High-Resolution Electron Diffraction of Hydrated Protein Crystals at Room Temperature.

Structural characterization is crucial to understanding protein function. Compared with X-ray diffraction methods, electron crystallography can be performed on nanometer-sized crystals and can provide additional information from the resulting Coulomb potential map. Whereas electron crystallography has successfully resolved three-dimensional structures of vitrified protein crystals, its widespread use as a structural biology tool has been limited. One main reason is the fragility of such crystals. Protein crystals can be easily damaged by mechanical stress, change in temperature, or buffer conditions as well as by electron irradiation. This work demonstrates a methodology to preserve these nanocrystals in their natural environment at room temperature for electron diffraction experiments as an alternative to existing cryogenic techniques. Lysozyme crystals in their crystallization solution are hermetically sealed via graphene-coated grids, and their radiation damage is minimized by employing a low-dose data collection strategy in combination with a hybrid-pixel direct electron detector. Diffraction patterns with reflections of up to 3 Å are obtained and successfully indexed using a template-matching algorithm. These results demonstrate the feasibility of in situ protein electron diffraction. The method described will also be applicable to structural studies of hydrated nanocrystals important in many research and technological developments.

Study of the Microstructure of Amorphous Silica Nanostructures Using High-Resolution Electron Microscopy, Electron Energy Loss Spectroscopy, X-ray Powder Diffraction, and Electron Pair Distribution Function.

Silica has many industrial (i.e., glass formers) and scientific applications. The understanding and prediction of the interesting properties of such materials are dependent on the knowledge of detailed atomic structures. In this work, amorphous silica subjected to an accelerated alkali silica reaction (ASR) was recorded at different time intervals so as to follow the evolution of the structure by means of high-resolution transmission electron microscopy (HRTEM), electron energy loss spectroscopy (EELS), and electron pair distribution function (e-PDF), combined with X-ray powder diffraction (XRPD). An increase in the size of the amorphous silica nanostructures and nanopores was observed by HRTEM, which was accompanied by the possible formation of Si-OH surface species. All of the studied samples were found to be amorphous, as observed by HRTEM, a fact that was also confirmed by XRPD and e-PDF analysis. A broad diffuse peak observed in the XRPD pattern showed a shift toward higher angles following the higher reaction times of the ASR-treated material. A comparison of the EELS spectra revealed varying spectral features in the peak edges with different reaction times due to the interaction evolution between oxygen and the silicon and OH ions. Solid-state nuclear magnetic resonance (NMR) was also used to elucidate the silica nanostructures.

A Comparison of a Direct Electron Detector and a High-Speed Video Camera for a Scanning Precession Electron Diffraction Phase and Orientation Mapping.

A scanning precession electron diffraction system has been integrated with a direct electron detector to allow the collection of improved quality diffraction patterns. This has been used on a two-phase α­ß titanium alloy (Timetal® 575) for phase and orientation mapping using an existing pattern-matching algorithm and has been compared to the commonly used detector system, which consisted of a high-speed video-camera imaging the small phosphor focusing screen. Noise is appreciably lower with the direct electron detector, and this is especially noticeable further from the diffraction pattern center where the real electron scattering is reduced and both diffraction spots and inelastic scattering between spots are weaker. The results for orientation mapping are a significant improvement in phase and orientation indexing reliability, especially of fine nanoscale laths of α-Ti, where the weak diffracted signal is rather lost in the noise for the optically coupled camera. This was done at a dose of ~19 e−/Å2, and there is clearly a prospect for reducing the current further while still producing indexable patterns. This opens the way for precession diffraction phase and orientation mapping of radiation-sensitive crystalline materials.

Reliable Characterization of Organic & Pharmaceutical Compounds with High Resolution Monochromated EEL Spectroscopy.

Organic and biological compounds (especially those related to the pharmaceutical industry) have always been of great interest for researchers due to their importance for the development of new drugs to diagnose, cure, treat or prevent disease. As many new API (active pharmaceutical ingredients) and their polymorphs are in nanocrystalline or in amorphous form blended with amorphous polymeric matrix (known as amorphous solid dispersion-ASD), their structural identification and characterization at nm scale with conventional X-Ray/Raman/IR techniques becomes difficult. During any API synthesis/production or in the formulated drug product, impurities must be identified and characterized. Electron energy loss spectroscopy (EELS) at high energy resolution by transmission electron microscope (TEM) is expected to be a promising technique to screen and identify the different (organic) compounds used in a typical pharmaceutical or biological system and to detect any impurities present, if any, during the synthesis or formulation process. In this work, we propose the use of monochromated TEM-EELS, to analyze selected peptides and organic compounds and their polymorphs. In order to validate EELS for fingerprinting (in low loss/optical region) and by further correlation with advanced DFT, simulations were utilized.

Novel TEM Microscopy and Electron Diffraction Techniques to Characterize Cultural Heritage Materials: From Ancient Greek Artefacts to Maya Mural Paintings.

To understand in-depth material properties, manufacturing, and conservation in cultural heritage artefacts, there is a strong need for advanced characterization tools that enable analysis down to the nanometric scale. Transmission electron microscopy (TEM) and electron diffraction (ED) techniques, like 3D precession electron diffraction tomography and ASTAR phase/orientation mapping, are proposed to study cultural heritage materials at nanoscale. In this work, we show how electron crystallography in TEM helps to determine precise structural information and phase/orientation distribution of various pigments in cultural heritage materials from various historical periods like Greek amphorisks, Roman glass tesserae, and pre-Hispanic Maya mural paintings. Such TEM-based methods can be an alternative to synchrotron techniques and can allow distinguishing accurately different crystalline phases even in cases of identical or very close chemical compositions at the nanometric scale.

Ultrafast Electron Diffraction Tomography for Structure Determination of the New Zeolite ITQ-58.

In this work a new ultrafast data collection strategy for electron diffraction tomography is presented that allows reducing data acquisition time by one order of magnitude. This methodology minimizes the radiation damage of beam-sensitive materials, such as microporous materials. This method, combined with the precession of the electron beam, provides high quality data enabling the determination of very complex structures. Most importantly, the implementation of this new electron diffraction methodology is easily affordable in any modern electron microscope. As a proof of concept, we have solved a new highly complex zeolitic structure named ITQ-58, with a very low symmetry (triclinic) and a large unit cell volume (1874.6 Å(3)), containing 16 silicon and 32 oxygen atoms in its asymmetric unit, which would be very difficult to solve with the state of the art techniques.

Electron microscopy and electron energy-loss spectroscopy study of Nd 1-x Sr x CoO 3-δ (0 ≤ x ≤ 1) system.

A solid solution of Nd 1-x Sr x CoO 3-δ (with x=0, 1/3, 2/3, and 1) has been prepared and characterized by a combination of X-ray diffraction, electron microscopy, and electron energy-loss spectroscopy (EELS). The structural characterization indicates that Nd-doped materials present an orthorhombic symmetry with a=√2xa p, b=√2xa p, and c=2xa p (a p refers to lattice parameter of simple cubic perovskite), while SrCoO2.5 has an orthorhombic symmetry with a=√2xa p, b=4xa p, and c=√2xa p. EELS analysis revealed that Co are in 3+ oxidation states but in different spin configurations.

Hafnium-silicon precipitate structure determination in a new heat-resistant ferritic alloy by precession electron diffraction techniques.

The structure determination of an HfSi4 precipitate has been carried out by a combination of two precession electron diffraction techniques: high precession angle, 2.2°, single pattern collection at eight different zone axes and low precession angle, 0.5°, serial collection of patterns obtained by increasing tilts of 1°. A three-dimensional reconstruction of the associated reciprocal space shows an orthorhombic unit cell with parameters a = 11.4 Å, b = 11.8 Å, c = 14.6 Å, and an extinction condition of (hkl) h + k odd. The merged intensities from the high angle precession patterns have been symmetry tested for possible space groups (SG) fulfilling this condition and a best symmetrization residual found at 18% for SG 65 Cmmm. Use of the SIR2011 direct methods program allowed solving the structure with a structure residual of 18%. The precipitate objects of this study were reproducibly found in a newly implemented alloy, designed according to molecular orbital theory.

Structure solution with three-dimensional sets of precessed electron diffraction intensities.

The use of the precession technique for obtaining three-dimensional (3D) sets of electron diffraction intensities suitable for structure solution is discussed. The minerals uvarovite and åkermanite have been used as testing structures. The electron diffraction data sets obtained on these samples retain an acceptable linear relation with calculated structure factor amplitudes. The quality of these data is suitable to solve both structures using direct methods opening the possibility to use 3D precession ED data for solving unknown mineral structures.

Direct electron crystallographic determination of zeolite zonal structures.

The prospect for improving the success of ab initio zeolite structure investigations with electron diffraction data is evaluated. First of all, the quality of intensities obtained by precession electron diffraction at small hollow cone illumination angles is evaluated for seven representative materials: ITQ-1, ITQ-7, ITQ-29, ZSM-5, ZSM-10, mordenite, and MCM-68. It is clear that, for most examples, an appreciable fraction of a secondary scattering perturbation is removed by precession at small angles. In one case, ZSM-10, it can also be argued that precession diffraction produces a dramatically improved 'kinematical' data set. There seems to no real support for application of a Lorentz correction to these data and there is no reason to expect for any of these samples that a two-beam dynamical scattering relationship between structure factor amplitude and observed intensity should be valid. Removal of secondary scattering by the precession mode appears to facilitate ab initio structure analysis. Most zeolite structures investigated could be solved by maximum entropy and likelihood phasing via error-correcting codes when precession data were used. Examples include the projected structure of mordenite that could not be determined from selected area data alone. One anomaly is the case of ZSM-5, where the best structure determination in projection is made from selected area diffraction data. In a control study, the zonal structure of SSZ-48 could be determined from selected area diffraction data by either maximum entropy and likelihood or traditional direct methods. While the maximum entropy and likelihood approach enjoys some advantages over traditional direct methods (non-dependence on predicted phase invariant sums), some effort must be made to improve the figures of merit used to identify potential structure solutions.

Ab initio determination of heavy oxide perovskite related structures from precession electron diffraction data.

Two complex perovskite-related structures were solved by ab initio from precession electron diffraction intensities. Structure models were firstly derived from HREM images and than have been confirmed independently using two and three-dimensional sets of precession intensities. Patterson techniques prove to be effective for ab initio structure resolution, specially in case of projections with no overlapping atoms. Quality of precession intensity data may be suitable enough to resolve unknown heavy oxide structures.

Ab initio determination of the framework structure of the heavy-metal oxide Cs(x)Nb2.54W2.46O14 from 100 kV precession electron diffraction data.

The present work deals with the ab initio determination of the heavy metal framework in Cs(x)(Nb, W)(5)O(14) from precession electron diffraction intensities. The target structure was first discovered by Lundberg and Sundberg [Ultramicroscopy 52 (1993) 429-435], who succeeded in deriving a tentative structural model from high-resolution electron microsopy (HREM) images. The metal framework of the compound was solved in this investigation via direct methods from hk0 precession electron diffraction intensities recorded with a Philips EM400 at 100 kV. A subsequent (kinematical) least-squares refinement with electron intensities yielded slightly improved co-ordinates for the 11 heavy atoms in the structure. Chemical analysis of several crystallites by EDX is in agreement with the formula Cs(0.44)Nb(2.54)W(2.46)O(14). Moreover, the structure was independently determined by Rietveld refinement from X-ray powder data obtained from a multi-phasic sample. The compound crystallises in the orthorhombic space group Pbam with refined lattice parameters a=27.145(2), b=21.603(2), and c=3.9463(3)A. Comparison of the framework structure from electron diffraction with the result from Rietveld refinement shows an average agreement for the heavy atoms within 0.09 A.