Study of local atomic order in amorphous materials in a computerized transmission electron microscope

Experimental results obtained by electron diffraction (ED) and extended electron energy loss fine structure (EXELFS) techniques to study the local atomic order in amorphous materials such as carbon, silicon, and its oxides are described. Potential applications of ED and EXELFS techniques and their limitations are also discussed.     

An electron microscopy three‐dimensional characterization of titania nanotubes

To characterize complex, three‐dimensional nanostructures, modern microscopy techniques are needed, such as electron tomography and focused ion beam (FIB) sectioning. The aim of this study was to apply these two techniques to characterize TiO₂ nanotubes in terms of their size, shape, volume, porosity, geometric surface area, and specific surface area (SSA). For these experiments, titania nanotubes were fabricated by means of the electrochemical oxidation of titanium at a voltage of 20 V for 2 hr followed by heat treatment at 450°C for 3 hr to change the amorphous structure into a crystalline anatase structure. The quantitative data obtained from the FIB and electron tomography reconstructions show a high similarity in porosity and some differences in SSA. These might be the result of differences in resolution between the two reconstruction techniques.     

Extended energy-loss fine structure spectroscopy of structural modifications in Nd2CuO4−δ at the oxygen K edge

The discovery of the superconducting electron-doped compound Nd₁₈₅Ce₀₁₅CuO_4?δ has stimulated great interest in its micro- and crystal structure, since the superconducting properties depend on parameters such as nonstoichiometry, phase composition, heat treatment and microstructure. The work presented herein is focused on the determination of the oxygen environment in the undoped parent compound Nd₂CuO₄ and in the structural modification Nd₂CuO₃₅ The analysis of the oxygen K (O 1s) edge extended electron energy-loss fine structure (EXELFS) of the tetragonal parent compound Nd₂CuO₄ and of the orthorhombic modification Nd₂CuO₃₅ is reported by using electron energy-loss spectroscopy in combination with transmission electron microscopy. Nd₂CuO₃₅ is produced by in situ heating and reduction of Nd₂CuO₄ in the transmission electron microscope. The EXELFS of the O 1s electron energy-loss edges is analysed with the classical extended X-ray absorption fine structure (EXAFS) treatment and compared with ab initio multiple scattering EXAFS calculations for both structural modifications. Highly accurate information on the local atomic environment of the oxygen atoms in Nd₂CuO₃₅ is obtained from EXELFS analysis using Nd₂CuO₄ as a standard. The results are in accordance with the structural data gained from X-ray diffraction analysis. This applies especially to the more complicated structure of Nd₂CuO₃₅ determined recently.     

Specimen preparation technique for high resolution transmission electron microscopy studies on model supported metal catalysts

A new technique is described which can be used for preparing transmission electron microscopy (TEM) specimens suitable for high resolution studies on supported metal catalysts. By conventional silicon processing techniques 200 × 200 μm² Si₃N₄ membranes on Si wafers are produced. These membranes are extremely flat and have a uniform thickness of 13 nm. They can be used as a support in various kinds of thin film deposition. A TiO₂ film, optimally structured with respect to the requirements for high resolution TEM work in TiO₂–metal cluster systems, is deposited on the Si₃N₄ layer. It consists of one monolayer of 10–25 nm TiO₂ crystallites. TiO₂ lattice images show that a line resolution down to 0.19 nm is possible. Examples of TiO₂–Pd and TiO₂–Rh are given using respectively photodeposition and impregnation reduction to produce l.5–4 nm metal clusters.     

Electron diffraction from micro- and nanoparticles of hydroxyapatite

Hydroxyapatite (HAP) obtained from aqueous solutions under different conditions has been examined by high-resolution transmission electron microscopy (HRTEM) and electron diffraction, including selected-area electron diffraction (SAED) and microdiffraction. A Philips CM300 field-emission gun electron microscope with a Schottky W/ZrO field-emission tip and a spherical aberration constant of 0.65 mm was used at 300 kV. The HAP crystals had different sizes, ranging from a few nanometres to a few micrometres. Single-crystal diffraction patterns have been obtained from the largest microcrystals using the conventional SAED technique. Assemblies of nanoparticles gave only broad diffuse rings. Nevertheless, microdiffraction with electron microprobes 3.5–10 nm in diameter clearly indicated the crystalline character of the nanoparticles in these assemblies. Experimental HRTEM images, Fourier transforms and calculated images exhibited the fine structure of the HAP crystals.     

Analytical scanning electron microscopy for solid surface

A scanning electron microscope of ultra-high-vacuum (UHV-SEM) with a field emission gun (FEG) is operated at the primary electron energies of from 100 eV to 3 keV. The instrument can form the images that contain information on surface chemical composition, chemical bonding state (electronic structure), and surface crystal structure in a microscopic resolution of several hundred angstroms (Å) using the techniques of scanning Auger electron microscope, scanning electron energy loss microscope, and scanning low-energy electron diffraction (LEED) microscope. A scanning tunneling microscope (STM) also has been combined with the SEM in order to obtain the atomic resolution for the solid surface. The instrumentation and examples of their applications are presented both for scanning LEED microscopy and STM.     

A mirror electron microscope for surface analysis

The principle of mirror microscopy has been adapted to provide a relatively low resolution surface microscope (<1000 ×), a large transfer width low energy electron diffractometer and a photoelectron analyser in k_|| space. A focused electron beam of ? 10 kV is decelerated through a Johansson lens, reflected in front of the sample and reaccelerated back through the lens to produce an electron image over a field of view of a few microns. The image can be interpreted as a micrograph of work function variations on the surface if other effects (geometry, magnetic field) are uniform. In the LEED mode, diffracted beams virtually retain their positions on the screen over the whole impact energy range used (0.160 V). Secondary electrons are preferentially focused around the lens-gun electro-optic axis, thus effectively filtering them out from the diffraction pattern. The design has an inherently large coherence length, of up to 10⁴ Å. Photoelectrons can similarly be imaged in k_|| space on the detector plane. The addition of energy filtering at the screen allows the two-dimensional Fermi surface to be imaged.     

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: The utility of methylamine tungstate stain and butvar support film in the study of macromolecules by transmission electron microscopy

The structure of ornithine decarboxylase (M_r ≈? 1.04 × 10⁶) from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.     

Research on precision‐calibration techniques for selected area electron diffraction patterns of pyrocarbon

The key techniques for determining orientation angle (OA) and interlayer space (d002) of pyrocarbon were investigated by analyzing selected area electron diffraction (SAED) patterns. A series of algorithms, which mainly include the five‐point center‐determined technique, the integral factor for the ellipse detection, the background subtraction operation and the Gaussian multipeak fitting algorithm, were designed for intensity sampling, data correction, and data fitting. The contribution ratio of the reflection intensity to the average d002 was considered. The algorithms were programmed and applied to evaluate SAED patterns of pyrocarbon in C/C composites by chemical vapor infiltration. Results showed that the proposed techniques can be effectively used to measure various SAED patterns, with a beam stop image or not, of pyrocarbon. The azimuthal intensities along the (002) arcs essentially obey the Gaussian distribution, although this is not obvious for the lower textural pyrocarbon. It is necessary for accurate OA to use the Gaussian multipeak fitting algorithm. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Direct imaging of local atomic ordering in a Pd-Ni-P bulk metallic glass using Cs-corrected transmission electron microscopy

In amorphous alloys, crystalline atomic clusters as small as 1-2 nm are frequently observed as local lattice fringe images by high-resolution electron microscopy (HREM). These clusters can be understood as local structures of amorphous alloys corresponding to "medium-range-order (MRO)". The MRO structure can be observed only under suitable defocusing conditions of the objective lens in HREM. A clear imaging of the MRO structure is difficult in conventional TEMs, mainly due to the delocalization of the image, caused mainly by the spherical aberration of the objective lens and eventually by the chosen defocus. In the present study, we have examined MRO in a Pd-based bulk metallic glass (Pd(40)Ni(40)P(20)) using a high-resolution TEM (acceleration voltage 200 kV) fitted with a spherical aberration constant corrector (Cs corrector) for aberration correction. We found that when Cs was close to zero and defocus values were near the Gaussian focus, MRO regions with an FCC-Pd structure could be clearly observed with a low image disturbance. Under these conditions, the phase-contrast transfer function was understood to act as an ideal filter function, which distinctly selects specific lattice periods of the FCC-Pd clusters. The obtained atomic images of the glass structure including the FCC-Pd clusters are in good agreement with those expected from image simulation according to our amorphous structure model. In this study, we have demonstrated that the Cs-corrected HREM is a powerful tool to directly image locally ordered structures in metallic glasses.     

A high-speed area detector for novel imaging techniques in a scanning transmission electron microscope

A scanning transmission electron microscope (STEM) produces a convergent beam electron diffraction pattern at each position of a raster scan with a focused electron beam, but recording this information poses major challenges for gathering and storing such large data sets in a timely manner and with sufficient dynamic range. To investigate the crystalline structure of materials, a 16×16 analog pixel array detector (PAD) is used to replace the traditional detectors and retain the diffraction information at every STEM raster position. The PAD, unlike a charge-coupled device (CCD) or photomultiplier tube (PMT), directly images 120–200 keV electrons with relatively little radiation damage, exhibits no afterglow and limits crosstalk between adjacent pixels. Traditional STEM imaging modes can still be performed by the PAD with a 1.1 kHz frame rate, which allows post-acquisition control over imaging conditions and enables novel imaging techniques based on the retained crystalline information. Techniques for rapid, semi-automatic crystal grain segmentation with sub-nanometer resolution are described using cross-correlation, sub-region integration, and other post-processing methods.     

Correlative light and electron microscopy for the analysis of cell division

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.     

Fundamental problems of imaging subsurface structures in the backscattered electron mode in scanning electron microscopy

In‐depth imaging of subsurface structures in scanning electron microscopy (SEM) is usually obtained by detecting backscattered electrons (BSE). For a layer‐by‐layer imaging in BSE microtomography, it is preferable to use an energy filtering of BSE. A simple approach is used to estimate the contrast by using backscattering coefficients of bulk materials and the maximum escape depths of the BSE. The contrast obtained by BSE energy filtering is about twice that of the standard BSE method by varying the acceleration voltage. The contrast decreases with increasing information depth. The information depth is about four times smaller than the electron range. The transmission of the spectrometer influences the minimum current of the order of 10^?8 A that is needed to get a contrast of 1%, for example.     

On the detection of thin intergranular films by electron microscopy

The presence of very thin (6–50 Å) films in integrated circuits and separating crystalline grains in ceramics, notably in zinc oxide varistors and in silicon nitride, has recently been reported using high resolution electron microscopy. The geometric conditions that must be met in order to observe such thin intergranular films using the microscopy techniques of bright-field imaging and lattice-fringe imaging, already adopted, are described. In addition, a method of defocus imaging is applied for the first time to the detection of thin films at grain boundaries.     

Direct imaging of paracrystalline phospholipid structure in the electron microscope

A long chain amphiphilic molecule—the phospholipid 1,2-dihexadecyl sn glycerophosphoethanolamine—has been crystallized epitaxially so that the interlamellar molecular periodicity is parallel to the substrate and hence normal to the electron beam in the electron microscope. This has permitted the direct resolution of the 55·6 Å lamellae in unstained crystals at room temperature. The lattice images have shown the presence of line dislocations and lenticular cracks in the crystals. Of significance to their biological properties is that the lattice is undulating with a periodicity of 0·1–0·5 μm. This would also account for the difficulties encountered by X-ray and electron diffraction techniques when examining these crystals.     

Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.     

Reflection electron energy-loss spectroscopy and imaging for surface studies in transmission electron microscopes.

A review is given on the techniques and applications of high-energy reflection electron energy-loss spectroscopy (REELS) and reflection electron microscopy (REM) for surface studies in scanning transmission electron microscopes (STEM) and conventional transmission electron microscopes (TEM). A diffraction method is introduced to identify a surface orientation in the geometry of REM. The surface dielectric response theory is presented and applied for studying alpha-alumina surfaces. Domains of the alpha-alumina (012) surface initially terminated with oxygen can be reduced by an intense electron beam to produce Al metal; the resistance to beam damage of surface domains initially terminated with Al+3 ions is attributed to the screening effect of adsorbed oxygen. Surface energy-loss near-edge structure (ELNES), extended energy-loss fine structure (EXELFS), and microanalysis using REELS are illustrated based on the studies of TiO2 and MgO. Effects of surface resonances (or channeling) on the REELS signal-to-background ratio are described. The REELS detection of a monolayer of oxygen adsorption on diamond (111) surfaces is reported. It is shown that phase contrast REM image content can be significantly increased with the use of a field emission gun (FEG). Phase contrast effects close to the core of a screw dislocation are discussed and the associated Fresnel fringes around a surface step are observed. Finally, an in situ REM experiment is described for studying atomic desorption and diffusion processes on alpha-alumina surfaces at temperatures of 1,300-1,400 degrees C.     

STM imaging of the complete bacterial cell sheath of Methanospirillum hungatei

Using a large range scanning tunnelling microscope (STM) we have obtained images of the complete outer cell wall of the archaebacterium Methanospirillum hungatei, deposited on a graphite substrate and over-coated with an Au or Pt evaporated film. The width of the collapsed cylindrical sheath structure was found to be 5000 Å ± ***10%, which agrees closely with the value from previously published electron microscope (EM) studies. The double thickness of the collapsed sheath was found to be 160 Å ± 10%, which is about 20% smaller than that from the EM results. Higher resolution STM images taken on top of the collapsed sheaths show corrugations running perpendicular to the cylinder axis and having widths which are multiples of ～ 30 Å, the minimum period expected from EM studies. The height of the corrugations have a minimum value of about 4 Å. The expected 2-D crystalline structure was not seen in the STM images.     

Thickness determination of ultra-thin films using backscattered electron spectra of a new toroidal electrostatic spectrometer

Backscattered electron spectroscopy offers detailed information for multilayer and subsurface-layer materials with distinct Z contrast: It can be used for the validation of Monte Carlo calculations, to obtain depth selective electron microtomographic images, and to determine the thickness of ultra thin films on bulk substrates. In this paper we describe a new energy-dispersive method for thickness determination of thin films on bulk aluminum using backscattered electron (BSE) spectra obtained by a polar, toroidal, electrostatic spectrometer. After a brief recapitulation of the spectrometer's geometry, the techniques for its energy calibration and the preparation of thin double-layer films are introduced. Backscattered electron measurements and thickness calibrations for Au and Cu films with thicknesses of 0–200 nm on bulk aluminum will be presented for various primary electron energies.