We evaluated the depth resolution of annular dark-field (ADF) scanning confocal electron microscopy (SCEM) with a stage-scanning system by observation of nanoparticles. ADF-SCEM is a three-dimensional (3D) imaging technique that we recently proposed. An ADF-SCEM instrument involves a pinhole aperture before a detector for rejecting electrons from the out-of-focal plane in a specimen and an annular aperture under the specimen for collecting only scattered electrons. The stage-scanning system enables us to directly obtain optical slice images perpendicular and parallel to an optical axis at a desired position. In particular, the parallel slices visualize the elongation of nanoparticles along the optical axis, which depends on the depth resolution. ADF-SCEM effectively reduced the elongation length of the nanoparticles sufficiently to demonstrate depth sectioning, in comparison with scanning transmission electron microscopy and bright-field SCEM. The experimentally obtained length was nearly equal to the theoretically estimated one from the probe size considering the experimental conditions. Furthermore, we applied this ADF-SCEM technique to analysis of the 3D position of catalytic nanoparticles on carbon nanostructures.

An electromechanical video camera coupler was developed to rotate a light microscope field of view (FOV) in real time without the need to physically rotate the stage or specimen. The device, referred to as the Camera Thetarotator, rotated microscopical views 240° to assist microscopists to orient specimens within the FOV prior to image capture. The Camera Thetarotator eliminated the effort and artifacts created when rotating photomicrographs using conventional graphics software. The Camera Thetarotator could also be used to semimanually register a dataset of histological sections for three-dimensional (3D) reconstruction by superimposing the transparent, real-time FOV to the previously captured section in the series. When compared to Fourier-based software registration, alignment of serial sections using the Camera Thetarotator was more exact, resulting in more accurate 3D reconstructions with no computer-generated null space. When software-based registration was performed after prealigning sections with the Camera Thetarotator, registration was further enhanced. The Camera Thetarotator expanded microscopical viewing and digital photomicrography and provided a novel, accurate registration method for 3D reconstruction. The Camera Thetarotator would also be useful for performing automated microscopical functions necessary for telemicroscopy, high-throughput image acquisition and analysis, and other light microscopy applications.

A high quality X-ray spectrum image of a 3.3 μm diameter sphere of K411 glass resting on a copper substrate was collected at 25 keV. The same sample configuration was modeled using the NISTMonte Monte Carlo simulation of electron and X-ray transport as is integrated into the quantitative X-ray microanalysis software package DTSA-II. The distribution of measured and simulated X-ray intensity compare favorably for all the major lines present in the spectra. The simulation is further examined to investigate the influence of angle-of-incidence, sample thickness, and sample diameter on the generated and measured X-ray intensity. The distribution of generated X-rays is seen to deviate significantly from a naive model which assumes that the distribution of generated X-rays is similar to bulk within the volume they share in common. It is demonstrated that the angle at which the electron beam strikes the sample has nonnegligible consequences. It is also demonstrated that within the volume that the bulk and particle share in common that electrons, which have exited and later reentered the particle volume, generate a significant fraction of the X-rays. Any general model of X-ray generation in particles must take into account the lateral spread of the scattered electron beam.

In this article, we use simulated and experimental data to explore how three operator-controllable parameters—(1) signal level, (2) detector resolution, and (3) number of factors chosen for analysis—affect quantitative analyses of scanning transmission electron microscopy–energy dispersive X-ray spectroscopy spectrum images processed by principal component analysis (PCA). We find that improvements in both signal level and detector resolution improve the precision of quantitative analyses, but that signal level is the most important. We also find that if the rank of the PCA solution is not chosen properly, it may be possible to improperly fit the underlying data and degrade the accuracy of results. Additionally, precision is degraded in the case when too many factors are included in the model.

We propose an image-based framework to evaluate the uncertainty in the estimation of the volume fraction of specific microstructures based on the observation of a single section. These microstructures consist of cubes organized on a cubic mesh, such as monocrystalline nickel base superalloys. The framework is twofold: a model-based stereological analysis allows relating two-dimensional image observations to three-dimensional microstructure features, and a spatial statistical analysis allows computing approximate confidence bounds while assessing the representativeness of the image. The reliability of the method is assessed on synthetic models. Volume fraction estimation variances and approximate confidence intervals are computed on real superalloy images in the context of material characterization.

Previous work has shown that focused ion beam (FIB) nanomachining can be effectively utilized for the cross-sectional analysis of polymers such as core-shell solid microspheres and hollow latex nanospheres. While these studies have clearly demonstrated the precise location selection and nanomachining control provided by the FIB technique, the samples studied consisted of only a single polymer. In this work, FIB is used to investigate bicomponent polymeric fiber systems by taking advantage of the component's differing sputter rates that result from their differing physical properties. An approach for cross sectioning and thus revealing the cross-sectional morphology of the polymeric components in a bicomponent polymeric fiber with the island-in-the-sea (I/S) structure is presented. The two I/S fibers investigated were fabricated using the melt spinning process and are composed of bicomponent combinations of linear low density polyethylene (LLDPE) and nylon 6 (PA6) or polylactic acid (PLA) and an EastONETM proprietary polymer. Topographical contrast as a result of differential sputtering and the high surface specificity and high signal-to-noise obtained using FIB-induced secondary electron imaging is shown to provide a useful approach for the rapid characterization of the cross-sectional morphology of bicomponent polymeric fibers without the necessity of staining or other sample preparation.

A computer-controlled procedure is outlined here that first determines the position of the amorphous-crystalline interface in an image. Subsequently, from a time series of these images, the velocity of the crystal growth front is quantified. The procedure presented here can be useful for a wide range of applications, and we apply the new approach to determine growth rates in a so-called fast-growth-type phase-change material. The growth rate (without nucleation) of this material is of interest for comparison with identical material used in phase-change random access memory cells. Crystal growth rates in the amorphous phase-change layers have been measured at various temperatures using in situ heating in a transmission electron microscope. Doped SbTe films (20 nm thick) were deposited on silicon nitride membranes, and samples with and without silicon oxide capping layer were studied. The activation energy for growth was found to be 3.0 eV. The samples without capping layer exhibit a nucleation rate that is an order of magnitude higher than the samples with a silicon oxide capping layer. This difference can be attributed to the partial oxidation of the phase-change layer in air. However, the growth rates of the samples with and without capping are quite comparable.

The defect and interfacial structure in a Fe3O4/BaTiO3 heteroepitaxial bilayer was investigated by scanning transmission electron microscopy. The results show that the Fe3O4 film grew epitaxially on BaTiO3. The orientation relationship between Fe3O4, BaTiO3 and MgO is [100]Fe3O4//[100]BaTi3O//[100]MgO and (010)Fe3O4//(010)BaTiO3//(010)MgO. An initial interfacial nucleation layer was formed that partially accommodated the lattice mismatch strain between BaTiO3 and MgO. This investigation indicates that the formation of this buffer layer provides a high-quality BaTiO3 surface for subsequent Fe3O4 growth, resulting in a semicoherent interface. The Fe3O4 surface is nearly atomically abrupt (roughness Rrms = 0.78 nm). The Fe3O4 film exhibits magnetic domains with a diameter in the range of 0.4–2 μm.

Deposition of calcium carbonate from groundwater was examined on a moss, Didymodon tophaceus, from a Niagara Escarpment waterfall. A spectrophotometer on a polarizing microscope was used for interferometry. A second-order blue interference with an interference minimum around 620 nm was found when moss cell spaces were fully calcified. Filled cell spaces were often surrounded by empty cell spaces. Complete calcification of whole leaflets resulted in progressively higher orders of interference colors and a positive shift in interference minima. Calcified leaflets finally became cemented together, but each retained a weak extinction when rotated. Small calcareous spherulites (mean diameter 15.7 ± 2.1 μm) were found between leaflets. Spherulites exhibited first-order white interference with a Maltese cross that rotated when the polarizer and analyzer were rotated in tandem. A Nikitin-Berek compensator was tilted at 5.5° to give an interference minimum at 600 nm in the optical axis. Quadrants of spherulites with radii more or less in line with the tilting axis of the compensator had lower (P < 0.001) interference minima (535 ± 27 nm) than quadrants with radii more or less perpendicular to the compensator (659 ± 15 nm), thus indicating a radial internal structure. Spherulites were tentatively identified as vaterite.

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or ▵1-90+▵267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Lightsource operating from 5 to 15 keV X-ray energy with 14 to 30 μm2 field of view has been used for high-resolution (30–40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 μm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30–40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51–54% of pure crystal density and plate-like areas had 44–53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies.

The adhesion of Candida albicans to the genital epithelium has not been fully investigated in vivo. The objective of this study was to evaluate the ultrastructural aspects of C. albicans adhesion in the lower genital system of female Wistar rats through scanning and transmission electron microscopy. The genital infection persisted until the end of the experiment, and all rats showed the same adhesion aspects. Various associated yeast/hyphae were observed in the lumen and adhered both at the vaginal and endocervical levels where the fungal filamentation process occurred. In the vaginal epithelium, closely adhered yeasts were observed as stretched strands bridging between yeasts and the epithelium surface. Different stages of the adhesion, where yeasts internalized into the epithelial cell inside a cytoplasmic vacuole, resembling endocytosis, and a wide fibrillar-floccular, glycocalyx-like layer on the yeasts were observed. On the endocervix, the adhesion occurred between the cilia. In the uterine body, only a yeast-like form was observed with superficial contact. This study reached the initial goal of demonstrating an experimental model for in vivo studies. Continuation of this line of research is important for studies of vulvovaginal candidiasis.

Irradiation of an amorphous layer of dried sodium phosphate buffer (pH = 7.0) by transmission electron microscopy (100–120 kV) causes rapid formation of numerous small spherical bubbles [10–100 Å (= 1–10 nm)] containing an unknown gas. Bubbling is detected even with the first low-dose exposure. In a thin layer (ca. 100–150 Å), bubbling typically goes through nucleation, growth, possible fusion, and end-state, after which further changes are not apparent; co-irradiated adjacent areas having a slightly smaller thickness never develop bubbles. In moderately thicker regions (ca. over 200 Å), there is no end-state. Instead, a complex sequence of microstructural changes is elicited during continued intermittent high-dose irradiation: nucleation, growth, early simple fusions, a second round of extensive multiple fusions, general reduction of matrix thickness (producing flattening and expansion of larger bubbles, occasional bubble fission, and formation of very large irregularly-shaped bubbles by a third round of compound fusion events), and slow shrinkage of all bubbles. The ongoing lighter appearance of bubble lumens, maintenance of their rounded shape, and extensive changes in size and form indicate that gas content continues throughout their surprisingly long lifetime; the thin dense boundary layer surrounding all bubbles is proposed to be the main mechanism for their long lifetime.

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, [email protected].