Elemental mapping in single-particle reconstructions by reconstructed electron energy-loss analysis

Transmission electron microscopy and related analytical techniques have been widely used to study the microstructure of different materials. However, few research works have been performed in the field of glasses, possibly due to the electron-beam irradiation damage. In this paper, we have developed a method based on electron energy loss spectroscopy (EELS) data acquisition and analyses, which enables determination of the boron speciation in a series of ternary alkali borosilicate glasses with constant molar ratios. A script for the fast acquisition of EELS has been designed, from which the fraction of BO4 tetrahedra can be obtained by fitting the experimental data with linear combinations of the reference spectra. The BO4 fractions (N4) obtained by EELS are consistent with those from 11B MAS NMR spectra, suggesting that EELS can be an alternative and convenient way to determine the N4 fraction in glasses. In addition, the boron speciation of a CeO2 doped potassium borosilicate glass has been analyzed by using the time-resolved EELS spectra. The results clearly demonstrate that the BO4 to BO3 transformation induced by the electron beam irradiation can be efficiently suppressed by doping CeO2 to the borosilicate glasses.

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

ABSTRACTTitanate ceramics have been proposed as candidate materials for immobilizing excess weapons plutonium. This study focuses on the characterization of a titanate-based ceramic through X-ray diffraction (XRD), electron probe microanalysis, and electron energy-loss spectroscopy (EELS). Three distinct phases have been identified, and their volume fraction was determined from element distribution maps using Scionimage-NIH Analysis software. This analysis revealed that the pyrochlore-group phase betafite (A2Ti2O7) forms the matrix of the ceramic and occupies 90.4% of the volume. Uniformly distributed in this matrix are perovskite (A2Ti2O6) and Hf-enriched rutile (TiO2), which account for 6.4 vol% and 3.1 vol%, respectively. The studied ceramic exhibits a very low porosity (0.3 vol%), which is characterized by small (<6 μm), rounded and isolated voids. In the studied ceramic, A-site cations are represented by Ca, rare earth elements, and Hf. The powder XRD pattern of the ceramic allowed refining the unit cell parameters for the cubic betafite, which is characterized by a cell edge of 10.132±0.003Å. The EELS data indicate that Ce is present as both Ce3+ and Ce4+ in betafite, whereas in perovskite, all Ce is trivalent.

EELS characterization of radiolytic products in frozen samples

The identification and differentiation of the mineral phases, hydroxyapatite (HA, Ca10(PO4)6(OH)2) and octacalcium phosphate (OCP, Ca8H2(PO4)6), remains challenging because of their similar composition and chemical structure. In this research, electron energy-loss spectroscopy (EELS) analyses revealed indicators to distinguish HA and OCP and these were applied to examine mineral development in enamel from mouse incisors. Reference EELS data for HA and OCP was established with commercial HA and synthesized OCP. An evaluation of electron damage and a mitigation strategy of multipass imaging was conducted, and the electron dose limitation of OCP was identified. New insights into the mechanism of electron beam damage on the apatite crystal were obtained. With the characterization of the energy-loss spectra and the EELS simulation, the oxygen K-edge was found to be one of the indicators for the differentiation of HA from OCP. The second indicator, the Ca/P ratio, was calculated with a calibrated experimental factor of K exp. Elemental mapping was done to establish the different Ca/P ratio of HA and OCP, and the boundary between these mineral forms. EELS analysis was performed on developing enamel in wild-type (WT) and Mmp20 knockout (KO) mice. This research establishes a protocol for EELS analysis on biological specimens and demonstrates the power and potential of EELS in biomaterial characterization.

Characterization of grain-boundary phases in Li7La3Zr2O12 solid electrolytes

A quasi 3-dimensional Monte Carlo simulation program, called SESAME, has been developed for the simulation of electron-matter interactions in totally arbitrary 2-dimensional structures. SESAME is designed for application in different analysis techniques, like scanning electron microscopy (SEM), electron probe microanalysis (EPMA), energy and wavelength dispersive X-ray spectroscopy (EDS/WDS), Auger electron spectroscopy (AES), transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), etc.. The program is extremely flexible in terms of simulation geometries as well as in terms of possessing the ability to handle the majority of electron-matter interaction processes. A modified quadtree discretization for geometry handling is used. Three different scattering models have been implemented to account for the great variety of possible applications.

Electron microscopy has been an important method for visualising biological structures and processes since the 1940s. The discovery of a practical vitreous-ice specimen-preparation technique in the mid-1980s [Adrian 1984] led to modern-day Cryogenic Electron Microscopy (Cryo-EM) which in recent years has become a major technique for studying the architecture of biological macromolecules. Many further instrumental and data-analysis improvements were established in the decades after the introduction of the “vitreous-ice” state of water. Especially the advent of direct electron detectors boosted the quality of the recorded data, allowing atomic-resolution information of biological complexes to be harvested in the early 2010s, developments that truly revolutionized the use of Cryo-EM in structural biology. Single-Particle Analysis (SPA) of isolated molecules, prepared in a thin layer of vitreous water, has proven a most successful approach in structural biology and now often supplants the use of classical techniques like X-ray crystallography, especially for large biological complexes. The ever-increasing number of researchers using Cryo-EM is reflected by the growing number of depositions in the Electron Microscopy Data Bank (EMDB). Explaining this methodology to a new generation of researchers has now become a priority. In writing this review we were reminded of some persistent confusions that emerged in the early days of Cryo-EM but that continue to muddle the field. A new problem with the prolific use of Graphic User Interfaces (GUIs), is that the underlying methodology is often no longer transparent to the users of these “black boxes”. Complicated procedures, well hidden behind a GUI window, may contain methodological flaws that the user must be aware of. The conquering of markets in this booming Cryo-EM field – crucial for developing new pharmaceuticals – must not prevail over scientific integrity. We here describe and critically review the principles of single-particle Cryo-EM. We warn for procedures that have gone astray and could generate serious problems especially in the quality-control of Single-Particle Cryogenic Electron Microscopy.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Quantitative electron energy loss spectroscopy in biology

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes. This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

New Approach To Quantitative Subcellular Imaging Of Phosphorus And Calcium Using Energy-filtered Transmission Electron Microscopy And Tomography

Single-particle reconstruction (SPR) in cryogenic electron microscopy (cryo-EM) aims at aligning and averaging two-dimensional micrographs to reconstruct a three-dimensional particle.How to reconstruct micrographs from heavy noise is a crucial point for achieving better micrograph quality, and thus many methods focus on noise removal. However, new problems such as over-smoothing often occur in their results due to failure in handling heavy noise well. This paper proposes a three-dimensional weighted nuclear norm minimization (3DWNNM) model for SPR in the cryo-EM task to address these issues. Specifically, we design a minimization solver based on the forward-backward splitting algorithm to tackle our model efficiently. Under certain conditions, this solution has an energy-decaying feature and performs exceptionally well in reconstruction. Numerical experiments fully demonstrate the effectiveness and the robustness of the proposed method.

The evolution of hydrogen from many hydrated cryo-preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm(-2) and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy-loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge-shaped specimens of frozen-hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen-hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy-loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation-sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen-based artefacts.

Focused Ion Beam (FIB): A novel technology for advanced application of micro- and nanoanalysis in geosciences and applied mineralogy