In this study the structure of collagen-chondroitin sulphate-hydroxyapatite porous composites is investigated by histochemical (Von Kossa staining), immunohistochemical, and transmission electron microscopy. Examination of composites on picrosirius red stained sections showed that polarization colors of collagen were generally in the range of orange-red. Immunofluorescence data indicate that chondroitin sulphate was either chemically incorporated into the bulk structure of collagen scaffolds or attached on surfaces of collagen bundles. Depending on the ratio between collagen:chondroitin sulphate:hydroxyapatite, von Kossa histochemical staining showed a progressive loading of collagen-chondroitin sulphate bundles with hydroxyapatite. Transmission electron microscopy studies have shown that composites contain mostly collagen fibrils aggregated with random orientation with very few collagen fibers showing the 67-nm banding pattern. Hydroxyapatite deposits of various sizes occurred among the collagen fibrils.

XFILM is a computer program for determining the thickness and composition of thin films on substrates and multilayers by electron probe microanalysis. In this study, we describe the X-ray emission model implemented in the latest version of XFILM and assess its reliability by comparing measured and calculated k-ratios from thin-film samples available in the literature. We present and discuss examples of applications of XFILM that illustrate the capabilities of the program.

Reactive multilayer thin films that undergo highly exothermic reactions are attractive choices for applications in ignition, propulsion, and joining systems. Ni/Al reactive multilayer thin films were deposited by dc magnetron sputtering with a period of 14 nm. The microstructure of the as-deposited and heat-treated Ni/Al multilayers was studied by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) in plan view and in cross section. The cross-section samples for TEM and STEM were prepared by focused ion beam lift-out technique. TEM analysis indicates that the as-deposited samples were composed of Ni and Al. High-resolution TEM images reveal the presence of NiAl in small localized regions. Microstructural characterization shows that heat treating at 450 and 700°C transforms the Ni/Al multilayered structure into equiaxed NiAl fine grains.

The recent advent of a novel design of in situ heating technology for electron microscopes has permitted unprecedented control of elevated temperature studies of catalytic materials, particularly when coupled with the sub-Ångström imaging performance of a modern aberration-corrected scanning transmission electron microscope (STEM). Using micro-electro-mechanical-systems (MEMS)-based Aduro™ heating chips from Protochips, Inc. (Raleigh, NC, USA) allows nearly instantaneous heating and cooling of catalyst powders, avoiding effects of temperature ramping as experienced with standard heating stages. The heating technology also provides stable operation limited only by the inherent drift in the microscope stage, thus allowing full image resolution to be achieved even at elevated temperatures. The present study details the use of both the high X-Y spatial resolution in both dark-field and simultaneous bright-field imaging, along with the high resolution in Z (depth sectioning) provided by the large probe incidence semiangle in the aberration-corrected instrument, to characterize the evolution of microstructure in a commercial Au/Fe2O3 water-gas shift catalyst during elevated temperature treatment. The phenomenon of Au diffusion to the surface of hematite support particles to form discrete crystalline Au nanoparticles in the 1–2 nm size range, after a prior leaching treatment to remove surface Au species has been characterized.

Although bacterial biofilms have been studied in detail, adhesion of Candida albicans and non-albicans species to an intrauterine contraceptive device (IUD) is not clear. The objective of this study was to evaluate aspects of imaging of the ultrastructure and viability of vaginal yeasts adhered to different parts of an IUD, through scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). We studied yeasts isolated from different patients with vulvovaginal candidiasis: C. albicans, C. glabrata, C. guillermondii, C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae. A suspension of the each yeast was prepared and incubated with IUD parts (tail, without copper, and copper-covered). SEM and CSLM showed that all the vaginal yeasts adhered to all the parts of the IUD and demonstrated viability, including 30 days after contact for C. albicans. Possibly irregularities of IUD surface contribute to the adherence process. Although all of the IUD parts contribute to retention of yeasts in the genital tract, high concentration of yeast cells on the tail may indicate the importance of this segment in maintaining the colonization by yeast cells because the tail forms a bridge between the external environment, the vagina that is colonized by yeast cells, and the upper genital tract where there is no colonization.

Lowering the electron energy in the transmission electron microscope allows for a significant improvement in contrast of light elements and reduces knock-on damage for most materials. If low-voltage electron microscopes are defined as those with accelerating voltages below 100 kV, the introduction of aberration correctors and monochromators to the electron microscope column enables Ångstrom-level resolution, which was previously reserved for higher voltage instruments. Decreasing electron energy has three important advantages: (1) knock-on damage is lower, which is critically important for sensitive materials such as graphene and carbon nanotubes; (2) cross sections for electron-energy-loss spectroscopy increase, improving signal-to-noise for chemical analysis; (3) elastic scattering cross sections increase, improving contrast in high-resolution, zero-loss images. The results presented indicate that decreasing the acceleration voltage from 200 kV to 80 kV in a monochromated, aberration-corrected microscope enhances the contrast while retaining sub-Ångstrom resolution. These improvements in low-voltage performance are expected to produce many new results and enable a wealth of new experiments in materials science.

While there has been rapid development of microfabrication techniques to produce high-resolution surface modifications on a variety of materials in the last decade, there is still a strong need to produce novel alternatives to induce guided tissue regeneration on dental implants. High-resolution microscopy provides qualitative and quantitative techniques to study cellular guidance in the first stages of cell-material interactions. The purposes of this work were (1) to produce and characterize the surface topography of isotropic and anisotropic microfabricated silica thin films obtained by sol-gel processing, and (2) to compare the in vitro biological behavior of human bone marrow stem cells on these surfaces at early stages of adhesion and propagation. The results confirmed that a microstamping technique can be used to produce isotropic and anisotropic micropatterned silica coatings. Atomic force microscopy analysis was an adequate methodology to study in the same specimen the sintering derived contraction of the microfabricated coatings, using images obtained before and after thermal cycle. Hard micropatterned coatings induced a modulation in the early and late adhesion stages of cell-material and cell-cell interactions in a geometry-dependent manner (i.e., isotropic versus anisotropic), as it was clearly determined, using scanning electron and fluorescence microscopies.

The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated.

Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images of Staphylococcus epidermidis ATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor, S. epidermidis ATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion, S. epidermidis ATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed for in vitro and/or in vivo applications.

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

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.

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.

The large variability in kidney morphology among fish makes it difficult to build a “universal” model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. Three-year-old specimens of both sexes were collected at four stages of their reproductive cycle. Kidney was processed for light and electron microscopy. The relative volumes of renal components, such as renal corpuscles and different nephron tubules, were estimated by stereological methods. Qualitatively, the general nephron structure of brown trout was similar to that described for other glomerular teleost species. Quantitatively, however, differences in the relative volume of some renal components were detected between sexes and among seasons. Particularly, highest values of vacuolized tubules and new growing tubules were observed after spawning, being more relevant in females. Despite seasonal changes, more linear correlations were found between those parameters and the reno-somatic index than the gonado-somatic index. Thus, we verified that some brown trout renal components undergo sex dependent seasonal variations, suggesting a morphological adaptation of the components to accomplish physiological needs. These findings constitute a baseline for launching studies to know which factors govern the morphological variations and their functional consequences.

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

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.

We describe the architecture of nitrifying bacteria biofilms immobilized on a three-dimensional (3D) polyurethane foam that permits efficient water flow through a bioreactor. The 3D spatial organization of immobilized bacterial colonies is characterized on three resolution levels with X-ray tomography, light confocal microscopy, and scanning electron microscopy (SEM). Using these techniques we demonstrate biofilm distribution in the foam and the existence of several modes of binding of bacteria to the foam. Computed X-ray tomography permits observation of the distribution of the biofilm in the whole open cellular polyurethane material volume and estimation of biofilm volume. SEM and confocal laser scanning microscopy techniques permit 3D visualization of biofilm structure. Three distinct immobilization patterns could be observed in the open cellular polyurethane material: (1) large irregular aggregates of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) spherical (pom-pom) aggregates of bacteria localized on the external surface of biofilm; and (3) biofilm threads adherent to the surface of polyurethane foam. Finally, we demonstrate that immobilized bacteria exhibit metabolic activity and growth.

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

For the transmission electron aberration-corrected microscope (TEAM) initiative of five U.S. Department of Energy laboratories in the United States, a correction system for the simultaneous compensation of the primary axial aberrations, the spherical aberration Cs, and the chromatic aberration Cc has been developed and successfully installed. The performance of the resulting Cc /Cs-corrected TEAM instrument has been investigated thoroughly. A significant improvement of the linear contrast transfer can be demonstrated. The information about the instrument one obtains using Young's fringe method is compared for uncorrected, Cs-corrected, and Cc /Cs-corrected instruments. The experimental results agree well with simulations. The conclusions might be useful to others in understanding the process of image formation in a Cc /Cs-corrected transmission electron microscope.