Arguably one of the most significant microscopy techniques of the past decade, electron backscatter diffraction (EBSD) has provided scientists and engineers with tremendous insight into the structure of crystalline materials. What started with basic observations of electron diffraction in the middle of the 20th century has grown into a mature technology that bridges the gap between the macro and micro length scales. EBSD has found a home in both the materials science and geological communities characterizing crystallographic texture and preferred orientations, residual strain, grain boundary character and networks, and identifying constituent phases. Advancements in computational power, camera technology, indexing algorithms, sample preparation, and dynamical simulations have made this possible.

Since the automation of the electron backscatter diffraction (EBSD) technique, EBSD systems have become commonplace in microscopy facilities within materials science and geology research laboratories around the world. The acceptance of the technique is primarily due to the capability of EBSD to aid the research scientist in understanding the crystallographic aspects of microstructure. There has been considerable interest in using EBSD to quantify strain at the submicron scale. To apply EBSD to the characterization of strain, it is important to understand what is practically possible and the underlying assumptions and limitations. This work reviews the current state of technology in terms of strain analysis using EBSD. First, the effects of both elastic and plastic strain on individual EBSD patterns will be considered. Second, the use of EBSD maps for characterizing plastic strain will be explored. Both the potential of the technique and its limitations will be discussed along with the sensitivity of various calculation and mapping parameters.

The pattern center of an electron backscatter diffraction (EBSD) image indicates the relative position of the image with reference to the interaction volume of the sample. As interest grows in high-resolution EBSD techniques, accurate knowledge of this position is essential for precise interpretation of the EBSD features. In a typical EBSD framework, Kikuchi bands are recorded on a phosphor screen. If the flat phosphor were instead shaped as a sphere, with its center at the specimen's electron interaction volume, then the incident backscattered electrons would form Kikuchi bands on that sphere with parallel band edges centered on great circles. In this article, the authors present a method of pattern center (PC) refinement that maps bands from the planar phosphor onto a virtual spherical screen and measures the deviation of bands from a great circle and from possessing parallel edges. Potential sources of noise and error, as well as methods for reducing these, are discussed. Finally, results are presented on the application of the PC algorithm to two types of simulated EBSD patterns and two experimental setups, and the resolution of the method is discussed.

Electron backscatter diffraction (EBSD) was employed to analyze the microstructure and texture of four different Alloy 82H gas tungsten arc welds that previously underwent static and dynamic modulus testing. The Young's moduli were shown to differ among the various welds, but also within each weld, dependent upon the direction measured. These differences were attributed to anisotropy of the crystallographic textures, which were described using inverse pole figures with respect to each of the weld's three orthogonal axes. The Young's modulus demonstrated a strong correlation with the texture, consistent with single crystal experiments. Sample directions containing a large population of {100} orientations had the lowest Young's modulus, while those with {111} grains possessed the highest. Microstructures with {110} textures were closer to the average modulus value of 207 GPa (30.0 Msi). X-ray diffraction texture measurements on four samples were used to verify the EBSD results.

Udimet 188 was subjected to thermomechanical processing (TMP) in an attempt to understand the effects of cold-rolling deformation on the microstructure and tensile-creep behavior. Commercially available sheet was cold rolled to varying amounts of deformation (between 5–35% reduction in sheet thickness) followed by a solution treatment at 1,464 K (1,191°C) for 1 h and subsequent air cooling. This sequence was repeated four times to induce a high-volume fraction of low-energy grain boundaries. The resultant microstructure was characterized using electron backscattered diffraction. The effect of the TMP treatment on the high-temperature [1,033–1,088 K (760–815°C)] creep behavior was evaluated. The measured creep stress exponents (6.0–6.8) suggested that dislocation creep was dominant at 1,033 K (760°C) for stresses ranging between 100–220 MPa. For stresses ranging between 25–100 MPa at 1,033 K (760°C), the stress exponents (2.3–2.8) suggested grain boundary sliding was dominant. A significant amount of grain boundary cracking was observed both on the surface and subsurface of deformed samples. To assess the mechanisms of crack nucleation, in situ scanning electron microscopy was performed during the elevated-temperature tensile-creep deformation. Cracking occurred preferentially along general high-angle grain boundaries (GHAB) and less than 25% of the cracks were found on low-angle grain boundaries (LAB) and coincident site lattice boundaries (CSLB). Creep rupture experiments were performed at T = 1,088 K (815°C) and σ = 165 MPa and the greatest average time-to-rupture was exhibited by the TMP sheet with the greatest fraction of LAB+CSLB. However, a clear correlation was not exhibited between the grain boundary character distribution and the minimum creep rates. The findings of this work suggest that although grain boundary engineering may be possible for this alloy, simply relating the fraction of grain boundary types to the creep resistance is not sufficient.

Strain-induced selective growth was investigated in a 1.5% temper-rolled Fe∼1%Si alloy using the electron backscatter diffraction (EBSD) technique. The EBSD technique was used to quantify the presence of orientation spreads within grains and to show that this particular case of selective growth can be directly related to differences in stored energy as reflected in the geometrically necessary dislocation content. The differences in stored energy were sufficient to give rise to selective growth as evidenced by bi-modal grain sizes.

Electron backscatter diffraction (EBSD) techniques were used to characterize “hot-rolled” quenched and partitioned microstructures produced via Gleeble thermal simulations representing a hot-strip cooling practice for steel. In particular, EBSD was utilized to positively identify the morphology and location of retained austenite, to qualitatively distinguish martensite from ferrite, and in an attempt to identify transition carbides. Large pools of retained austenite and some thin films were accurately indexed; however, there was some disparity between austenite volume fractions measured by EBSD and those measured by X-ray diffraction. Due to similarities between the crystal structures of martensite and ferrite (body centered tetragonal versus body centered cubic, respectively), martensite could not be distinguished from ferrite by indexing of diffraction patterns; however, martensite could qualitatively be distinguished from ferrite by regions of low image quality based on the very high dislocation density of martensite.

An open source software package dedicated to processing stored electron backscatter patterns is presented. The package gives users full control over the type and order of operations that are performed on electron backscatter diffraction (EBSD) patterns as well as the results obtained. The current version of EBSD-Image (www.ebsd-image.org) offers a flexible and structured interface to calculate various quality metrics over large datasets. It includes unique features such as practical file formats for storing diffraction patterns and analysis results, stitching of mappings with automatic reorganization of their diffraction patterns, and routines for processing data on a distributed computer grid. Implementations of the algorithms used in the software are described and benchmarked using simulated diffraction patterns. Using those simulated EBSD patterns, the detection of Kikuchi bands in EBSD-Image was found to be comparable to commercially available EBSD systems. In addition, 24 quality metrics were evaluated based on the ability to assess the level of deformation in two samples (copper and iron) deformed using 220 grit SiC grinding paper. Fourteen metrics were able to properly measure the deformation gradient of the samples.

Copper, gold, and tungsten thin films have been exposed to 30 kV Ga+ ion irradiation, and the resulting microstructural modifications are studied as a function of ion dose. The observed microstructural changes include texture development with respect to the easy channeling direction in the target, and in the case of Cu, an additional intermetallic phase is produced. Texture development in these target materials is a function of the starting materials grain size, and these changes are not observed in large grained materials. The accepted models of differential damage driven grain growth are not supported by the results of this study. The implications of this study to the use of focused ion beam tools for sample preparation are discussed.

A series of multislice simulations to quantify the effect of various degrees of order, composition, and thickness on the electron diffracted intensities were performed using the L10 FePt system as the case study. The dynamical diffraction studies were done in both a convergent electron beam diffraction and selected area electron diffraction condition. The L10 symmetry demonstrated some peculiar challenges in the simulation, in particular between the {111} plane normal and the ⟨111⟩ direction, which are not equivalent because of tetragonality. A hybrid weighting function atom of Fe-Pt was constructed to account for S < 1 or nonequiatomic compositions. This statistical approach reduced the complexity of constructing a crystal with the probability that a particular atom was at a particular lattice site for a given order parameter and composition. Considerations of accelerating voltage, convergent angle, and thermal effects are discussed. The simulations revealed significant differences in intensity ratios between films of various compositions but equivalent unit cell numbers and degree of order.

Eight FePt thin film specimens of various thicknesses, compositions, and order parameters have been analyzed to determine the robustness and fidelity of multislice simulations in determining the chemical order parameter via electron diffraction (ED). The shape of the simulated curves depends significantly on the orientation and thickness of the specimen. The ED results are compared to kinematical scattering order parameters, from the same films, acquired from synchrotron X-ray diffraction (XRD). For the specimens analyzed with convergent beam electron diffraction conditions, the order parameter closely matched the order parameter as determined by the XRD methodology. However, the specimens analyzed by selected area electron diffraction conditions did not show good agreement. This has been attributed to substrate effects that hindered the ability to accurately quantify the intensity values of the superlattice and fundamental reflections.

X-ray elemental mapping and X-ray spectrum imaging are powerful microanalytical tools. However, their scope is often limited spatially by the raster area of a scanning electron microscope or microprobe. Limited sampling size becomes a significant issue when large area (>10 cm2), heterogeneous materials such as concrete samples or others must be examined. In such specimens, macro-scale structures, inclusions, and concentration gradients are often of interest, yet microbeam methods are insufficient or at least inefficient for analyzing them. Such requirements largely exclude the samples of interest presented in this article from electron probe microanalysis. Micro X-ray fluorescence–X-ray spectrum imaging (μXRF-XSI) provides a solution to the problem of macro-scale X-ray imaging through an X-ray excitation source, which can be used to analyze a variety of large specimens without many of the limitations found in electron-excitation sources. Using a mid-sized beam coupled with an X-ray excitation source has a number of advantages, such as the ability to work at atmospheric pressure and lower limits of detection owing to the absence of electron-induced bremsstrahlung. μXRF-XSI also acts as a complement, where applicable, to electron microbeam X-ray output, highlighting areas of interest for follow-up microanalysis at a finer length scale.

We demonstrate for the first time that multivariate statistical analysis techniques can be applied to atom probe tomography data to estimate the chemical composition of a sample at the full spatial resolution of the atom probe in three dimensions. Whereas the raw atom probe data provide the specific identity of an atom at a precise location, the multivariate results can be interpreted in terms of the probabilities that an atom representing a particular chemical phase is situated there. When aggregated to the size scale of a single atom (∼0.2 nm), atom probe spectral-image datasets are huge and extremely sparse. In fact, the average spectrum will have somewhat less than one total count per spectrum due to imperfect detection efficiency. These conditions, under which the variance in the data is completely dominated by counting noise, test the limits of multivariate analysis, and an extensive discussion of how to extract the chemical information is presented. Efficient numerical approaches to performing principal component analysis (PCA) on these datasets, which may number hundreds of millions of individual spectra, are put forward, and it is shown that PCA can be computed in a few seconds on a typical laptop computer.

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

Labeling of organelles for microscopy is achieved generally by specific dyes that either accumulate in a cellular compartment such as cyanine dyes in mitochondria or are only fluorescent under specific conditions such as the low pH in the lysosome. Here we demonstrate that Cy5—a fluorescent molecule that does not enter cells by itself—can be loaded into cells by attaching a short oligonucleotide. This very inexpensive labeling procedure can be done in the presence of serum. Therefore, very sensitive cell types should also be amenable to this procedure, and longer observations can be achieved compared to other commercially available dyes as the labeling reagent does not need to be washed out. This also points to the pitfall of using fluorescently labeled oligonucleotides for live cell imaging where the oligonucleotide is supposed to detect a specific target sequence in its subcellular distribution.

The objective of this study was to evaluate the influence of a cola soft drink (CSD) and coffee on the microhardness and surface roughness of composite resin. Fifty cavities were prepared on the vestibular surface of bovine incisors and restored with nanoparticulate resin. The teeth were divided into five groups (n = 10): group A (control), immersion in artificial saliva (AS) for 14 days; group B, immersion in coffee for 15 min (3×/day) for 7 days followed by immersion in AS for another 7 days; group C, immersion in CSD for 15 min (3×/day) for 7 days followed by immersion in AS for another 7 days; group D, immersion in AS for 7 days, immersion in coffee for 15 min (3×/day) for 7 days; group E, immersion in AS for 7 days, immersion in CSD for 15 min (3×/day) for 7 days. After the immersion periods the specimens were analyzed for their microhardness and surface roughness. The data were subjected to analysis of variance (ANOVA) followed by t-test with 5% significance. Group A presented the highest average microhardness and lowest surface roughness, so it was possible to conclude that the consumption of CSD and coffee alters the microhardness and surface roughness of new restorations.

To date, there are limited studies on loggerhead turtle (Caretta caretta) eggshell ultrastructure and its elemental composition. Eggs were collected from turtle nests immediately after oviposition and post hatching. Three eggshell layers were recognized. The outer calcareous layer consists of loose nodular units of different shapes and sizes with loose attachment between the units, resulting in numerous spaces and openings. Each unit consists of CaCO3 crystals in aragonite (99%) and calcite (1%). The middle layer has several strata with numerous openings connecting the calcareous and the inner shell membrane. Crystallites of the middle layer are a mix of amorphous material with aragonite (62%) and calcite (38%). The inner shell membrane has numerous reticular fibers mixed predominantly with halite (NaCl) and small amounts of sylvite. Thermogravimetry analysis of the calcareous showed a low exothermic peak at 425°C, which corresponds to a transitional phase from aragonite to calcite. A high endothermic peak at 814°C corresponds to decomposition of calcite CaCO3 to CaO and CO2. Electron diffraction confirmed the presence of NaCl halite crystal. A significant difference was found in the percentage of elements and crystal configurations in the three layers. This study has value in assessing the emergence success in this endangered species.

Morphology of foliar trichomes was analyzed in Quercus variabilis by electron microscopy and three-dimensional surface profiling. Leaves from suppressed or dominant sprouts of the oak species were collected after a forest fire to unravel the effects of the disturbance factor on sprouting of the oak species. Scanning electron microscopy revealed two types of trichomes depending on the leaf surface. The trichomes on the adaxial surface were branched and constricted, and possessed a single row of thin-walled cells with a collapsed morphology (glandular branched uniseriate trichomes). Meanwhile, the trichomes on the abaxial surface were star-shaped, unfused with each other, and had 6 to 10 rays (nonglandular simple stellate trichomes). An apparent proliferation of trichomes was evident on the adaxial surface of the dominant sprouts. Uniseriate trichomes could be discernable as an elevation from the surface by white light scanning interferometry. By transmission electron microscopy, thin and convoluted cell wall, degenerated cytoplasm, and a single row of cells were characteristic of the trichomes on the adaxial surface. The thick cell walls of the mature trichomes on the abaxial surface represented the nonglandular nature. This is the first report on the morphological and ultrastructural characterization of foliar trichomes of the oak species.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to Nan Yao, nyao@Princeton.edu.