel’s importance, are used to define a novel saliency loss in the GAN cost function. Experimental results show the resulting SR images have perceptual quality very close to the original images and perform better than competing methods that do not weigh pixels according to their importance. When used for retinal vasculature segmentation, our SR images result in accuracy levels close to those obtained when using the original images.

Structural enzymology using X-ray free electron lasers

Abstract: Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis s-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-s-lactamase and of the ceftriaxone bound form were obtained at 2.8 A and 2.4 A resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Orbital angular momentum light in microscopy.

Abstract: Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application.This article is part of the themed issue 'Optical orbital angular momentum'.

Electron ptychographic microscopy for three-dimensional imaging

Abstract: Knowing the three-dimensional structural information of materials at the nanometer scale is essential to understanding complex material properties. Electron tomography retrieves three-dimensional structural information using a tilt series of two-dimensional images. In this paper, we report an alternative combination of electron ptychography with the inverse multislice method. We demonstrate depth sectioning of a nanostructured material into slices with 0.34 nm lateral resolution and with a corresponding depth resolution of about 24–30 nm. This three-dimensional imaging method has potential applications for the three-dimensional structure determination of a range of objects, ranging from inorganic nanostructures to biological macromolecules. Three-dimensional ptychographic imaging with electrons has remained a challenge because, unlike X-rays, electrons are easily scattered by atoms. Here, Gao et al. extend multi-slice methods to electrons in the multiple scattering regime, paving the way to nanometer-scale 3D structure determination with electrons.

Tomographic diffractive microscopy with isotropic resolution

Abstract: Microscopy techniques allowing observation of unlabeled samples have recently experienced a regain of interest. In particular, approaches based on recording of the optical field diffracted by the specimen, in amplitude and phase, have proven their capacities for biological investigations. When combined with variations of specimen illumination, tomographic acquisitions are possible. One limitation of previously developed approaches is the anisotropic resolution, characteristic of all transmission microscopes. In this context, an instrument, characterized by isotropic high-resolution 3D imaging capabilities, is still awaited. For the first time, to the best of our knowledge, we have developed tomographic diffractive microscopy combining specimen rotation and illumination rotation, which delivers images with (almost) isotropic resolution below 200 nm. The method is illustrated by observations of nanoscopic fiber tips, microcrystals and pollens, and should be helpful for characterizing freestanding natural (diatoms, spores, red or white blood cells, etc.) or artificial samples.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

Abstract: X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. By working with cells that have been rapidly frozen without the use of chemical fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.

How cryo-electron microscopy and X-ray crystallography complement each other.

Abstract: With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms.

Superresolution via structured illumination quantum correlation microscopy

Abstract: We propose to use intensity correlation microscopy in combination with structured illumination to image quantum emitters that exhibit antibunching with a spatial resolution reaching far beyond the Rayleigh limit Combining intensity measurements and intensity autocorrelations up to order m creates an effective PSF with an FWHM shrunk by the factor m Structured illumination microscopy, on the other hand, introduces a resolution improvement of factor 2 by use of the principle of moire fringes Here, we show that for linear low-intensity excitation and linear optical detection, the simultaneous use of both techniques leads to a theoretically unlimited resolution power, with the improvement scaling favorably as m+m, dependent on the correlation order m Hence, this technique should be of interest in microscopy for imaging a variety of samples, including biological ones We present the underlying theory and simulations, demonstrating the highly increased spatial superresolution, and point out the requirements for an experimental implementation

Super-resolution optical microscopy for studying membrane structure and dynamics

Abstract: Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Super-resolution Microscopy - Applications in Plant Cell Research.

Abstract: Most of the present knowledge about cell organization and function is based on molecular and genetic methods as well as cytological investigations. While electron microscopy allows identifying cell substructures until a resolution of ~1nm, the resolution of fluorescence microscopy is restricted to ~200 nm due to the diffraction limit of light. However, the advantage of this technique is the possibility to identify and co-localize specifically labelled structures and molecules. The recently developed super-resolution microscopy techniques, such as Structured Illumination Microscopy (SIM), Photoactivated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM) and Stimulated Emission Depletion (STED) microscopy allow analysing structures and molecules beyond the diffraction limit of light. Recently, there is an increasing application of these techniques in cell biology. This review evaluates and summarizes especially the data achieved until now in analysing the organization and function of plant cells, chromosomes and interphase nuclei using super-resolution techniques.

meV resolution in laser-assisted energy-filtered transmission electron microscopy

Abstract: The electronic, optical, and magnetic properties of quantum solids are determined by their low-energy (< 100 meV) many-body excitations. Dynamical characterization and manipulation of such excitations relies on tools that combine nm-spatial, fs-temporal, and meV-spectral resolution. Currently, phonons and collective plasmon resonances can be imaged in nanostructures with sub-nm and 10s meV space/energy resolution using state-of-the-art energy-filtered transmission electron microscopy (TEM), but only under static conditions, while fs-resolved measurements are common but lack spatial or energy resolution. Here, we demonstrate a new method of spectrally resolved photon-induced near-field electron microscopy (SRPINEM) that allows us to obtain nm-fs-resolved maps of nanoparticle plasmons with an energy resolution determined by the laser linewidth (20 meV in this work), and not limited by electron beam and spectrometer energy spreading. This technique can be extended to any optically-accessible low-energy mode, thus pushing TEM to a previously inaccessible spectral domain with an unprecedented combination of space, energy and temporal resolution.

Effect of CT image size and resolution on the accuracy of rock property estimates

Abstract: In order to study the effect of the micro-CT scan resolution and size on the accuracy of up-scaled digital rock property estimation of core samples Bentheimer sandstone images with the resolution varying from 0.9 μm to 24 μm are used. We statistically show that the correlation length of the pore-to-matrix distribution can be reliably determined for the images with the resolution finer than 9 voxels per correlation length and the representative volume for this property is about 153 correlation length. Similar resolution values for the statistically representative volume are also valid for the estimation of the total porosity, specific surface area, mean curvature and topology of the pore space. Only the total porosity and the number of isolated pores are stably recovered, whereas geometry and the topological measures of the pore space are strongly affected by the resolution change. We also simulate fluid flow in the pore space and estimate permeability and tortuosity of the sample. The results demonstrate that the representative volume for the transport property calculation should be greater than 50 correlation lengths of pore-to-matrix distribution. On the other hand, permeability estimation based on the statistical analysis of equivalent realizations shows some weak influence of the resolution on the transport properties. The reason for this might be that the characteristic scale of the particular physical processes may affect the result stronger than the model (image) scale.

Mapping Elastic Properties of Heterogeneous Materials in Liquid with Angstrom-Scale Resolution

Abstract: Fast quantitative mapping of mechanical properties with nanoscale spatial resolution represents one of the major goals of force microscopy. This goal becomes more challenging when the characterization needs to be accomplished with subnanometer resolution in a native environment that involves liquid solutions. Here we demonstrate that bimodal atomic force microscopy enables the accurate measurement of the elastic modulus of surfaces in liquid with a spatial resolution of 3 A. The Young’s modulus can be determined with a relative error below 5% over a 5 orders of magnitude range (1 MPa to 100 GPa). This range includes a large variety of materials from proteins to metal–organic frameworks. Numerical simulations validate the accuracy of the method. About 30 s is needed for a Young’s modulus map with subnanometer spatial resolution.

Achieving High Spatial Resolution Surface Plasmon Resonance Microscopy with Image Reconstruction

Abstract: Surface plasmon resonance microscopy (SPRM) is a powerful platform for biomedical imaging and molecular binding kinetics analysis. However, the spatial resolution of SPRM along the plasmon propagation direction (longitudinal) is determined by the decaying length of the plasmonic wave, which can be as large as tens of microns. Different methods have been proposed to improve the spatial resolution, but each at the expense of decreased sensitivity or temporal resolution. Here we present a method to achieve high spatial resolution SPRM based on deconvolution of complex field. The method does not require additional optical setup and improves the spatial resolution in the longitudinal direction. We applied the method to image nanoparticles and achieved close-to-diffraction limit resolution in both longitudinal and transverse directions.