Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.

The following papers are samples of ongoing Portuguese contributions to the field of microscopy research. They were first communicated at the 43rd Annual Meeting of the Portuguese Society for Microscopy (Sociedade Portuguesa de Microscopia, SPMicros) held in Porto, October 30–31, 2008, and, together with most of the other communications, were published as extended abstracts in the Proceedings of the meeting (Microsc. Microanal. 16, Suppl. S3). This was the second time that the abstracts of the SPMicros Annual Meetings were made available worldwide through our collaborations with Microscopy and Microanalysis; although throughout its long history, the society has always kept Portuguese and English records of the communications either in abstract books or as journal supplements.

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.

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 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.

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.

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.

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.

A simple fluorescence microscopy technique is developed and presented to investigate heterogeneities in emission intensity and quenching responses of luminescence sensors and to measure diffusion and permeation coefficients of oxygen in polymers. Most luminescence oxygen sensors do not follow linearity of the Stern-Volmer (SV) equation due to heterogeneity of luminophore in the polymer matrix. To circumvent this limitation, inverted fluorescence microscopy is utilized in this work to investigate the SV response of the sensors at the micron scale. It was found that intensity is higher in regions where the luminophore is aggregated, but the response is poorer to oxygen concentration. In contrast, the nearly homogeneous regions exhibit linearity with high SV constants. In these diffusion experiments, oxygen concentration was measured by luminescence changes in regions with high SV constants and good linearity. Two diffusion experiments were performed—termed film-on-sensor and accumulation-in-volume techniques. A new Fick's law based quasi-steady-state diffusion model was developed and combined with the SV equation to obtain effective permeation coefficients for the accumulation-in-volume technique. Using these experimental techniques, oxygen diffusion properties in free-standing Teflon polymer films, cast silicon elastomers, and cast polydimethylsiloxane films containing different weight percentages of zeolite were determined with good precision.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

The product and direct role of the rssC gene of Serratia marcescens is unknown. For unraveling the role of the rssC gene, atomic force microscopy has been used to identify the surfaces of intact S. marcescens wild-type CH-1 cells and rssC mutant CH-1ΔC cells. The detailed surface topographies were directly visualized, and quantitative measurements of the physical properties of the membrane structures were provided. CH-1 and CH-1ΔC cells were observed before and after treatment with lysozyme, and their topography-related parameters, e.g., a valley-to-peak distance, mean height, surface roughness, and surface root-mean-square values, were defined and compared. The data obtained suggest that the cellular surface topography of mutant CH-1ΔC becomes rougher and more precipitous than that of wild-type CH-1 cells. Moreover, it was found that, compared with native wild-type CH-1, the cellular surface topography of lysozyme-treated CH-1 was not changed profoundly. The product of the rssC gene is thus predicted to be mainly responsible for fatty-acid biosynthesis of the S. marcescens outer membrane. This study represents the first direct observation of the structural changes in membranes of bacterial mutant cells and offers a new prospect for predicting gene expression in bacterial cells.

Polyurethane catheters made of Pellethane 2363-80AE® were treated with a low temperature plasma developed for the decontamination of reusable polymer devices in hospitals. We investigated the modifications of the polymer surface by studying the topographic modifications, the chemical modifications, and their consequences on the wettability and bacterial adhesion. This study showed that plasma treatment modified the topography and grafted oxygen and nitrogen species onto the surface, resulting in an increase in the surface polarity. This effect could be correlated to the number of nitrogen atoms interacting with the surface. Moreover, this study demonstrated the significance of multiscale heterogeneities, and the complexity of industrial medical devices made from polymers. Their surface can be heterogeneous, and they contain additives that can migrate and change the surface composition.

This in vitro study evaluated the marginal gap at the composite tooth/resin interface in class V cavities under the influence of two insertion techniques and a curing system by means of atomic force microscopy (AFM). Forty enamel and dentin cavities were prepared on the buccal surface in bovine teeth with quadratic forms measuring 2 mm × 2 mm and depth of 1.5 mm. The teeth were then divided into four groups: group A, 10 cavities were restored in one increment, light cured by halogen light; group B, 10 cavities filled with bulk filling, light cured by the light emitting diodes (LED); group C, 10 cavities were restored by the incremental technique, light cured by halogen light; group D, 10 cavities were restored by the incremental technique, light cured by the LED. The teeth underwent the polishing procedure and were analyzed by AFM for tooth/restoration interface evaluation. The data were compared between groups using the nonparametric Kruskall-Wallis and Mann-Whitney tests (p < 0.05). The results showed a statistically significant difference between groups A and B and groups A and C. It was concluded that no insertion and polymerization technique was able to completely seal the cavity.

A promising novel type of electrostatic phase plate for transmission electron microscopy (TEM) is presented. The phase plate consists of a single microcoaxial cable-like rod with its electrode exposed to the undiffracted electrons. The emerging field is used to shift the phase of the undiffracted electrons with respect to diffracted electrons. The design overcomes the drawback of the spatial frequency-blocking ring electrode of the Boersch phase plate. First, experimental phase-contrast images are presented for PbSe and Pt nanoparticles with clearly varying phase contrast, which depends on the applied voltage and resulting phase shift of the unscattered electrons. With the new phase-plate design, we show for the first time the reconstruction of an object wave function based on a series of only three experimental phase-contrast TEM images obtained with an electrostatic phase plate.

Scanning transmission electron microscope (STEM) images of three-dimensional (3D) samples were simulated. The samples consisted of a micrometer(s)-thick substrate and gold nanoparticles at various vertical positions. The atomic number (Z) contrast as obtained via the annular dark-field detector was generated. The simulations were carried out using the Monte Carlo method in the CASINO software (freeware). The software was adapted to include the STEM imaging modality, including the noise characteristics of the electron source, the conical shape of the beam, and 3D scanning. Simulated STEM images of nanoparticles on a carbon substrate revealed the influence of the electron dose on the visibility of the nanoparticles. The 3D datasets obtained by simulating focal series showed the effect of beam broadening on the spatial resolution and on the signal-to-noise ratio. Monte Carlo simulations of STEM imaging of nanoparticles on a thick water layer were compared with experimental data by programming the exact sample geometry. The simulated image corresponded to the experimental image, and the signal-to-noise levels were similar. The Monte Carlo simulation strategy described here can be used to calculate STEM images of objects of an arbitrary geometry and amorphous sample composition. This information can then be used, for example, to optimize the microscope settings for imaging sessions where a low electron dose is crucial for the design of equipment, or for the analysis of the composition of a certain specimen.

We describe a feedback mechanism in the gas cascade amplification process used in magnetic immersion lens environmental scanning electron microcopy (ESEM). Feedback dominates gas gain under the conditions typically used for ultra-high-resolution ESEM and gives rise to novel dependencies of the imaging signal and noise on microscope operating parameters. It is ascribed tentatively to the generation of free electrons upon de-excitation of metastable species in the gas cascade. The results have implications for optimization of ESEM systems for applications such as critical dimension metrology and real-time imaging of nanostructure growth by gas mediated electron beam induced deposition.

Despite the many demonstrated applications of factor analysis (FA) in analyzing hyperspectral materials images, FA does have inherent mathematical limitations, preventing it from solving certain materials characterization problems. A notable limitation of FA is its parsimony restriction, referring to the fact that in FA the number of components cannot exceed the chemical rank of a dataset. Clustering is a promising alternative to FA for the phase classification of hyperspectral materials images. In contrast with FA, the phases extracted by clustering do not have to be parsimonious. Clustering has an added advantage in its insensitivity to spectral collinearity that can result in phase mixing using FA. For representative energy dispersive X-ray spectroscopy materials images, namely a solder bump dataset and a braze interface dataset, clustering generates phase classification results that are superior to those obtained using representative FA-based methods. For the solder bump dataset, clustering identifies a Cu-Sn intermetallic phase that cannot be isolated using FA alone due to the parsimony restriction. For the braze interface sample that has collinearity among the phase spectra, the clustering results do not exhibit the physically unrealistic phase mixing obtained by multivariate curve resolution, a commonly utilized FA algorithm.

Quantitative X-ray microanalysis of thick samples is usually performed by measuring the characteristic X-ray intensities of each element in a sample and in corresponding standards. The ratio of the measured intensities from the unknown material to that from the standard is related to the concentration using the ZAF or ϕ(ρz) equations. Under optimal conditions, accuracies approaching 1% are possible. However, all the experimental conditions must remain the same during the sample and standard measurements. This is not possible with cold field emission scanning electron microscopes (FE-SEMs) where beam current can fluctuate around 5% in its stable regime. Very little work has been done on variable beam current conditions (Griffin, B.J. & Nockolds, C.E., Scanning13, 307–312, 1991), and none relating to cold FE-SEM applications. To address this issue, a new method was developed using a single spectral measurement. It is similar in approach to the Cliff-Lorimer method developed for the analytical transmission electron microscope. However, corrections are made for X rays generated from thick specimens using the ratio of the characteristic X-ray intensities of two elements in the same material. The proposed method utilizes the ratio of the intensity of a characteristic X-ray normalized by the sum of X-ray intensities of all the elements measured for the sample, which should also reduce the amplitude of error propagation. Uncertainties in the physical parameters of X-ray generation are corrected using a calibration factor that must be previously acquired or calculated. As an example, when this method was applied to the calculation of the composition of Au-Cu National Institute of Standards and Technology standards measured with a cold field emission source SEM, relative accuracies better than 5% were obtained.

A simple algorithm is developed and implemented to eliminate ambiguities, in both statistical analyses of orientation data (e.g., orientation averaging) and electron backscattered diffraction (EBSD) orientation map visualization, caused by symmetrically equivalent orientations and the wrap-around or umklapp effect. Using crystal symmetry operators and the lowest Euclidian-distance criterion, the orientation of each pixel within a grain is redefined. An advantage of this approach is demonstrated for direct determination of the representative orientation of a grain within an EBSD map by mean, median, or quaternion-based averaging methods that can be further used within analyses or visualization of misorientation or geometrically necessary dislocation (GND) density. If one also considers the lattice curvature tensor, five components of the dislocation density tensor—corresponding to a part of the GND content—may be inferred. The methodology developed is illustrated using EBSD orientation data obtained from the fatigue crack-tips/wakes in aerospace aluminum alloys 2024-T351 and 7050-T7451.