The new generation of energy-dispersive X-ray (EDX) detectors with higher count rates than ever before, paves the way for a new approach to quantitative elemental analysis in the scanning transmission electron microscope. Here we demonstrate a method of calculating partial cross sections for use in quantifying EDX data, beneficial especially because of the simplicity of its implementation. Applying this approach to acid-leached PtCo catalyst nanoparticles leads to quantitative determination of the Pt surface enrichment.

In this work we investigated the chemical composition and structure of (K0.5Na0.5)NbO3 (KNN) single crystals grown by the solid state crystal growth method. The optical, scanning, and transmission electron microscopies were employed for the analysis of the chemical homogeneity and domain structure of the KNN crystal. No compositional inhomogeneities within experimental error were encountered in the KNN single crystals. The domain structure of the KNN single crystal, with a monoclinic unit cell, is composed of large 90° domains of up to 100 μm width, which further consist of smaller 180° domains with widths from 50 to 300 nm.

Here we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 μm wide and up to 10 μm long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.Abbreviations: EM: electron microscope; L/P: lactate/pyruvate; QT: QuickTime; CCCP: carbonyl cyanide 3-chloro-phenylhydrazone; CMXRos, chloromethyl-X-rosamine; Rh123, Rhodamine 123; RTM, real time microscope

Confocal Raman microspectroscopy (CRM) has emerged as a powerful approach to visualize the compositional distribution in lignocellulosic biomass of cell walls. In this work, the applicability of CRM for imaging the topochemical correlation between lignin and hydroxycinnamic acids (HCA) in the Miscanthus sinensis internode was explored. Model compound [p-coumaric acid (PCA) and ferulic acid (FA)] analysis indicated that the band region from 1,152 to 1,197 cm−1 can be used to characterize the distribution of HCA. Raman images calculated by integrating over the area intensity of characteristic spectral regions showed heterogeneous distribution of lignin and HCA at cellular and sub-cellular level. When overlaying the Raman image of lignin and HCA distribution, it was found that these two polymers were co-located in the middle lamella and secondary wall of corresponding cells. Raman images for the band intensity ratio (1,173 cm−1/1,603 cm−1) indicated a clear association between lignin and HCA distribution within morphologically distinct cell wall layers of sclerenchyma fibers and the parenchyma. This is the first time that the spatial correlation between lignin and HCA concentration has been illustrated by a microspectroscopy imaging approach. The results are of importance in extending the current understanding of lignin and aromatics topochemistry in herbaceous biomass.

Energy resolution is one of the most important parameters in electron energy-loss spectroscopy. This is especially true for measurement of surface plasmon resonances, where high-energy resolution is crucial for resolving individual resonance peaks, in particular close to the zero-loss peak. In this work, we improve the energy resolution of electron energy-loss spectra of surface plasmon resonances, acquired with a monochromated beam in a scanning transmission electron microscope, by the use of the Richardson–Lucy deconvolution algorithm. We test the performance of the algorithm in a simulated spectrum and then apply it to experimental energy-loss spectra of a lithographically patterned silver nanorod. By reduction of the point spread function of the spectrum, we are able to identify low-energy surface plasmon peaks in spectra, more localized features, and higher contrast in surface plasmon energy-filtered maps. Thanks to the combination of a monochromated beam and the Richardson–Lucy algorithm, we improve the effective resolution down to 30 meV, and evidence of success up to 10 meV resolution for losses below 1 eV. We also propose, implement, and test two methods to limit the number of iterations in the algorithm. The first method is based on noise measurement and analysis, while in the second we monitor the change of slope in the deconvolved spectrum.

Three-phase three-dimensional (3D) microstructural reconstructions of lithium-ion battery electrodes are critical input for 3D simulations of electrode lithiation/delithiation, which provide a detailed understanding of battery operation. In this report, 3D images of a LiCoO2 electrode are achieved using focused ion beam-scanning electron microscopy (FIB-SEM), with clear contrast among the three phases: LiCoO2 particles, carbonaceous phases (carbon and binder) and the electrolyte space. The good contrast was achieved by utilizing an improved FIB-SEM sample preparation method that combined infiltration of the electrolyte space with a low-viscosity silicone resin and triple ion-beam polishing. Morphological parameters quantified include phase volume fraction, surface area, feature size distribution, connectivity, and tortuosity. Electrolyte tortuosity was determined using two different geometric calculations that were in good agreement. The electrolyte tortuosity distribution versus position within the electrode was found to be highly inhomogeneous; this will lead to inhomogeneous electrode lithiation/delithiation at high C-rates that could potentially cause battery degradation.

A system for illuminating a sample in situ with visible and ultraviolet light inside a transmission electron microscope was devised to study photocatalysts. There are many mechanical and optical factors that must be considered when designing and building such a system. Some of the restrictions posed by the electron microscope column are significant, and care must be taken not to degrade the microscope's electron-optical performance or to unduly restrict the other capabilities of the microscope. We discuss the nature of the design considerations, as well as the practical implementation and characterization of a solution. The system that has been added to an environmental transmission electron microscope includes a high brightness broadband light source with optical filters, a fiber to guide the light to the sample, and a mechanism for precisely aligning the fiber tip.

Quantitative X-ray microanalysis of thick samples is usually performed by measuring the characteristic X-ray intensities of each element in a sample and in corresponding standards. The ratio of the measured intensities from the unknown material to that from the standard is related to the concentration using the ZAF or ϕ(ρz) equations. Under optimal conditions, accuracies approaching 1% are possible. However, all the experimental conditions must remain the same during the sample and standard measurements. This is not possible with cold field emission scanning electron microscopes (FE-SEMs) where beam current can fluctuate around 5% in its stable regime. Very little work has been done on variable beam current conditions (Griffin, B.J. & Nockolds, C.E., Scanning13, 307–312, 1991), and none relating to cold FE-SEM applications. To address this issue, a new method was developed using a single spectral measurement. It is similar in approach to the Cliff-Lorimer method developed for the analytical transmission electron microscope. However, corrections are made for X rays generated from thick specimens using the ratio of the characteristic X-ray intensities of two elements in the same material. The proposed method utilizes the ratio of the intensity of a characteristic X-ray normalized by the sum of X-ray intensities of all the elements measured for the sample, which should also reduce the amplitude of error propagation. Uncertainties in the physical parameters of X-ray generation are corrected using a calibration factor that must be previously acquired or calculated. As an example, when this method was applied to the calculation of the composition of Au-Cu National Institute of Standards and Technology standards measured with a cold field emission source SEM, relative accuracies better than 5% were obtained.

Segregation of Bismuth to Triple Junctions in Copper

Nb–silicide in situ composites have great potential for high-temperature turbine applications. Nb–silicide composites consist of a ductile Nb-based solid solution together with high-strength silicides, such as Nb5Si3 and Nb3Si. With the appropriate addition of alloying elements, such as Ti, Hf, Cr, and Al, it is possible to achieve a promising balance of room-temperature fracture toughness, high-temperature creep performance, and oxidation resistance. In Nb–silicide composites generated from metal-rich binary Nb-Si alloys, Nb3Si is unstable and experiences eutectoid decomposition to Nb and Nb5Si3. At high Ti concentrations, Nb3Si is stabilized to room temperature, and the eutectoid decomposition is suppressed. However, the effect of both Ti and Hf additions in quaternary alloys has not been investigated previously. The present article describes the discovery of a low-temperature eutectoid phase transformation during which (Nb)3Si decomposes into (Nb) and (Nb)5Si3, where the (Nb)5Si3 possesses the hP16 crystal structure, as opposed to the tI32 crystal structure observed in binary Nb5Si3. The Ti and Hf concentrations were adjusted over the ranges of 21 to 33 (at.%) and 7.5 to 33 (at.%) to understand the effect of bulk composition on the phases present and the eutectoid phase transformation.

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.

Local magnification effects and trajectory overlaps related to the presence of a second phase (clusters) are key problems and still open issues in the assessment of quantitative composition data in three-dimensional atom probe tomography (APT) particularly for tiny solute-enriched clusters. A model based on the distribution of distance of first nearest neighbor atoms has been developed to exhibit the variations in the apparent atomic density in reconstructed volumes and to correct compositions that are biased by local magnification effects. This model was applied to both simulated APT reconstructions and real experimental data and shows an excellent agreement with the expected composition of clusters.

Structure and Bonding at NiFATAL ERROR -- Missing abstract.ndash;ZrO2 (Cubic) Interfaces Formed by the Reduction of a NiOFATAL ERROR -- Missing abstract.ndash;ZrO2 (Cubic) Composite

Micro-electro-mechanical systems (MEMS)-based heating holders offer exceptional control of temperature and heating/cooling rates for transmission electron microscopy experiments. The use of such devices is relatively straightforward for nano-particulate samples, but the preparation of specimens from bulk samples by focused ion beam (FIB) milling presents significant challenges. These include: poor mechanical integrity and site selectivity of the specimen, ion beam damage to the specimen and/or MEMS device during thinning, and difficulties in transferring the specimen onto the MEMS device. Here, we describe a novel FIB protocol for the preparation and transfer of specimens from bulk samples, which involves a specimen geometry that provides mechanical support to the electron-transparent region, while maximizing the area of that region and the contact area with the heater plate on the MEMS chip. The method utilizes an inclined stage block that minimizes exposure of the chip to the ion beam during milling. This block also allows for accurate and gentle placement of the FIB-cut specimen onto the chip by using simultaneous electron and ion beam imaging during transfer. Preliminary data from Si and Ag on Si samples are presented to demonstrate the quality of the specimens that can be obtained and their stability during in situ heating experiments.

Biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM). They can be visualized by scanning electron microscopy (SEM), but the requisite SEM sample preparation can modify the biofilm morphology. Here, four different approaches to prepare biofilms of hydrated Staphylococcus aureus for SEM imaging are compared. In order of increasing cooling effectiveness these are: (1) drying in air; (2) plunging in liquid nitrogen; (3) plunging in liquid ethane; and (4) high pressure freezing with liquid nitrogen. These different methods give rise to markedly different biofilm morphologies, which are revealed by cryo-SEM imaging. Significantly, high-pressure frozen biofilms exhibit a rich network of nanoscale ECM fibers surrounding individual bacteria throughout the biofilm thickness. This structure is entirely lost when similar biofilms are dried in air, and it is substantially modified when these biofilms are plunged into liquid nitrogen or liquid ethane.

High-angle annular dark-field and annular bright-field imaging experiments were carried out on an aberration-corrected transmission electron microscope. These techniques have been demonstrated on thin films of complex oxides Ba3.25La0.75Ti3O12 and on LaB6. The results show good agreement between theory and experiments, and for the case of LaB6 they demonstrate the detection of contrast from the B atoms in the annular bright-field images. Elemental mapping with electron-energy-loss spectroscopy has been used to deduce the distribution of Cr and Fe in a thin film of the complex oxide Bi2(Fe1/2Cr3/2)O6 at the unit cell level and the changes in the near-edge structure within the inequivalent regions in the crystalline unit cell. Energy-filtered images in the low-loss region of the energy-loss spectrum show contrast and resolution consistent with the modulation of the signals from elastic scattering. High-resolution contrast, mediated by phonon scattering, is observed for interband transitions. The limitations in terms of detection and signal are discussed.

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.