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

We have investigated the changes produced in single-element and two-layer transmission electron microscope (TEM) specimens irradiated by an intense nanometer-sized electron probe, such as that produced in a field-emission or aberration-corrected TEM. These changes include hole formation and the accumulation of material within the irradiated area. The results are discussed in terms of mechanisms, including electron-beam sputtering and surface diffusion. Strategies for minimizing the effect of the beam are considered.

Here, we describe the development of an inexpensive and versatile manipulation system for in situ experiments in a field emission scanning electron microscope based on a parallel-guiding plate-spring mechanism and low cost materials. The system has been tested for a wide range of applications, such as collecting, moving, and positioning particles, fabricating atomic force microscopy tips based on carbon nanotubes, and characterizing individual nanobjects. The nanomanipulation results demonstrate that there are many opportunities for the use of physical manipulation in the bottom-up approach to fabrication of nanodevices.

Structural features like defects or heterointerfaces in crystals or amorphous phases give rise to different local patterns in high-resolution electron micrographs or object wave functions. Pattern recognition techniques can be used to identify these typical patterns that constitute the image itself, as was already demonstrated for compositional changes in isostructural heterostructures, where the patterns within unit cells of the lattice were analyzed. To extend such analyses to more complex materials, we examined patterns in small circular areas centered on intensity maxima of the image. Nonsupervised clustering, namely, Ward's clustering method, was applied to these patterns. In two examples, a highly defective ZnMnTe layer on GaAs and a tunnel magneto resistance device, we demonstrate how typical patterns are identified by this method and how these results can be used for a further investigation of the microstructural properties of the sample.

A calibration procedure for the detection efficiency of energy dispersive X-ray spectrometers (EDS) used in combination with scanning electron microscopy (SEM) for standardless electron probe microanalysis (EPMA) is presented. The procedure is based on the comparison of X-ray spectra from a reference material (RM) measured with the EDS to be calibrated and a reference EDS. The RM is certified by the line intensities in the X-ray spectrum recorded with a reference EDS and by its composition. The calibration of the reference EDS is performed using synchrotron radiation at the radiometry laboratory of the Physikalisch-Technische Bundesanstalt. Measurement of RM spectra and comparison of the specified line intensities enables a rapid efficiency calibration on most SEMs. The article reports on studies to prepare such a RM and on EDS calibration and proposes a methodology that could be implemented in current spectrometer software to enable the calibration with a minimum of operator assistance.

It has been shown, by imaging gold (200) planes, that it is possible to achieve better than 0.20-nm structural resolution in cryo-transmission electron microscopy (cryo-TEM). This has been done using commercially available cryo equipment and using a 300-kV field emission gun (FEG) TEM. The images of 15-nm gold particles embedded in amorphous frozen water clearly show the (111) planes (separated by 0.235 nm) in gold. Fourier transform demonstrates the presence of (200) planes in the image, proving a resolution of better than 0.20 nm. The experimental results are supported by image simulations using the multislice method. These simulations suggest that it should be possible to achieve the same resolution even in smaller particles and particles of lighter elements. The crucial experimental problem to overcome is keeping the thickness of the amorphous film low and to work at low electron dose conditions.

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In a conventional transmission electron microscope system, the resolution is regarded as an absolute limitation, that is, 0.2 nm in theory and 0.6 nm in sections of biological materials. However, in an oversampled system, this limitation can be broken. In the present study, 60-nm-thick Epon sections from a mouse kidney were used. From these sections tight junctions located in the distal tubule were selected as test objects. Sets of up to 15 electron microscope images of the same target were recorded on negatives at ×10,000, ×13,000, and ×63,000, respectively. The recorded films were digitized using a light microscope equipped with a digital camera. In each set the images were expanded, aligned, and merged into a more highly resolved output image. Each output image revealed details in the tight junction, which were not visible at the original magnifications. Two different sizes of colloidal gold particles (10 nm and 1 nm) conjugated with an immunoglobin G (IgG) served as references. With this improvement of resolution, it becomes possible to inspect some barely visible biologic (virus) particles and structures, such as glycogen and free ribosomes in their native environment.

A combination of transmission electron microscopy (TEM) and in situ tensile testing in an environmental scanning electron microscopy (ESEM) was used to evaluate the static bulk and dynamic surface morphologies of medical polyurethanes. TEM results showed phase-separated hard segment and soft segment structures. Surface morphology as a function of strain was studied using ESEM in conjunction with a tensometer.

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

Image quality (IQ) maps constructed from electron backscatter diffraction data provide useful visualizations of microstructure. The contrast in these maps arises from a variety of sources, including phase, strain, topography, and grain boundaries. IQ maps constructed using various IQ metrics are compared to identify the most prominent contrast mechanism for each metric. The conventional IQ metric was found to provide the superior grain boundary and strain contrast, whereas an IQ metric based on the average overall intensity of the diffraction patterns was found to provide better topological and phase contrast.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.

Electron energy loss spectra in conjunction with near-edge fine structures of purely stoichiometric niobium monoxide (NbO) and niobium pentoxide (Nb2O5) reference materials were recorded. The structures of the niobium oxide reference materials were checked by selected area electron diffraction to ensure a proper assignment of the fine structures. NbO and Nb2O5 show clearly different energy loss near-edge fine structures of the Nb-M4,5 and -M2,3 edges and of the O-K edge, reflecting the specific local environments of the ionized atoms. To distinguish the two oxides in a quantitative manner, the intensities under the Nb-M4,5 as well as Nb-M2,3 edges and the O-K edge were measured and their ratios calculated. k-factors were also derived from these measurements.

This issue of Microscopy and Microanalysis presents nine of the articles from the 9th Workshop of the European Microbeam Analysis Society (EMAS) on “Modern Developments and Applications in Microbeam Analysis” held in conjunction with the 3rd Meeting of the International Union of Microbeam Analysis Societies (IUMAS) in Florence, Italy, on May 22–26, 2005.

The resolution-limiting aberrations of round electromagnetic lenses can now be successfully overcome via the use of multipole element “aberration correctors.” The installation and performance of a hexapole-based corrector (CEOS GmbH) integrated on the probe-forming side of a JEOL 2200FS FEG STEM/TEM is described. For the resolution of the microscope not to be severely compromised by its environment, a new, specially designed building at Oak Ridge National Laboratory has been built. The Advanced Microscopy Laboratory was designed with the goal of providing a suitable location for aberration-corrected electron microscopes. Construction methods and performance of the building are discussed in the context of the performance of the microscope. Initial performance of the microscope on relevant specimens and modifications made to eliminate resolution-limiting conditions are also discussed.

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Ultramicrotomy, the technique of cutting nanometers-thin slices of material using a diamond knife, was applied to prepare transmission electron microscope (TEM) specimens of nanoporous poly(methylsilsesquioxane) (PMSSQ) thin films. This technique was compared to focused ion beam (FIB) cross-section preparation to address possible artifacts resulting from deformation of nanoporous microstructure during the sample preparation. It was found that ultramicrotomy is a successful TEM specimen preparation method for nanoporous PMSSQ thin films when combined with low-energy ion milling as a final step. A thick, sacrificial carbon coating was identified as a method of reducing defects from the FIB process which included film shrinkage and pore deformation.

Planar defects in a polycrystalline diamond film were studied by high-resolution transmission electron microscopy (HRTEM) and high-resolution scanning transmission electron microscopy (STEM). In both modes, sub-Ångström resolution was achieved by making use of two aberration-corrected systems; a TEM and a STEM CS-corrected microscope, each operated at 300 kV. For the first time, diamond in 〈110〉 zone-axis orientation was imaged in STEM mode at a resolution that allows for resolving the atomic dumbbells of carbon at a projected interatomic distance of 89 pm. Twin boundaries that show approximately the Σ3 CSL structure reveal at sub-Ångström resolution imperfections; that is, local distortions, which break the symmetry of the ideal Σ3 type twin boundary, are likely present. In addition to these imperfect twin boundaries, voids on the atomic level were observed. It is proposed that both local distortions and small voids enhance the mechanical toughness of the film by locally increasing the critical stress intensity factor.

Nuclear factor–kappa B (NF-κB) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-κBs, which prevent entry into the nucleus. Following cellular stimulation, the I-κBs are rapidly degraded, activating NF-κB. The active form of NF-κB rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-κB is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-κB (p65 subunit, p65-NF-κB) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-κB protein in the nucleus determined biochemically. The appearance of p65-NF-κB in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-κB can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

Silicon-germanium thin films have been analyzed by EDS microanalysis in a field emission gun scanning transmission electron microscope (FEG-STEM) equipped with a high angular dark-field detector (STEM/HAADF). Several spectra have been acquired in the same homogeneous area of the cross-sectioned sample by drift-corrected linescan acquisitions. The Ge concentrations and the local film thickness have been obtained by using a previously described Monte Carlo based “two tilt angles” method. Although the concentrations are in excellent agreement with the known values, the resulting confidence intervals are not as good as expected from the precision in beam positioning and tilt angle position and readout offered by our state-of-the-art microscope. The Gaussian shape of the SiKα and GeKα X-ray intensities allows one to use the parametric bootstrap method of statistics, whereby it becomes possible to perform the same quantitative analysis in sample regions of different compositions and thicknesses, but by doing only one measurement at the two angles.