Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

This study compared dentinal tubule density and diameter of human primary and permanent teeth at different depths of the coronal dentin. Crowns of eight primary second molars and eight permanent third molars were serially sectioned into three disks of ~0.5 mm thickness (superficial, middle, and deep layers), perpendicular to the long axis. Tubule density and diameter were evaluated in 2,000× and 3,000× magnifications by scanning electron microscopy. Data obtained were subjected to two-way repeated measures ANOVA and Tukey's post hoc test (α = 0.05). Tubule density was greater in primary teeth compared with permanent ones, regardless of depth (primary: 124,329 ± 43,594 mm2; permanent: 45,972 ± 21,098 mm2). In general, the tubule density increased as the dentin depth increased, except to the superficial and middle layers from permanent teeth. Tubule diameter was larger in the dentin layer close to the pulp chamber (superficial: 2.4 ± 0.07 μm; middle: 3.70 ± 0.06 μm; deep: 4.28 ± 0.04 μm). No difference was observed between primary (3.48 ± 0.81 μm) and permanent teeth (3.47 ± 0.73 μm). The tubule diameter increases as the dentin depth increases for primary and permanent teeth; however, the tubule density is higher in primary teeth.

A method that enables high precision extraction of transmission electron microscope (TEM) specimens in low contrast materials has been developed. The main idea behind this work is to produce high contrast markers on both sides of and close to the area of interest. The markers are filled during the depositing of the protective layer. The marker material can be of either Pt or C depending on which one gives the highest contrast. It is thereby possible to distinguish the location of the area of interest during focused ion beam (FIB) milling and ensure that the TEM sample is extracted precisely at the desired position. This method is generally applicable and enables FIB/scanning electron microscope users to make high quality TEM specimens from small features and low contrast materials without a need for special holders. We explain the details of this method and illustrate its potential by examples from three different types of materials.

A fully automatic approach to locate polyomavirus particles in transmission electron microscopy images is presented that can localize intact particles, many damaged capsids, and an acceptable percentage of superposed ones. Performance of the approach is quantified in 25 electron micrographs containing nearly 390 particles and compared with the interpretation of the micrographs by two independent electron microscopy experts. All parameterization is based on the particle expected dimensions. This approach uses indicators calculated from the local co-occurrence matrix of gray levels to assess the textured pattern typical of polyomavirus and prune the initial set of candidates. In more complicated backgrounds, about 2–10% of the elements survive. A restricted set of the accepted points is used to evaluate the typical average and variance and to reduce the set of survivors accordingly. These intermediate points are evaluated using (i) a statistical index concerning the radiometric distribution of a circular neighborhood around the centroid of each candidate and (ii) a structural index resuming the expected morphological characteristics of eight radial intensity profiles encompassing the area of the possible particle. This hierarchical approach attains 90% efficiency in the detection of entire virus particles, tolerating a certain lack of differentiation in the borders and a certain amount of shape alterations.

African Swine Fever Virus (ASFV) is one of the most threatening agents of domestic pig diseases, without a vaccine or treatment, being its control exclusively based on compulsive sanitary measures. Until now, ASFV was thought to perform its viral life cycle within the cytoplasm although recent evidences indicate the presence of viral DNA material inside the host cell nucleus promoting ASFV reclassification into the Nucleocytoplasmic Large DNA Viruses group (NCLDV). So far, no studies have been performed regarding ASFV genome replication phenomena or the host nuclear compartments morphology during cellular infection. Therefore, we aim to unveil the spatiotemporal localization of ASFV DNA replication and the distribution patterns of three subnuclear domains (PML, speckles and coiled bodies), in order to improve knowledge on host nucleus-viral interactions.

For the viral DNA replication foci staining, Vero cells were synchronized at a G2/M stage with nocodazole (250 ng/ml, 24h). Following synchronization, cells were grown on glass coverslips (5,0x104 cells/cm2) and infected with ASFV-Ba71V isolate (1h adsorption period with a multiplicity of infection of 5). Afterwards, at specific time-points of viral infection, BrdU (150 µM/ml) was added for a short pulse (30 min) and immediate fixation was performed. For BrdU and ASFV immunodetection, the following primary and secondary antibodies were used: sheep polyclonal anti-BrdU (GTX21893, Genetex, USA; 1:100), an in-house clarified swine anti-ASFV whole-serum (1:100); Alexa Fluor 594 donkey anti-sheep IgG (A-11016, Life Technologies, USA; 1:500) and a FITC rabbit anti-swine IgG (ab6773, Abcam, UK; 1:400). For PML, speckles and coiled nuclear bodies immunolabeling a rabbit polyclonal anti-PML (ab53773, Abcam; 1:100), a goat polyclonal anti-SC35 (sc-10252, Santa Cruz Biotech, USA; 1:50) and a rabbit popyclonal anti-coilin (sc-32860, Santa Cruz Biotech; 1:50) were used, while DyLight 594 donkey anti-rabbit IgG (ab98490, Abcam; 1:500) and Alexa Fluor 594 chicken anti-goat IgG (A-21468, Molecular Probes, 1:400) were used as the secondary antibodies. Microscopic analysis of cells was performed with a Leica epifluorescence microscope (model DM R HC, Germany). De novo synthesized viral DNA was identified in a scattered nuclear localization (discreet dots) during the initial period of infection (Fig. 1, row 1), whereas in a later phase of infection (8h pi), most of the BrdU signal accumulated in the cytoplasmic viral factory (Fig. 1, row 2). PML bodies and nuclear speckles presented a morphological enlargement and a decreasing number in ASFV infected Vero cells (Fig. 2, rows 1 and 2). In an opposite manner, the coiled bodies increased their number during infection (Fig.2, row 3).

Our results provide the first evidence that ASFV DNA replication also occurs inside the host nucleus. Nuclear discrete replication foci, at the initial onset of the viral infection, contrast to an accumulation of synthesized viral DNA inside the bigger cytoplasmic viral factory, suggesting that these two distinct patterns are related to the viral life cycle demands. The early PML reorganization can probably be related with cellular antiviral defense mechanisms, given that DNA damage response and p53 are activated during ASFV infection. The low number of nuclear speckles observed in ASFV-infected cells, associated with their enlargement, is most possibly related to the relocation of host splicing factors, since ASFV genome lacks intronic regions.

This study was supported by Fundação para a Ciência e Tecnologia through the Project (PTDC/CVT/105630/2008) and the PhD fellowship (SFRH/BD/65532/2009).

As the presence of Sc2O3 and CeO2 is known to largely enhance the ionic conductivity in the temperature range of 600–800°C, compared with the conventional yttria-stabilized ZrO2, Sc2O3&CeO2-stabilized ZrO2 provide its applicability as electrolytes in solid oxide fuel cells. The current study introduces the methodology to synthesize Sc2O3&CeO2-stabilized ZrO2 powders by using co-precipitation technique or high-temperature hydrothermal reaction, and further describes the structural characterization of the zirconia powders synthesized by the above-mentioned two methods. The co-precipitation technique was found to allow obtaining powders of cubic phase, whereas high-temperature hydrothermal synthesis results in the presence of a monoclinic phase as well. The scanning transmission electron microscope observations also confirm that the size of the synthesized ZrO2 powders in this study is found to be much smaller than that of commercially available powders.

Imaging of latex particles, especially those with low glass transition temperature (Tg) has been a challenging issue. Different sample preparation methods for characterization of the morphology of a poly(n-butyl acrylate)/polystyrene two-phase latex are discussed and compared in this study. A method via hydroxyethyl cellulose embedding combined with ruthenium tetraoxide (RuO4) staining for scanning transmission electron microscope (STEM) observation is developed. By using this method, the spherical shape of latex particles can be maintained without deformation. The degree of incorporation of RuO4 into latex particles and cellulose matrix is different, which makes latex particles readily identifiable from cellulose matrix under STEM. A series of latexes with different structures such as copolymer latex and organic-inorganic hybrid latex were also successfully investigated by this method. The results indicate this specimen preparation method can be applied to study the morphology of a wide range of latex systems.

Focused ion beam (FIB) milling using high-energy gallium ions is widely used in the preparation of specimens for transmission electron microscopy (TEM). However, the energetic ion beam induces amorphization on the edge of specimens during milling, resulting in a mischievous influence on the clearness of high-quality transmission electron micrographs. In this work, the amorphization induced by the FIB milling was investigated by TEM for three kinds of materials, metallic materials in bulk shape, and semiconductive and electronic ceramic materials as a substrate for the deposition of thin films.

Electron Rutherford backscattering (ERBS) is a new technique that could be developed into a tool for materials analysis. Here we try to establish a methodology for the use of ERBS for materials analysis of more complex samples using bone minerals as a test case. For this purpose, we also studied several reference samples containing Ca: calcium carbonate (CaCO3) and hydroxyapatite and mouse bone powder. A very good understanding of the spectra of CaCO3 and hydroxyapatite was obtained. Quantitative interpretation of the bone spectrum is more challenging. A good fit of these spectra is only obtained with the same peak widths as used for the hydroxyapatite sample, if one allows for the presence of impurity atoms with a mass close to that of Na and Mg. Our conclusion is that a meaningful interpretation of spectra of more complex samples in terms of composition is indeed possible, but only if widths of the peaks contributing to the spectra are known. Knowledge of the peak widths can either be developed by the study of reference samples (as was done here) or potentially be derived from theory.

Three different bioactive materials suitable as dentine substitutes in tooth repair have been studied: glass-ionomer cement, particulate bioglass, and calcium-silicate cement. On 15 permanent human molars, Class V cavities were prepared and the bottom of each cavity was de-mineralized by an artificial caries gel. After the de-mineralization, the teeth were restored with: (1) Bioglass®45S5 and ChemFil® Superior; (2) Biodentine™ and ChemFil® Superior; and (3) ChemFil® Superior for a complete repair. The teeth were stored for 6 weeks in artificial saliva, then cut in half along the longitudinal axis: the first half was imaged in a scanning electron microscope (SEM) and the other half was embedded in resin and analyzed by SEM using energy-dispersive X-ray analysis. The glass-ionomer and the bioglass underwent ion exchange with the surrounding tooth tissue, confirming their bioactivity. However, the particle size of the bioglass meant that cavity adaptation was poor. It is concluded that smaller particle size bioglasses may give more acceptable results. In contrast, both the glass-ionomer and the calcium-silicate cements performed well as dentine substitutes. The glass-ionomer showed ion exchange properties, whereas the calcium silicate gave an excellent seal resulting from its micromechanical attachment.

Animal skin pigments, often of undisclosed biochemical nature, have many functions besides signalling and camouflage: they also play a protective role against environmental stressors, from radiation and oxidative stress–inducers to predators and parasites. Most pigments are not fully synthetized by animals but rather acquired from vegetable feed and often biotransformed. These intriguing pigments called the attention of researchers in the past, such as the awkward green “chlorophylloid” pigments of the marine invertebrate Bonellia, first described in the 19th century. For decades, however, research has come to a stand–still. The marine annelid Eulalia viridis (Phyllodocidae: Polychaeta), although common throughout NW Europe, has so far received little attention to the origin and function of its bright green colouration. In fact, with the exception of part of its digestive system even the basic histological description of this animal is absent, as for marine annelids in general.

Adult wild E. viridis were subjected to a comprehensive microscopy study employing several fixation and staining techniques for bright–field (BF), transmission electron (TEM) and UV–epifluorescence microscopy in order to disclose the pigment cells’ microanatomy and arrangement and to infer the potential origin of the pigments. Stains for bright–field microscopy included Haematoxylin and Eosin (HE), Alcian Blue–Periodic Acid/Schiff’s–Haematoxylin (AB–PAS–H) and Bronner’s Sudan Black. Samples were either embedded in paraffin (BF and UV microscopy) or LR White resin (BF and TEM).

The high complexity of the animals’ integument was confirmed, in accordance to the scarcely known polychaete microanatomy. The green colouration is chiefly caused by the presence of specialized skin cells containing minute pigment vesicles (Fig. 1A). These pigment cells are compressed between multiple types of skin cells and possess microvilli that protrude through the integument’s polysaccharide–based cuticle. The vesicles themselves appear attached to the cells’ tonofilaments (Fig. 1B). The vesicles are densely packed within pigment cells (Fig. 2A) and are not fluorescent, which should exclude the existence of active chlorophyll within (Fig. 2B). From this data is may be inferred that the pigments are chlorophyll–derived substances (chlorins/pheophorbides), similar to what was observed in the polychaete Chaetopterus variopedatus. Oddly enough, while the latter is a filter–feeder, thus able to sequester microalgae from the water column, E. viridis is a foraging scavenger that feeds mostly on the flesh of other invertebrates. Furthermore, the animal inhabits nude rocky shores and not algal beds where its green colour would be a clear advantage, which renders further intriguing the adaptative value of this feature, however, due to some known biocidal properties of these pigments; defence should not be overruled.

P.M. Costa acknowledges the Portuguese Science and Technology Foundation (FCT) for the grant SFRH/BPD/72564/2010. The authors also thank L. Ascensão (FCUL) for the important assistance.

A method for the fabrication of a wedge-shaped thin NiO lamella by focused ion beam is reported. The starting sample is an oxidized bulk single crystalline, ⟨100⟩ oriented, Ni commercial standard. The lamella is employed for the determination, by analytical electron microscopy at 200 kV of the experimental k(O-Ni) Cliff-Lorimer (G. Cliff & G.W. Lorimer, J Microsc103, 203–207, 1975) coefficient, according to the extrapolation method by Van Cappellen (E. Van Cappellen, Microsc Microstruct Microanal1, 1–22, 1990). The result thus obtained is compared to the theoretical k(O-Ni) values either implemented into the commercial software for X-ray microanalysis quantification of the scanning transmission electron microscopy/energy dispersive spectrometry equipment or calculated by the Monte Carlo method. Significant differences among the three values are found. This confirms that for a reliable quantification of binary alloys containing light elements, the choice of the Cliff-Lorimer coefficients is crucial and experimental values are recommended.

This in vitro study evaluated the effect of light curing unit (LCU) type, mouthwashes, and soft drink on chemical degradation of a nanofilled resin composite. Samples (80) were divided into eight groups: halogen LCU, HS—saliva (control); HPT—Pepsi Twist®; HLC—Listerine®; HCP—Colgate Plax®; LED LCU, LS—saliva (control); LPT—Pepsi Twist®; LLC—Listerine®; LCP—Colgate Plax®. The degree of conversion analysis and the measure of the peak area at 2,930 cm−1 (organic matrix) of resin composite were done by Fourier-transform Raman spectroscopy (baseline, after 7 and 14 days). The data were subjected to multifactor analysis of variance (ANOVA) at a 95% confidence followed by Tukey's HSD post-hoc test. The DC ranged from 58.0% (Halogen) to 59.3% (LED) without significance. Differences in the peak area between LCUs were found after 7 days of storage in S and PT. A marked increase in the peak intensity of HLC and LLC groups was found. The soft-start light-activation may influence the chemical degradation of organic matrix in resin composite. Ethanol contained in Listerine® Cool Mint mouthwash had the most significant degradation effect. Raman spectroscopy is shown to be a useful tool to investigate resin composite degradation.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

During heart valve development, epithelial–mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

Icosahedral nucleo-cytoplasmic large DNA viruses (NCLDV)-like viruses, which form inclusions in the erythrocyte cytoplasm of reptiles, were previously presented as candidates for a new genus of the Iridoviridae family. The present work describes the distribution of infected lizard hosts and ultrastructural characteristics of the viral inclusions of NCLDV-like viruses from Portugal and adjacent locations in Spain. Giemsa-stained blood smears of 235 Lacerta schreiberi from Portugal and Spain, 571 Lacerta monticola from the mountain Serra da Estrela (Portugal), 794 Podarcis hispanica from several localities in Portugal and Spain, and 25 Lacerta dugesii from Madeira Island, were studied. Infection in L. schreiberi was only found in mountain populations, up to 30% in Serra da Estrela and 9–11% elsewhere. It was absent in lizards from lowlands. Prevalence of infection among L. monticola in Serra da Estrela was 10%; infected lizards were found during March to July and October but not in August and September. Infection in P. hispanica was below 3.3%. Only one infected specimen of L. dugesii was identified by light microscopy. Ultrastructural examination of infected samples revealed that the inclusions are virus assembly sites of icosahedral cytoplasmic iridovirus-like virions. Virions from different host species have different ultrastructural features and probably represent different related viruses.

Theories have been put forward on the etiology of sialoliths; however, a comprehensive understanding of their growth mechanisms is lacking. In an attempt to fill this gap, the current study has evaluated the internal architecture and growth patterns of a set of 30 independent specimens of sialoliths characterized at different scales by computed microtomography and electron microscopy. Tomography reconstructions showed cores in most of the sialoliths. The cores were surrounded by concentric or irregular patterns with variable degrees of mineralization. Regardless of the patterns, at finer scales the sialoliths consisted of banded and globular structures. The distribution of precipitates in the banded structures is compatible with a Liesegang–Ostwald phenomenon. On the other hand, the globular structures appear to arise from surface tension effects and to develop self-similar features as a result of a viscous fingering process. Electron diffraction patterns demonstrated that Ca- and P-based electrolytes crystallize in a structure close to that of hydroxyapatite. The organic matter contained sulfur with apparent origin from sulfated components of secretory material. These results cast new light on the mechanisms involved in the formation of sialoliths.