A novel imaging mode for high-resolution transmission electron microscopy is described. It is based on the adjustment of a negative value of the spherical aberration CS of the objective lens of a transmission electron microscope equipped with a multipole aberration corrector system. Negative spherical aberration applied together with an overfocus yields high-resolution images with bright-atom contrast. Compared to all kinds of images taken in conventional transmission electron microscopes, where the then unavoidable positive spherical aberration is combined with an underfocus, the contrast is dramatically increased. This effect can only be understood on the basis of a full nonlinear imaging theory. Calculations show that the nonlinear contrast contributions diminish the image contrast relative to the linear image for a positive-CS setting whereas they reinforce the image contrast relative to the linear image for a negative-CS setting. The application of the new mode to the imaging of oxygen in SrTiO3 and YBa2Cu3O7 demonstrates the benefit to materials science investigations. It allows us to image directly, without further image processing, strongly scattering heavy-atom columns together with weakly scattering light-atom columns.

Atom probe tomography is a technique for the nanoscale characterization of microstructural features. Analytical techniques have been developed to estimate the size, composition, and other parameters of features as small as 1 nm from the atom probe tomography data. These methods are outlined and illustrated with examples of yttrium-, titanium-, and oxygen-enriched particles in a mechanically alloyed, oxide-dispersion-strengthened steel.

The performance of the pulsed-laser atom probe can be limited by both instrument and specimen factors. The experiments described in this article were designed to identify these factors so as to provide direction for further instrument and specimen development. Good agreement between voltage-pulsed and laser-pulsed data is found when the effective pulse fraction is less than 0.2 for pulsed-laser mode. Under the conditions reported in this article, the thermal tails of the peaks in the mass spectra did not show any significant change when produced with either a 10-ps or a 120-fs pulsed-laser source. Mass resolving power generally improves as the laser spot size and laser wavelength are decreased and as the specimen tip radius, specimen taper angle, and thermal diffusivity of the specimen material are increased. However, it is shown that two of the materials used in this study, aluminum and stainless steel, depend on these factors differently. A one-dimensional heat flow model is explored to explain these differences. The model correctly predicts the behavior of the aluminum samples, but breaks down for the stainless steel samples when the tip radius is large. A more accurate three-dimensional model is needed to overcome these discrepancies.

Quantitative EPMA (electron probe microanalysis) intensity measurements require an accurate correction for the X-ray continuum (or background) created by the Bremsstrahlung effect from the primary electron beam. This X-ray continuum, as measured on a wavelength-dispersive spectrometer at any particular wavelength, is primarily a function of the mean atomic number of the material being analyzed. One can calibrate the dependence of the continuum on mean atomic number by measuring and curve fitting the X-ray intensities at the analytical peak in pure elements, oxides, and binary compound standards that do not contain any of the analyte or any interfering elements and use that calibration to calculate the X-ray background correction. For unknown samples, the mean atomic number is determined from the elemental concentrations calculated by the ZAF or φ(ρz) matrix correction, and the fit regression coefficients are used iteratively to calculate the actual background correction. Over a large range of mean atomic number we find that the dependence of the continuum intensity on mean atomic number is well described by a second-order polynomial fit. In the case of low-energy X-ray lines (<1 to 2 keV), this fit is significantly improved by correcting the X-ray continuum intensities for absorption. For major and most minor element analyses, the improved mean atomic number background correction procedure presented in this paper is accurate and robust for a wide variety of samples. Empirical mean atomic number background data are presented for a typical 10-element silicate and a 15-element sulfide analytical set up that demonstrate the validity of the technique as well as some potential limitations.

The aberration-corrected scanning transmission electron microscope has great sensitivity to environmental or instrumental disturbances such as acoustic, mechanical, or electromagnetic interference. This interference can introduce distortions to the images recorded and degrade both signal noise and resolution performance. In addition, sample or stage drift can cause the images to appear warped and leads to unreliable lattice parameters being exhibited. Here a detailed study of the sources, natures, and effects of imaging distortions is presented, and from this analysis a piece of image reconstruction code has been developed that can restore the majority of the effects of these detrimental image distortions for atomic-resolution data. Example data are presented, and the performance of the restored images is compared quantitatively against the as-recorded data. An improvement in apparent resolution of 16% and an improvement in signal-to-noise ratio of 30% were achieved, as well as correction of the drift up to the precision to which it can be measured.

We apply a deep convolutional neural network segmentation model to enable novel automated microstructure segmentation applications for complex microstructures typically evaluated manually and subjectively. We explore two microstructure segmentation tasks in an openly available ultrahigh carbon steel microstructure dataset: segmenting cementite particles in the spheroidized matrix, and segmenting larger fields of view featuring grain boundary carbide, spheroidized particle matrix, particle-free grain boundary denuded zone, and Widmanstätten cementite. We also demonstrate how to combine these data-driven microstructure segmentation models to obtain empirical cementite particle size and denuded zone width distributions from more complex micrographs containing multiple microconstituents. The full annotated dataset is available on materialsdata.nist.gov.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Cathodoluminescence (CL) microscopy and spectroscopy are luminescence techniques with widespread applications in geosciences. Many rock-forming and accessory minerals show CL characteristics, which can be successfully used in geoscientific research. One of the most spectacular applications is the visualization of growth textures and other internal structures that are not discernable with other analytical techniques. In addition, information from CL imaging and spectroscopy can be used for the reconstruction of processes of mineral formation and alteration to provide information about the real structure of minerals and materials, and to prove the presence and type of lattice incorporation of several trace elements. In the present article, an overview about CL properties of selected minerals is given, and several examples of applications discussed. The presented data illustrate that best results are achieved when luminescence studies are performed under standardized conditions and combined with other analytical techniques with high sensitivity and high spatial resolution.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

We propose a framework for indexing of grain and subgrain structures in electron backscatter diffraction patterns of polycrystalline materials. We discretize the domain of a dynamical forward model onto a dense grid of orientations, producing a dictionary of patterns. For each measured pattern, we identify the most similar patterns in the dictionary, and identify boundaries, detect anomalies, and index crystal orientations. The statistical distribution of these closest matches is used in an unsupervised binary decision tree (DT) classifier to identify grain boundaries and anomalous regions. The DT classifies a pattern as an anomaly if it has an abnormally low similarity to any pattern in the dictionary. It classifies a pixel as being near a grain boundary if the highly ranked patterns in the dictionary differ significantly over the pixel’s neighborhood. Indexing is accomplished by computing the mean orientation of the closest matches to each pattern. The mean orientation is estimated using a maximum likelihood approach that models the orientation distribution as a mixture of Von Mises–Fisher distributions over the quaternionic three sphere. The proposed dictionary matching approach permits segmentation, anomaly detection, and indexing to be performed in a unified manner with the additional benefit of uncertainty quantification.

In situ scanning transmission electron microscopy (STEM) through liquids is a promising approach for exploring biological and materials processes. However, options for in situ chemical identification are limited: X-ray analysis is precluded because the liquid cell holder shadows the detector and electron energy-loss spectroscopy (EELS) is degraded by multiple scattering events in thick layers. Here, we explore the limits of EELS in the study of chemical reactions in their native environments in real time and on the nanometer scale. The determination of the local electron density, optical gap, and thickness of the liquid layer by valence EELS is demonstrated. By comparing theoretical and experimental plasmon energies, we find that liquids appear to follow the free-electron model that has been previously established for solids. Signals at energies below the optical gap and plasmon energy of the liquid provide a high signal-to-background ratio regime as demonstrated for LiFePO4 in an aqueous solution. The potential for the use of valence EELS to understand in situ STEM reactions is demonstrated for beam-induced deposition of metallic copper: as copper clusters grow, EELS develops low-loss peaks corresponding to metallic copper. From these techniques, in situ imaging and valence EELS offer insights into the local electronic structure of nanoparticles and chemical reactions.

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25–40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

The recent developments of phase-contrast synchrotron imaging techniques have been of great interest for paleontologists, providing three-dimensional (3D) tomographic images of anatomical structures, thereby leading to new paleobiological insights and the discovery of new species. However, until now, it has not been used on features smaller than 5–7 μm voxel size in fossil bones. Because much information is contained within the 3D histological architecture of bone, including an ontogenetic record, crucial for understanding the paleobiology of fossil species, the application of phase-contrast synchrotron tomography to bone at higher resolutions is potentially of great interest. Here we use this technique to provide new 3D insights into the submicron-scale histology of fossil and recent bones, based on the development of new pink-beam configurations, data acquisition strategies, and improved processing tools. Not only do the scans reveal by nondestructive means all of the major features of the histology at a resolution comparable to that of optical microscopy, they provide 3D information that cannot be obtained by any other method.

Three-dimensional (3D) analysis is an essential tool to obtain quantitative results from 3D datasets. Considerable progress has been made in 3D imaging techniques, resulting in a growing need for more flexible, complete analysis packages containing advanced algorithms. At the Centre for X-ray Tomography of the Ghent University (UGCT), research is being done on the improvement of both hardware and software for high-resolution X-ray computed tomography (CT). UGCT collaborates with research groups from different disciplines, each having specific needs. To meet these requirements the analysis software package, Morpho+, was developed in-house. Morpho+ contains an extensive set of high-performance 3D operations to obtain object segmentation, separation, and parameterization (orientation, maximum opening, equivalent diameter, sphericity, connectivity, etc.), or to extract a 3D geometrical representation (surface mesh or skeleton) for further modeling. These algorithms have a relatively short processing time when analyzing large datasets. Additionally, Morpho+ is equipped with an interactive and intuitive user interface in which the results are visualized. The package allows scientists from various fields to obtain the necessary quantitative results when applying high-resolution X-ray CT as a research tool to the nondestructive investigation of the microstructure of materials.

We present a microfluidic system that maintains liquid flow in a specimen chamber for scanning transmission electron microscope (STEM) imaging. The specimen chamber consists of two ultrathin silicon nitride windows supported by silicon microchips. They are placed in a specimen holder that seals the sample from the vacuum in the electron microscope and incorporates tubing to and from the sample connected to a syringe pump outside the microscope. Using results obtained from fluorescence microscopy of microspheres flowing through the system, an equation to characterize the liquid flow through the system was calibrated. Gold nanoparticles of diameters of 30 and 100 nm moving in liquid were imaged with a 200 kV STEM. It was concluded that despite strong influences from Brownian motion, and sensitivity to small changes in the depth of the bypass channel, the electron microscopy flow data matched the calculated flow speed within an order of magnitude. The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic or bacterial cells, to certain stimuli.

A new approach for the simulation of dynamic electron backscatter diffraction (EBSD) patterns is introduced. The computational approach merges deterministic dynamic electron-scattering computations based on Bloch waves with a stochastic Monte Carlo (MC) simulation of the energy, depth, and directional distributions of the backscattered electrons (BSEs). An efficient numerical scheme is introduced, based on a modified Lambert projection, for the computation of the scintillator electron count as a function of the position and orientation of the EBSD detector; the approach allows for the rapid computation of an individual EBSD pattern by bi-linear interpolation of a master EBSD pattern. The master pattern stores the BSE yield as a function of the electron exit direction and exit energy and is used along with weight factors extracted from the MC simulation to obtain energy-weighted simulated EBSD patterns. Example simulations for nickel yield realistic patterns and energy-dependent trends in pattern blurring versus filter window energies are in agreement with experimental energy-filtered EBSD observations reported in the literature.

Artifacts in the field evaporation behavior of small precipitates have limited the accuracy of atom probe tomography analysis of clusters and precipitates smaller than 2 nm. Here, we report on specific observations of reconstruction artifacts that were obtained in case of precipitates with radii less than 10 nm in Al alloys, focusing particularly on a shift that appears in the relative positioning of matrix and precipitate atoms. We show that this chemically dependent behavior, referred to as “chromatic aberration,” is due to the electrostatic field above the emitter and the variations in field evaporation of the elements constituting the precipitates.

The ability to map plastic deformation around high strain gradient microstructural features is central in studying phenomena such as fatigue and stress corrosion cracking. A method for the visualization of plastic deformation in electron back-scattered diffraction (EBSD) data has been developed and is described in this article. This technique is based on mapping the intragrain misorientation in polycrystalline metals. The algorithm maps the scalar misorientation between a local minimum misorientation reference pixel and every other pixel within an individual grain. A map around the corner of a Vickers indentation in 304 stainless steel was used as a test case. Several algorithms for EBSD mapping were then applied to the deformation distributions around air fatigue and stress corrosion cracks in 304 stainless steel. Using this technique, clear visualization of a deformation zone around high strain gradient microstructural features (crack tips, indentations, etc.) is possible with standard EBSD data.

A method for phase analysis, similar to the Rietveld method in X-ray diffraction, was not developed for electron diffraction (ED) in the transmission electron microscope (TEM), mainly due to the dynamic nature of ED. Nowadays, TEM laboratories encounter many thin samples with grain size in the 1–30 nm range, not too far from the kinematic ED conditions. This article describes a method that performs (semi)quantitative phase analysis for nanocrystalline samples from selected area electron diffraction (SAED) patterns. Fractions of the different nanocrystalline components are determined from rotationally symmetric ring patters. Both randomly oriented nanopowders and textured nanopowders, observed from the direction of the texture axis produce such SAED patterns. The textured fraction is determined as a separate component by fitting the spectral components, calculated for the previously identified phases with a priori known structures, to the measured distribution. The Blackman correction is applied to the set of kinematic diffraction lines to take into account dynamic effects for medium grain size. Parameters of the peak shapes and the other experimental parameters are refined by exploring the parameter space with the help of the Downhill-SIMPLEX. Part I presents the principles, while future publication of Parts II and III will elaborate on current implementation and will demonstrate its usage by examples, respectively.