Orientation averaging of electron backscattered diffraction data

The use of data averaging to improve the angular precision of electron backscattered diffraction (EBSD) maps is discussed. It is shown that orientations may be conveniently and rapidly averaged using the four Euler-symmetric parameters which are coefficients of a quaternion representation. The processing of EBSD data requires the use of an edge preserving filter and a modified Kuwahara filter has been successfully implemented and tested. Three passes of such a filter have been shown to reduce orientation noise by a factor of ∼10. Application of the method to deformed and recovered aluminium alloys has shown that such data processing enables small subgrain misorientation (< 0.5°) to be detected reliably.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

Backscattered electron imaging of titanium dioxide in frozen hydrated preparations

Backscattered electron (BSE) imaging was used to study ultrafine TiO₂ crystals distribution in a test cream. The cream was fast frozen, cryofractured and observed uncoated at low temperature. The BSE detector was a microchannel plate. The results demonstrate that up-to-date photoprotective preparations can be investigated by this technique.     

Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

Backscattered electron imaging of cultured cells: application to electron probe X-ray microanalysis using a scanning electron microscope

We report a simple method to study the elemental content in cultured human adherent cells by electron probe X-ray microanalysis with scanning electron microscopy. Cells were adapted to grow on polycarbonate tissue culture cell inserts, washed with distilled water, plunge-frozen with liquid nitrogen and freeze-dried. Unstained, freeze-dried cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron microscopy. With backscattered electron imaging it was possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray microanalysis was used simultaneously to determine the elemental content in cultured cells at the cellular level. In addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings demonstrate that backscattered electron imaging offers a powerful method to examine whole, freeze-dried cultured cells for scanning electron probe X-ray microanalysis.     

Backscattered electron imaging of immunogold-labeled and silver-enhanced microtubules in cultured mammalian cells

This technique permits the visualization of microtubules in situ by employing silver-enhanced immunogold labeling and backscattered electron imagery. For best results, monolayer cultures of PtK₂ cells are lysed with Triton X-100 in a microtubule stabilizing buffer, fixed with 1% glutaraldehyde, reduced with NaBH₄, incubated with monoclonal antitubulin and 5-nm gold-labeled anti-IgG, silver enhanced, freeze dried, lightly coated with aluminum, and examined in an SEM equipped with a backscattered electron detector. A high contrast view of the entire microtubule complex of each cell is obtained. Microtubules in freeze-dried preparations have relatively smooth surfaces, whereas those in critical point dried preparations are more irregular or beaded. At high magnifications, an unstained inner core of each microtubule can be resolved. Backscattered electron imaging appears to be a promising technique for localizing cytoskeletal proteins and other intracellular antigens that can be labeled with immunogold and enhanced with silver.     

First combined electron backscatter diffraction and transmission electron microscopy study of grain boundary structure of deformed quartzite

The structures of boundaries in a deformed and dynamically recovered and recrystallized quartz polycrystal (mylonite) were characterized by transmission electron microscopy, after the misorientation angles across the same grain boundaries had been analysed using electron backscatter diffraction in a scanning electron microscope. In this new approach, a specific sample area is mapped with electron backscatter diffraction, and the mapped area is then attached to a foil, and by the ion beam thinned for transmission electron microscopy analysis. Dislocations in grain boundaries were recognized as periodic and parallel fringes. The fringes associated with dislocations are observed in boundaries with misorientations less than 9°, whereas such fringes cannot be seen in the boundaries with misorientations larger than 17°. Some boundaries with misorientations between 9° and 17° generally have no structures associated with dislocation. One segment of a boundary with a misorientation of 13.5° has structures associated with dislocations. It is likely that the transition from low‐angle to high‐angle boundaries occurs at misorientations ranging from approximately 9° to 14°. Change in the grain boundary structure presumably influences the mobility of the boundaries. In the studied deformed quartz vein, a relative dearth of boundaries between misorientation angles of θ = 2° and θ = 15° has previously been reported, and high‐angle boundaries form cusps where they intersect low‐angle boundaries, suggesting substantial mobility of high‐angle boundaries.     

Secondary and backscattered electron imaging of weathered chromian spinel

Secondary (SE) and backscattered electron (BSE) imaging as well as x-ray microanalysis have demonstrated that the weathering of chromian spinel occurs as a progressive form of alteration. Numerous chemical discriminant analysis methods based on the composition of chromian spinel are used to locate valuable deposits of minerals. These methods will be misleading if the correct interpretation of the weathering of chromian spinel and the subsequent pattern of changes in its mineral chemistry are not properly assessed using scanning electron microscopy. This assessment is vital in understanding the geological processes involved and the economic potential of any indicated deposit. Minerals such as chromian spinel, pyrope garnet, and picroilmenite are considered to be highly resistant to weathering and abrasion and are therefore useful in the search for associated valuable deposits of diamond, nickel, platinum, and gold. Known as indicator minerals, they are usually present in relatively large concentrations compared with the target mineral (e.g., diamond) and form large and often subtle dispersion anomalies adjacent to the deposit. Chromian spinel has long been regarded as a stable indicator mineral; however, detailed SE and BSE imaging indicates that many of the chromian spinels that are routinely examined using scanning electron microscopes (SEM) and microprobes are extensively altered. Secondary electron and BSE imaging of weathered chromian spinel in a normal SEM provides valuable data on the form and chemical style of the alteration. Secondary electron imaging of weathered chromian spinel in the environmental SEM (ESEM) not only enhances the difference in atomic number between unaltered and altered areas but also allows high-resolution imaging of these very fine replacement textures.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Progressive steps in the development of electron backscatter diffraction and orientation imaging microscopy

Ten years ago electron backscatter diffraction (EBSD) became available to a wider group active in materials research. This paper highlights some of the more significant developments in camera technology and software developments that have arisen since then. The use of slow‐scan charge couple device cameras for phase identification and rapid determination of orientation image micrographs is reviewed. The current limiting spatial resolution of the technique is shown to be less than 10 nm. A procedure for improving lattice spacing measurement by utilizing the full resolution of the camera is described with experimental measurements on silicon and nickel showing relative errors of plus/minus 3%. An investigation of partially recrystallized aluminium shows how the recrystallized fraction can be extracted with confidence but that the mapping of substructure in the highly deformed regions is questionable. Phase identification is described for complex cases in which the phase data tabulated in standard databases do not correspond to what is observed in the EBSD patterns. Phase mapping in a complex mineral in which chemical data and EBSD data are collected simultaneously is shown to be improved by recording both the chemical and the EBSD data into computer memory and proceeding with the phase discrimination and orientation measurement in off‐line analysis.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

Characterizing the deformed state in Al-0.1 Mg alloy using high-resolution electron backscattered diffraction

The application of high resolution electron backscatter diffraction (EBSD) in a field emission gun scanning electron microscope to the characterization of a deformed aluminium alloy is discussed and the results are compared with those obtained by transmission electron microscopy. It is shown that the adequate spatial resolution, accompanied by the improvement in angular resolution to ~0.5° that can be achieved by data processing, together with the extensive quantitative data obtainable, make EBSD a suitable method for characterizing the cell or subgrain structures in deformed aluminium. The various methods of analysing EBSD data to obtain subgrain sizes are discussed and it is concluded that absolute subgrain reconstruction is the most accurate.     

Low angle boundary migration of shot‐peened pure nickel investigated by electron channeling contrast imaging and electron backscatter diffraction

Study on recrystallization of deformed metal is important for practical industrial applications. Most of studies about recrystallization behavior focused on the migration of the high‐angle grain boundaries, resulting in lack of information of the kinetics of the low angle grain boundary migration. In this study, we focused on the migration of the low angle grain boundaries during recrystallization process. Pure nickel deformed by shot peening which induced plastic deformation at the surface was investigated. The surface of the specimen was prepared by mechanical polishing using diamond slurry and colloidal silica down to 0.02 μm. Sequential heat treatment under a moderate annealing temperature facilitates to observe the migration of low angle grain boundaries. The threshold energy for low angle boundary migration during recrystallization as a function of misorientation angle was evaluated using scanning electron microscopy techniques. A combination of electron channeling contrast imaging and electron backscatter diffraction was used to measure the average dislocation density and a quantitative estimation of the stored energy near the boundary. It was observed that the migration of the low angle grain boundaries during recrystallization was strongly affected by both the stored energy of the deformed matrix and the misorientation angle of the boundary. Through the combination of electron channeling contrast imaging and electron backscatter diffraction, the threshold stored energy for the migration of the low angle grain boundaries was estimated as a function of the boundary misorientation.     

Identification of growth increments in the shell of the bivalve mollusc Arctica islandica using backscattered electron imaging

Annually resolved growth increments in the shell of the bivalve mollusc Arctica islandica have previously been used in combination with geochemical measurements to successfully construct high-resolution proxy records of past marine environmental conditions. However, to ensure the accuracy of these paleoenvironmental reconstructions it is essential that the annual growth series of increments within the examined shells are reliably identified, and can be distinguished from spurious lines caused by nonannual perturbations such as those resulting from storm disturbance. The current methods used for identifying the growth increment series are sometimes compromised because of ambiguity that results from the employed preparation methods. Here it is shown that backscattered electron imaging of polished shell cross sections may be used to clearly discriminate between the two compositionally and structurally distinct increments that comprise 1 year of outer shell growth. This method, involving minimal specimen preparation, is likely to be primarily useful as a validation technique of particular value in cases where increment identification using existing methods is difficult or ambiguous.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

Application of electron backscatter diffraction to the study of phase transformations: present and possible future

This paper first underlines the main advantages, use and limitations of the electron backscatter diffraction technique from the viewpoint of phase transformations. To get a deeper understanding of physical mechanisms involved in phase transformations, several evolutions are now in progress to get an insight into both three-dimensional and real-time information. Two of them, in particular, improvement of data collection versus improvement of data processing are discussed in the second part of this paper.     

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.     

User‐independent EBSD parameters to study the progress of recovery and recrystallization in Cu–Zn alloy during in situ heating

Microstructural evolution of cold‐rolled Cu–5%Zn alloy during in situ heating inside field‐emission scanning electron microscope was utilized to obtain user‐independent parameters in order to trace the progress of static recovery and recrystallization. Electron back‐scattered diffraction (EBSD)‐based orientation imaging microscopy was used to obtain micrographs at various stages of in situ heating. It is shown that unlike the pre‐existing methods, additional EBSD‐based parameter can be used to trace the progress of recovery and recrystallization, which is not dependent on user input and hence less prone to error. True strain of 0.3 was imposed during cold rolling of alloy sample. Rolled sample was subjected to in situ heating from room temperature to 500°C (～0.58 Tm) with soaking time of 10 min, at each of the intermediate temperatures viz. 100, 200, 300, 400 and 450°C. After reaching 500°C, the sample was kept at this temperature for a maximum duration of around 15 h. The sample showed clear signs of recovery for temperature up to 450°C, and at 500°C, recrystallization started to take place. Recrystallization kinetics was moderate, and full recrystallization was achieved in approximately 120 min. We found that EBSD parameter, namely, band contrast intensity can be used as an extra handle to map out the progress of recrystallization occurring in the sample. By contrast, mean angular deviation can be used to understand the evolution of recovery in samples. The parameters mentioned in the current study, unlike other pre‐existing methods, can also be used for mapping local microstructural transformations due to recovery and recrystallization. We discuss the benefits and limitations in using these additional handles in understanding the changes taking place in the material during in situ heating.     

Analytical transmission electron microscopy and electron diffraction for characterization of multiphase Ni-P-Ti surface layer on Ti-6Al-4V alloy

The microstructure, chemical and phase composition of the hard Ni‐P‐Ti layer formed on the Ti‐6Al‐4V alloy after duplex surface treatment were investigated by light microscopy, X‐ray diffraction, scanning electron microscopy and analytical/high‐resolution transmission electron microscopy. These investigations showed that the improved mechanical and tribological properties of the surface‐treated alloy were related to the presence of a multilayered microstructure containing several phases from the Ni‐Ti‐P‐Al system.