Electron-excited X-ray mapping is a key operational mode of the scanning electron microscope (SEM) equipped with energy dispersive X-ray spectrometry (EDS). The popularity of X-ray mapping persists despite the significant time penalty due to the relatively low output count rates, typically less than 25 kHz, that can be processed with the conventional EDS. The silicon drift detector (SDD) uses the same measurement physics, but modifications to the detector structure permit operation at a factor of 5–10 times higher than conventional EDS for the same resolution. Output count rates as high as 500 kHz can be achieved with 217 eV energy resolution (at MnKα). Such extraordinarily high count rates make possible X-ray mapping through the method of X-ray spectrum imaging, in which a complete spectrum is captured at each pixel of the scan. Useful compositional data can be captured in less than 200 s with a pixel density of 160 × 120. Applications to alloy and rock microstructures, ultrapure materials with rare inclusions, and aggregate particles with complex chemistry illustrate new approaches to characterization made practical by high-speed X-ray mapping with the SDD.Note: The Siegbahn notation for characteristic X-rays is commonly used in the field of electron beam X-ray spectrometry and will be used in this article. The equivalent IUPAC notation is indicated in parentheses at the first use.In this article, the following arbitrary definitions will be used when referring to concentration (C) ranges: major: C > 0.1 (10 wt%), minor: 0.01 ≤ C ≤ 0.1 (1–10 wt%), and trace: C < 0.01 (1 wt%).

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

Instructions for contributors

Copyright information

Disclaimer

Subscription information

Editorial office

Office of publication

Advertising sales & production

With a vertical resolution of 0.1 nm, atomic force microscopy (AFM) height measurements can be used to determine accurately the diameter of single-walled carbon nanotubes (SWNT) with the assumption that they have circular cross sections. The aim of this article is to draw attention to the need to optimize operating parameters in tapping mode for quantitative AFM height (diameter) analysis of SWNTs. Using silicon tip/cantilever assemblies with force constants ranging from 0.9 to 40 N m−1, we examined the effect of applied force on the apparent diameter of SWNT wrapped with a 29-residue amphiphilic α-helical peptide. A decrease in apparent height (SWNT diameter) with increasing applied force was observed for the higher force constant cantilevers. Cantilevers having force constants of 0.9 and 3 N m−1 demonstrated minimal vertical sample compression with increasing applied force. The effects of AFM image pixel density and scan speed on the measured height (diameter) of SWNTs were also assessed.

Wedge polishing was used to prepare one-dimensional Si n-p junction and Si p-channel metal-oxide-silicon field effect transistor (pMOSFET) samples for precise and quantitative electrostatic potential analysis using off-axis electron holography. To avoid artifacts associated with ion milling, cloth polishing with 0.02-μm colloidal silica suspension was used for final thinning. Uniform thickness and no significant charging were observed by electron holography analysis for samples prepared entirely by this method. The effect of sample thickness was investigated and the minimum thickness for reliable results was found to be ∼160 nm. Below this thickness, measured phase changes were smaller than expected. For the pMOSFET sample, quantitative analysis of two-dimensional electrostatic potential distribution showed that the metallurgical gate length (separation between two extension junctions) was ∼54 nm, whereas the actual gate length was measured to be ∼70 nm by conventional transmission electron microscopy. Thus, source and drain junction encroachment under the gate was 16 nm.

A comprehensive three-dimensional (3D) microanalysis procedure using a combined scanning electron microscope (SEM)/focused ion beam (FIB) system equipped with an energy-dispersive X-ray spectrometer (EDS) has been developed. The FIB system was used first to prepare a site-specific region for X-ray microanalysis followed by the acquisition of an electron-beam generated X-ray spectral image. A small section of material was then removed by the FIB, followed by the acquisition of another X-ray spectral image. This serial sectioning procedure was repeated 10–12 times to sample a volume of material. The series of two-spatial-dimension spectral images were then concatenated into a single data set consisting of a series of volume elements or voxels each with an entire X-ray spectrum. This four-dimensional (three real space and one spectral dimension) spectral image was then comprehensively analyzed with Sandia's automated X-ray spectral image analysis software. This technique was applied to a simple Cu-Ag eutectic and a more complicated localized corrosion study where the powerful site-specific comprehensive analysis capability of tomographic spectral imaging (TSI) combined with multivariate statistical analysis is demonstrated.

Development of the epicardium is critical to proper heart formation. It provides all of the precursor cells that form the coronary system and supplies signals that stimulate cardiac myocyte proliferation. The epicardium forms from mesothelial cells associated with the septum transversum and is referred to as the proepicardium (PE). Two different methods by which these PE cells colonize the developing heart have been described. In avians, PE cells form a bridge to the heart over which PE cells migrate onto the heart. In fish and mammals, PE cells form vesicles of cells that detach from the mesothelium, float through the pericardial cavity, and attach to the heart. A previous study of rat PE development investigated this process at the histological level. Protein markers have been developed since this study. Thus, we investigated this important developmental process coupled with these new markers using other visualization techniques such as scanning electron microscopy (SEM) and confocal microscopy. Finally, a novel, three-dimensional (3-D) culture system was used to confirm the identity of the PE cells. In this study, we found convincing evidence that the rat PE cells directly attach to the heart in a manner similar to that observed in avians.

Methods for accurate and automated determination of the coefficients of the wave aberration function are compared with particular emphasis on measurements of higher order coefficients in corrected instruments. Experimental applications of aberration measurement to the determination of illumination isoplanicity and high precision local refinement of restored exit waves are also described.

Electron energy loss spectroscopy (EELS), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy have been used to study iron catalysts for Fischer–Tropsch synthesis. When silica-containing iron oxide precursors are activated in flowing CO, the iron phase segregates into iron carbide crystallites, leaving behind some unreduced iron oxide in an amorphous state coexisting with the silica binder. The iron carbide crystallites are found covered by characteristic amorphous carbonaceous surface layers. These amorphous species are difficult to analyze by traditional catalyst characterization techniques, which lack spatial resolution. Even a surface-sensitive technique such as XPS shows only broad carbon or iron peaks in these catalysts. As we show in this work, EELS allows us to distinguish three different carbonaceous species: reactive amorphous carbon, graphitic carbon, and carbidic carbon in the bulk of the iron carbide particles. The carbidic carbon K edge shows an intense “π*” peak with an edge shift of about 1 eV to higher energy loss compared to that of the π* of amorphous carbon film or graphitic carbon. EELS analysis of the oxygen K edge allows us to distinguish the amorphous unreduced iron phase from the silica binder, indicating these are two separate phases. These results shed light onto the complex phase transformations that accompany the activation of iron catalysts for Fischer–Tropsch synthesis.

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

Table of Contents

Degradation processes occurring at the surface and in the bulk of Cu-based amorphous alloys during cathodic hydrogen charging were used for promoting the catalytic activity of such alloys. These processes modifying the structure, composition, and morphology of the substrate proved to be useful methods for transforming Cu-Hf and inactive Cu-Ti amorphous alloy precursors into active and durable catalysts. Indeed, their catalytic activity for dehydrogenation of 2-propanol increased up to a conversion level of ∼60% at selectivities to acetone of about 99% for Cu-Ti and to conversion of ∼90% at selectivities of ∼95% for Cu-Hf. Previous attempts carried out by aging in air or hydrogen charging from the gas phase resulted in a maximum conversion level up to 15% for Cu-Hf and up to 3% for Cu-Ti. High resolution Auger spectroscopy allowed changes occurring during the activation process to be identified, namely, the formation of small Cu particles on the HfO2 surface and the formation of highly porous particles containing mostly Cu and some Ti and O (Cu-Ti-O) on a Cu-Ti substrate. Differences in the chemistry and structure of both catalysts are discussed, and the implications for catalytic function are considered. A probable configuration of active sites on the Cu-Ti-O/Ti-O-Cu catalyst for dehydrogenation of 2-propanol is proposed.

Registration form.

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.Note: M. Rizzo and K. Segawa performed this research at Vanderbilt University (same address as Piston). W. Zipfel performed research at Cornell University.

A new Monte Carlo program, Win X-ray, is presented that predicts X-ray spectra measured with an energy dispersive spectrometer (EDS) attached to a scanning electron microscope (SEM) operating between 10 and 40 keV. All the underlying equations of the Monte Carlo simulation model are included. By simulating X-ray spectra, it is possible to establish the optimum conditions to perform a specific analysis as well as establish detection limits or explore possible peak overlaps. Examples of simulations are also presented to demonstrate the utility of this new program. Although this article concentrates on the simulation of spectra obtained from what are considered conventional thick samples routinely explored by conventional microanalysis techniques, its real power will be in future refinements to address the analysis of sample classifications that include rough surfaces, fine structures, thin films, and inclined surfaces because many of these can be best characterized by Monte Carlo methods. The first step, however, is to develop, refine, and validate a viable Monte Carlo program for simulating spectra from conventional samples.

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 μm. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

A fixed-bed reactor has been designed and constructed for ex situ transmission electron microscopy (TEM) studies of heterogeneous catalysts. The ex situ facility exposes a fully prepared TEM sample on a grid to actual process conditions (e.g., temperature, pressure, gas composition, etc.) by placing the grid at the exit section of a conventional fixed-bed reactor. A unique reactor design allows grid transfer into the electron microscope and back into the reactor again under a controlled (inert) environment, thus allowing time-resolved monitoring of catalyst morphology changes under realistic, well-controlled conditions. This facility stands completely independent of the TEM. Thus, no special TEM modifications are required and long-term ex situ studies do not impact microscope utilization. The utility of the facility is demonstrated via the oxidation of intermediate size (∼20–∼80 nm) supported copper particles.

Exit wave restoration using focus series of images has become a widely used technique to improve image resolution and interpretation. To understand the effects of the imaging approximations used, we have critically compared the specimen exit wave functions restored using the efficient linear Wiener filter, with a general nonlinear maximum likelihood method.

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

The bulk morphology and surface features that developed upon precipitation on micrometer-size calcite powders and millimeter-size cleavage fragments were imaged by three different microscopic techniques: field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) of Pt-C replicas, and atomic force microscopy (AFM). Each technique can resolve some nanoscale surface features, but they offer different ranges of magnification and dimensional resolutions. Because sample preparation and imaging is not constrained by crystal orientation, FE-SEM and TEM of Pt-C replicas are best suited to image the overall morphology of microcrystals. However, owing to the decoration effect of Pt-C on the crystal faces, TEM of Pt-C replicas is superior at resolving nanoscale surface structures, including the development of new faces and the different microtopography among nonequivalent faces in microcrystals, which cannot be revealed by FE-SEM. In conjunction with SEM, Pt-C replica provides the evidence that crystals grow in diverse and face-specific modes. The TEM imaging of Pt-C replicas has nanoscale resolution comparable to AFM. AFM yielded quantitative information (e.g., crystallographic orientation and height of steps) of microtopographic features. In contrast to Pt-C replicas and SEM providing three-dimensional images of the crystals, AFM can only image one individual cleavage or flat surface at a time.