The UFeB4 phase present in different alloys of the B–Fe–U system was studied by powder X-ray diffraction (PXRD) and scanning electron microscopy complemented with energy-dispersive spectroscopy and electron backscattered diffraction (EBSD). The PXRD data showed that the ternary compound crystallized adopting essentially the YCrB4-type structure. However, microstructural observations revealed that under high undercooling conditions the UFeB4 phase exhibits a random distribution of defects parallel to, which are consistently associated with intense higher-order Laue zone rings in EBSD patterns. Indexation of the EBSD patterns showed that the defective structure is compatible with an intergrowth of YCrB4- and ThMoB4-type layers according to the (010)YCrB4//(110)ThMoB4 and [001]YCrB4//[001]ThMoB4 orientation relation previously reported for an analogous compound. Magnetic studies indicated that the annealed UFeB4 compound has a paramagnetic behavior in the 2–300 K temperature range.

The ordered structures in different doping levels (x = 0.1, 0.15, 0.2, 0.25, 0.3) of yttrium doped ceria (YDC, Ce(1−x)YxO2−δ) electrolytes were investigated by electron diffraction, high-resolution transmission electron microscopy (TEM), scanning TEM, and electron energy loss spectroscopy. Oxygen vacancy ordering was experimentally confirmed within the ordered structures. With increasing the doping level, the concentration of trivalent Ce cations was increased in YDC samples and such trivalent Ce cations were supposed to mainly exist in the ordered structures. Based on our electron microscopic observation and microanalysis, a crystal model for the ordered structures is proposed.

These in-depth courses will be held Monday-Thursday from 1:00 PM to 3:00 PM.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.

We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.

Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).

Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

We investigated the microstructural evolution of Sn96.4Ag2.8Cu0.8 solder through in situ heating transmission electron microscopy observations. As-soldered bump consisted of seven layers, containing the nanoeutectic lamella structure of AuSn and Au5Sn phases, and the polygonal grains of AuSn2 and AuSn4, on Au-plated Cu bond pads. Here, we found that there are two nanoeutectic lamellar layers with lamella spacing of 40 and 250 nm. By in situ heating above 140°C, the nanoeutectic lamella of AuSn and Au5Sn was decomposed with structural degradation by sphering and coarsening processes of the lamellar interface. At the third layer neighboring to the lamella layer, on the other hand, Au5Sn particles with a zig-zag shape in AuSn matrix became spherical and were finally dissipated in order to minimize the interface energy between two phases. In the other layers except both lamella layers, polycrystal grains of AuSn2 and AuSn4 grew by normal grain growth during in situ heating. The high interface energy of nanoeutectic lamella and polygonal nanograins, which are formed by rapid solidification, acted as a principal driving force on the microstructural change during the in situ heating.

Nd:11Fe:Ti alloys prepared by arc melting followed by splat quenching and annealing have been investigated by electron microscopy. The as-cast microstructure evidenced an α-Fe(Ti) → NdFe11Ti → Nd2(Fe,Ti)17 solidification sequence compatible with a cascade of peritectic reactions. The Nd2(Fe,Ti)17 phase was not detected in the microstructure of the splat-quenched materials, but after annealing the ternary compound grains consisted of a mixture of ThMn12-type and Th2Zn17-type structures exhibiting a consistent (020)1:12//(003)2:17 and [100]1:12//[110]2:17, orientation relation, with the invariant plane sitting at (022)1:12//(333)2:17. A series of 3D microdiffraction experiments carried out on grains presenting a random distribution of planar defects has been used to map the reciprocal space of the intergrown phases.

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

Human epidermal growth factor receptor (HER2) is a tumor biomarker that when overexpressed and/or amplified is associated with a poor prognosis for women with breast cancer. This specific tumor subtype is eligible for a specific immunotherapy that increases survival period. However, in feline oncology, only a few studies have been performed on molecular characterization of feline (fHER2) in feline mammary carcinoma (FMC), and the available data are inconsistent. In this study, fHER2 protein levels and gene status in FMC were evaluated by immunohistochemistry and in situ hybridization. After being optimized, these techniques revealed that fHER2 is overexpressed in 33% of FMC cases, although fHER2 and fTOP2A gene amplification could not be observed. Our results support the possibility of using FMC as a natural model for comparative oncology. Additional data obtained may also improve the diagnostics, and consequently the treatment, of this type of tumor in veterinary medicine.

Morphological studies of helminths by scanning electron microscopy are generally limited to the external topography of the organisms. In this work, we present a simple technique using ethanol as a cryoprotectant without postfixation in osmium tetroxide that allows for observation of the inner organization of helminths and preserves cellular structures. We tested the technique in three helminths: Echinostoma paraensei, Cruzia tentaculata, and Hassalstrongylus epsilon. The results show that this technique could potentially be applied to study the morphology, ultrastructure, and taxonomy of helminths.

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Fluorescence resonance energy transfer (FRET) microscopy is a powerful technique to quantify dynamic protein-protein interactions in live cells. Total internal reflection fluorescence (TIRF) microscopy can selectively excite molecules within about 150 nm of the glass-cell interface. Recently, these two approaches were combined to enable high-resolution FRET imaging on the adherent surface of living cells. Here, we show that interference fringing of the coherent laser excitation used in TIRF creates lateral heterogeneities that impair quantitative TIRF-FRET measurements. We overcome this limitation by using a two-dimensional scan head to rotate laser beams for donor and acceptor excitation around the back focal plane of a high numerical aperture objective. By setting different radii for the circles traced out by each laser in the back focal plane, the penetration depth was corrected for different wavelengths. These modifications quell spatial variations in illumination and permit calibration for quantitative TIRF-FRET microscopy. The capability of TIRF-FRET was demonstrated by imaging assembled cyan and yellow fluorescent protein–tagged HIV-Gag molecules in single virions on the surfaces of living cells. These interactions are shown to be distinct from crowding of HIV-Gag in lipid rafts.

In this paper we describe the preparation of fluorescent mesoporous silica nanoparticles (MSNs) for traceable drug delivery systems. The nanoparticles were prepared following a sol–gel procedure, incorporating a modified perylenediimide dye in the silica structure. Transmission electron microscopy and scanning electron microscopy show that the nanoparticles are monodispersed, with a spheroid shape and a raspberry-type surface morphology. The hybrid MSNs are robust, maintaining the mesoporous structure after template removal, with a pore diameter above 2 nm. A polymer shell was synthesized from the external surface of the hybrid nanoparticles by atom transfer radical polymerization, showing temperature-switchable collapsed/expanded conformation control. The fluorescent properties of the perylenediimide dye incorporated in the MSN pore walls are intact, and internalization in HEK293 cells shows that the nanoparticles are efficiently dispersed in the cytosol. These results show that the mesoporous fluorescent hybrid nanoparticles are an excellent platform for development of a traceable drug delivery system.

Aganaspis daci and Aganaspis pelleranoi (Hymenoptera: Figitidae) are important parasitoids of fruit flies. Here we studied, with light and scanning electron microscopy, aspects of their morphology that could help with plans to mass rear and thus contribute to improved pest control (preimaginal phases) and to shed light on parasitoid-pest relationships (sensillar equipment). The two species present a stalked egg, eucoiliform first and second-instar larvae and hymenopteriform third instar and mature larvae. The first instar presents tegumental differentiations in the mesoma and first metasomal segment in A. daci, but not in A. pelleranoi, while unlike other figitids, neither species displays setae in the mesosomal processes. Second and third instar and mature larvae present tegumental differentiations in A. daci, but not in A. pelleranoi. The moniliform (female) and filiform (male) antennae of A. daci and A. pelleranoi harbor seven types of sensilla, four of them (sensilla campaniformia, sensilla coeloconica type II, and two types of sensilla trichoidea) described here for the first time in Cynipoidea. The largest sensilla were the multiporous placoid sensilla, which were smaller and more numerous in A. pelleranoi. Species also differed to some extent in morphology of sensilla coeloconica. Observations on the ovipositor revealed the presence of coeloconic sensilla on Valva I in both species.

Stereological analysis has been coupled with transmission electron microscope (TEM) orientation mapping to investigate the grain boundary character distribution in nanocrystalline copper thin films. The use of the nanosized (<5 nm) beam in the TEM for collecting spot diffraction patterns renders an order of magnitude improvement in spatial resolution compared to the analysis of electron backscatter diffraction patterns in the scanning electron microscope. Electron beam precession is used to reduce dynamical effects and increase the reliability of orientation solutions. The misorientation distribution function shows a strong misorientation texture with a peak at 60°/[111], corresponding to the Σ3 misorientation. The grain boundary plane distribution shows {111} as the most frequently occurring plane, indicating a significant population of coherent twin boundaries. This study demonstrates the use of nanoscale orientation mapping in the TEM to quantify the five-parameter grain boundary distribution in nanocrystalline materials.

We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.

In contrast to symmetric division in mitosis, mammalian oocyte maturation is characterized by asymmetric cell division that produces a large egg and a small polar body. The asymmetry results from oocyte polarization, which includes spindle positioning, migration, and cortical reorganization, and this process is critical for fertilization and the retention of maternal components for early embryo development. Although actin dynamics are involved in this process, the molecular mechanism underlying this remained unclear until the use of confocal microscopy and live cell imaging became widespread in recent years. Information obtained through a PubMed database search of all articles published in English between 2000 and 2012 that included the phrases “oocyte, actin, spindle migration,” “oocyte, actin, polar body,” or “oocyte, actin, asymmetric division” was reviewed. The actin nucleation factor actin-related protein 2/3 complex and its nucleation-promoting factors, formins and Spire, and regulators such as small GTPases, partitioning-defective/protein kinase C, Fyn, microRNAs, cis-Golgi apparatus components, myosin/myosin light-chain kinase, spindle stability regulators, and spindle assembly checkpoint regulators, play critical roles in asymmetric cell division in oocytes. This review summarizes recent findings on these actin-related regulators in mammalian oocyte asymmetric division and outlines a complete signaling pathway.

The charging effects of microfibrils of sciatic nerve tissues due to electron irradiation are investigated using electron holography. The phenomenon that the charging effects are enhanced with an increase of electron intensity is visualized through direct observations of the electric potential distribution around the specimen. The electric potential at the surface of the specimen could be quantitatively evaluated by simulation, which takes into account the reference wave modulation due to the long-range electric field.