A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Different approaches using microscopy image analysis procedures were employed for characterization of activated sludge systems. The approaches varied mainly on the type of visualization and acquisition method used for collection of data. In this context, this study focused on the comparison of the two most common acquisition methods: bright field and phase-contrast microscopy. Images were acquired from seven different wastewater treatment plants for a combined period of two years. Advantages and disadvantages of each acquisition technique and the results are discussed. Bright field microscopy proved to be more simple and inexpensive and provided the best overall results.

The posterior esophagus of Bulla striata, running from the gizzard to the stomach, was investigated with light and electron microscopy to obtain new data for a comparative analysis of the digestive system in cephalaspidean opisthobranchs. In this species, the posterior esophagus can be divided into two regions. In the first, the epithelium is formed by columnar cells with apical microvilli embedded in a cuticle. Many epithelial and subepithelial secretory cells are present in this region. In both, electron-lucent secretory vesicles containing filaments and a peripheral round mass of secretory material fill the cytoplasm. These acid mucus-secreting cells may also contain a few dense secretory vesicles. In the second part of the posterior esophagus, the cuticle is absent and the epithelium is ciliated. In this region, epithelial cells may contain larger lipid droplets and glycogen reserves. Subepithelial secretory cells are not present, and in epithelial secretory cells the number of dense vesicles increases, but most secretory cells still contain some electron-lucent vesicles. These cells secrete a mixture of proteins and acid polysaccharides and should be considered seromucous. The secretory cells of the posterior esophagus are significantly different from those previously reported in the anterior esophagus of this herbivorous species.

The properties of activated sludge systems can be characterized using image analysis procedures. When these systems operate with high biomass content, accurate sludge characterization requires samples to be diluted. Selection of the best image acquisition magnification is directly related to the amount of biomass screened. The aim of the present study was to survey the effects of dilution and magnification on the assessment of aggregated and filamentous bacterial content and structure using image analysis procedures. Assessments of biomass content and structure were affected by dilutions. Therefore, the correct operating dilution requires careful consideration. Moreover, the acquisition methodology comprising a 100× magnification allowed data on aggregated and filamentous biomass to be determined and smaller aggregates to be identified and characterized, without affecting the accuracy of lower magnifications regarding biomass representativeness.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

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.

Tilted illumination exit-wave restoration is compared for two aberration-corrected instruments at different accelerating voltages. The experimental progress of this technique is also reviewed and the significance of off-axial aberrations examined. Finally, the importance of higher order aberration compensation combined with careful correction of the lower order aberrations is highlighted.

Aging is the single most significant risk factor for erectile dysfunction (ED), leading to structural modification of cavernous tissue and altering expression of vascular growth factors. The angiopoietin/Tie2 system has been recently considered as a potential target for therapy of vascular disorders, including ED. Hence, the aim of this study was to analyze expression of angiopoietin1 (Ang1), angiopoietin2 (Ang2), and their receptor Tie2 in corpus cavernosum (CC) of rat during aging (6, 12, 18, and 24 months). The expression of Ang1, Ang2, and Tie2 was studied by immunohistochemistry and immunofluorescence, followed by semiquantification after Western blotting. Both Ang1 and Ang2 were localized mainly in perivascular smooth muscle and endothelial cells, while Tie2 was strictly detected at the vascular endothelium. A significant decrease in Ang2's expression was observed at 12 months when compared with 6-month-old rats, a tendency that reverses in older animals. No significant differences were demonstrated for Ang1 or Tie2, which is consistent with their constitutive expression in CC. The ratios Ang1/Tie2 and Ang2/Tie2 were also calculated and both decrease during aging, while no marked variation was observed for Ang1/Ang2. Our results suggest that the angiopoietin/Tie2 system participate in the vascular maintenance and remodeling of the CC during aging.

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.

Plants have suberized cells that act as protective interfaces with the environment or between different plant tissues. A lamellar structure of alternating dark and light bands has been found upon transmission electron microscopy (TEM) observation of cork cells and considered a typical feature of the suberized secondary wall. We observed cork cells from periderms of Quercus suber, Quercus cerris, Solanum tuberosum, and Calotropis procera by TEM after uranyl acetate and lead citrate staining. A lamellated structure was observed in S. tuberosum and C. procera but not in Q. suber and Q. cerris where the suberized cell wall showed a predominantly hyaline aspect with only a dark dotted staining. Removal of suberin from Q. suber cells left a thinner secondary wall that lost the translucent aspect. We hypothesize that the species' specific chemical composition of suberin will result in different three-dimensional macromolecular development and in a different spatial location of lignin and other aromatics. A lamellated ultrastructure is therefore not a general feature of suberized cells.

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.

Localization microscopy techniques based on localizing single fluorophore molecules now routinely achieve accuracies better than 30 nm. Unlike conventional optical microscopies, localization microscopy experiments do not generate an image but a list of discrete coordinates of estimated fluorophore positions. Data display and analysis therefore generally require visualization methods that translate the position data into conventional images. Here we investigate the properties of several widely used visualization techniques and show that a commonly used algorithm based on rendering Gaussians may lead to a 1.44-fold loss of resolution. Existing methods typically do not explicitly take sampling considerations into account and thus may produce spurious structures. We present two additional visualization algorithms, an adaptive histogram method based on quad-trees and a Delaunay triangulation based visualization of point data that address some of these deficiencies. The new visualization methods are designed to suppress erroneous detail in poorly sampled image areas but avoid loss of resolution in well-sampled regions. A number of criteria for scoring visualization methods are developed as a guide for choosing among visualization methods and are used to qualitatively compare various algorithms.

In this article, it is shown that focused ion beam (FIB) systems can be used to study the inner structure of flax fibers, the use of which as a reinforcing material in polymer composites still draws much interest from multiple disciplines. This technique requires none of the specific preparations necessary for scanning electron microscopy or transmission electron microscopy studies. Irradiation experiments performed on FIB prepared cross sections with very low Ga+ ion beam currents revealed the softer material components of fibers. Thus, it confirmed the presence of pectin-rich layers at the interfaces between the fibers of a bundle, but also allowed the precise localization of such layers within the secondary cell wall. Furthermore, it suggested new insights on the transition modes between the sublayers of the secondary cell wall.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Tumors are supported by the development of a unique vascular bed. We used fractal dimension (Db) and image analysis to quantify differences in the complexity of the vasculature in normal intestinal submucosa and intestinal polyps. ApcMin/+ mice and wild-type mice were perfused with a curable latex compound, intestines sectioned, and images collected via confocal microscopy. The images were analyzed and area (A), perimeter (P), and integrated optical density (IOD) of the normal and tumor vascular beds were measured. The Db, a quantitative descriptor of morphological complexity, was significantly greater for the polyp vasculature from ApcMin/+ mice than controls. This indicates that the polyp microvasculature is more chaotic than that of the controls, while the IOD and average vascular density values displayed no differences. This suggests the mass of blood volume is equivalent in normal and polyp microvasculature. The lower vascular area-perimeter ratios expressed by the polyp microvasculature suggest it is composed of smaller, more tortuous vessels. These data demonstrate that fractal analysis is applicable for providing a quantitative description of vascular complexity associated with angiogenesis occurring in normal or diseased tissue. Application of Db, IOD, and average density provides a clearer quantification of the complex morphology associated with tissue microvasculature.

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.

The aberration-corrected scanning transmission electron microscope can provide information on nanostructures with sub-Ångström image resolution. The relatively intuitive interpretation of high-angle annular dark-field (HAADF) imaging technique makes it a popular tool to image a variety of samples and finds broad applications to characterizing nanostructures, especially when combined with electron energy-loss spectroscopy and X-ray energy-dispersive spectroscopy techniques. To quantitatively interpret HAADF images, however, requires full understanding of the various types of signals that contribute to the HAADF image contrast. We have observed significant intensity enhancement in HAADF images, and large expansion of lattice spacings, of surface atoms of atomically flat ZnO surfaces. The surface-resonance channeling effect, one of the electron-beam channeling phenomena in crystalline nanostructures, was invoked to explain the observed image intensity enhancement. A better understanding of the effect of electron beam channeling along surfaces or interfaces on HAADF image contrast may have implications for quantifying HAADF images and may provide new routes to utilize the channeling phenomenon to study surface structures with sub-Ångström spatial resolution.

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.