Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been considered a classical glycolytic enzyme involved exclusively in cytosolic energy production. Several recent studies, however, have demonstrated that GAPDH is a multifunctional protein whose presence and activity can be regulated by disease states and/or experimental manipulation. Expression levels of GAPDH have been shown to be altered in certain tumors as well as in proliferating and differentiating cells. Since dedifferentiation and proliferation of smooth muscle cells (SMCs) are important features of human atherosclerosis, we have characterized the expression profile of GAPDH in the SMCs of atherosclerotic plaques and its putative interrelationship with the synthetic/proliferative status of these cells utilizing the proliferating cell nuclear antigen (PCNA) antibody, a valuable marker of cell proliferation. Western blot data revealed that GAPDH was significantly upregulated in atherosclerotic plaque specimens. Immunohistochemical stains demonstrated that GAPDH accumulated in the nucleus of dedifferentiated SMCs that also showed positive immunoreactivity for PCNA, but remained cytoplasmatic in the contractile SMCs (PCNA-negative), thus reflecting the proliferative, structural and synthetic differences between them. We suggest that, in human atherosclerotic plaque, GAPDH might exert additional functions that are independent of its well-documented glycolytic activity and might play key roles in development of the disease.

Mycotoxins, such as aflatoxin (AF), fumonisin B1, zearalenone (ZEA), and deoxynivalenol (DON), are commonly found in many food commodities. Mycotoxins have been shown to increase DNA methylation levels in a human intestinal cell line. We previously showed that the developmental competence of oocytes was affected in mice that had been fed a mycotoxin-containing diet. In this study, we explored possible mechanisms of low mouse oocyte developmental competence after mycotoxin treatment in an epigenetic modification perspective. Mycotoxin-contaminated maize (DON at 3,875 μg/kg, ZEA at 1,897 μg/kg, and AF at 806 μg/kg) was included in diets at three different doses (mass percentage: 0, 15, and 30%) and fed to mice for 4 weeks. The fluorescence intensity analysis showed that the general DNA methylation levels increased in oocytes from high dose mycotoxin-fed mice. Mouse oocyte histone methylation was also altered. H3K9me3 and H4K20me3 level increased in oocytes from mycotoxin-fed mice, whereas H3K27me3 and H4K20me2 level decreased in oocytes from mycotoxin-fed mice. Thus, our results indicate that naturally occurring mycotoxins have effects on epigenetic modifications in mouse oocytes, which may be one of the reasons for reduced oocyte developmental competence.

This study examines histometrical changes induced by sodium arsenite (SA), as an environmental pollutant, and investigates the protective effect of α-tocopherol on ovaries of SA-treated rats during the prenatal stage until sexual maturity. Rats were classified into groups: control, SA (8 ppm/day), α-tocopherol (100 ppm/day), and SA+α-tocopherol. Treatment was performed from pregnancy until maturation when the rats and ovaries were weighed. The Cavalieri method was used to estimate volume of the ovaries, cortex, medulla, and corpus luteum. The mean diameter of oocytes, granulosa cells, and nuclei were measured and volume was estimated using the Nucleator method. The number of oocytes and thickness of the zona pellucida (ZP) were determined using an optical dissector and orthogonal intercept method, respectively. SA reduced the body and ovary weight, the number of secondary, antral and Graafian oocytes, volume of the ovaries, cortex, medulla and corpus luteum, mean diameter and volume of oocytes in primordial and primary follicles, mean diameter and volume of oocyte nuclei in all types of follicles, and mean thickness of the ZP in secondary and antral follicles. Also, the mean diameter and volume of granulosa cells and their nuclei in antral and Graafian follicles decreased significantly. Vacuolization and vascular congestion in the corpus luteum and an increase in the number of atretic oocytes were seen in the SA group. Most of these parameters were unchanged from the control level in the SA+α-tocopherol group. It was concluded that α-tocopherol supplementation reduced the toxic effects of SA exposure on ovarian tissue in rats.

Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.

Scanning electron microscopy (SEM) of scales in six species of the fish genus Channa revealed certain features relevant to taxonomic significance. The location of focus, inter-radial distance and width of circuli, inter-circular space, width of radii, shape and size of lepidonts, etc. were found to be different in different species. The importance of SEM of scales in poorly understood taxonomy and phylogeny of the fish genus Channa is discussed with the help of relevant literature. Further, the role of SEM of fish scales for taxonomic applications is discussed in detail.

Cell motility involves a number of strategies that cells use in order to seek nutrients, escape danger, and fulfill morphogenetic roles. Here we present a methodology to quantify morphological changes and their relationship with signaling events from time-lapse imaging microscopy experiments, in order to characterize physiological and pathological processes. To this aim, the stationary spatial pattern of signaling events is determined through an intracellular fluorescent probe, and it is related with the frequency and entity of morphodynamic events, which are in turn quantified through a stochastic approach: two pseudoimages are obtained from a time series of moving cells that describe the probability that a pixel belongs to the cell, and the probability that a pixel is subject to a dynamic event. The simultaneous construction of these maps permits visualization of hot spots of dynamic events, i.e., zones of formation of membrane protrusions and retractions and their relationship with the signaling events reported by the specific probe employed. The method is tested on spontaneous movement of cells, trasfected with redox-sensitive yellow fluorescent protein, in which the distribution of the hot spots and its change upon expression of constitutively active Rac (V12-Rac), is related to the distribution of oxidized spots.

Two methods are especially suited for tomographic imaging with histological detail of macroscopic samples that consist of multiple tissue types (bone, muscle, nerve or fat): Light sheet (based) fluorescence microscopy (LSFM) and micro-computed tomography (micro-CT). Micro-CT requires staining with heavy chemical elements (and thus fixation and sometimes dehydration) in order to make soft tissue imageable when measured alongside denser structures. LSMF requires fixation, decalcification, dehydration, clearing and staining with a fluorescent dye. The specimen preparation of both imaging methods is prone to shrinkage, which is often not mentioned, let alone quantified. In this paper the presence and degree of shrinkage are quantitatively identified for the selected preparation methods/stains. LSFM delivers a volume shrinkage of 17% for bone, 56% for muscle and 62% for brain tissue. The three most popular micro-CT stains (phosphotungstic acid, iodine with potassium iodide, and iodine in absolute ethanol) deliver a volume shrinkage ranging from 10 to 56% for muscle and 27–66% for brain, while bone does not shrink in micro-CT preparation.

The purpose of this study was to evaluate the ability of two dentin adhesive systems to induce remineralization in the bonded dentin interface after in vitro thermo-cycling. Dentin surfaces were treated with two different adhesive approaches: (1) 37% phosphoric acid (PA) plus an “etch-and-rinse” dentin adhesive (single bond, SB) (PA+SB) or (2) application of a “self-etch” dentin adhesive (Clearfil SE bond, SEB). Three groups were established: (i) 24 h or (ii) 3 m storage, and (iii) specimens submitted to thermal cycling (100,000 cy/5 and 55ºC). Atomic force microscopy imaging/nanoindentation, Raman spectroscopy/cluster analysis with dye-assisted confocal laser scanning microscopy (CLSM) evaluation and Masson’s trichrome staining assessments were implemented for characterization. Thermo-cycling increased nanohardness in PA+SB at the hybrid layer (HL) and in SEB at the bottom of the HL (BHL). Young’s modulus increased at both the HL and BHL in SEB and at the HL in PA+SB, after thermal stress. Cluster analysis demonstrated an augmentation of the mineral–matrix ratio in thermo-cycled specimens. CLSM showed a decrease of both micropermeability and nanoleakage after thermo-cycling in PA+SB, and were completely absent in SEB. Trichrome staining reflected a scarce demineralized front in PA+SB after thermo-cycling and total remineralization in SEB.

This study evaluated the influence of fluoride mouth rinses and repolishing on the superficial morphology and color stability of nanofilled resin. About 150 specimens were prepared and polished using aluminum oxide discs for 15 s with a pressure of 2 kg. The experimental groups were divided according to the immersion medium (artificial saliva, 0.5% sodium fluoride, Fluordent Reach, Oral B, Fluorgard) and repolishing procedure (without and with). The specimens were continuously immersed for 1 week. Thereafter, half of each sample was repolished. A color reading was performed after 24 h of immersion in the artificial saliva baseline, after continuous immersion, and after repolishing. The superficial morphology was examined using scanning electron microscopy (SEM) in a qualitative way. Color change (∆E) data were submitted to a mixed analysis of variance using a Shapiro–Wilk test (p>0.05 for the different immersion media) and Sidak’s test (p<0.05 for the differences between groups). In the interaction between the repolishing and the immersion media, Fluorgard showed a statistical difference between the ∆E values with and without repolishing (p<0.0001). On the SEM observations, both Fluordent Reach and Fluorgard caused degradation of the superficial resinous matrix of the composite after continuous immersion. This matrix was removed after repolishing.

This study evaluated the cleaning efficacy of a root canal system by Nd:YAG laser and rotary instruments. Sixty single-rooted human teeth were divided into four experimental groups (n=15). In the first group the teeth were prepared with a step-back technique using conventional K-files. In the second and third groups, tooth preparation was carried out using Nd:YAG laser and rotary NiTi instruments, respectively. Teeth in the fourth group were prepared by combined laser and rotary methods. The smear layer remaining on canal walls was then assessed by scanning electron microscopy in the coronal, middle, and apical portions. The comparison of smear layer removal efficacy between groups was carried out by Kruskal–Wallis and Mann–Whitney U tests. The mean grades of smear layer removal in rotary-laser, rotary, laser and step-back techniques were 1.34±0.18, 2.2±0.28, 1.91±0.25, and 2.42 ±0.19, respectively. On the whole, differences between rotary-laser and rotary groups, step-back, and the three other techniques (rotary, laser, and rotary-laser) were significant at p=0.034. Based on the findings of this study, the cleaning efficacy of rotary, laser, and rotary-laser techniques were better than the step-back technique and the combined laser and rotary technique was the most efficient method.

This work presents a systematic study that evaluates the feasibility and reliability of local band gap measurements of Cu(In,Ga)Se2 thin films by valence electron energy-loss spectroscopy (VEELS). The compositional gradients across the Cu(In,Ga)Se2 layer cause variations in the band gap energy, which are experimentally determined using a monochromated scanning transmission electron microscope (STEM). The results reveal the expected band gap variation across the Cu(In,Ga)Se2 layer and therefore confirm the feasibility of local band gap measurements of Cu(In,Ga)Se2 by VEELS. The precision and accuracy of the results are discussed based on the analysis of individual error sources, which leads to the conclusion that the precision of our measurements is most limited by the acquisition reproducibility, if the signal-to-noise ratio of the spectrum is high enough. Furthermore, we simulate the impact of radiation losses on the measured band gap value and propose a thickness-dependent correction. In future work, localized band gap variations will be measured on a more localized length scale to investigate, e.g., the influence of chemical inhomogeneities and dopant accumulations at grain boundaries.

A study by electron microscopy techniques of the structural and compositional properties of AlxGa1-xN/GaN nanowire (NW) heterostructures on Si(111) is presented. AlxGa1-xN depositions grown without catalyst by plasma-assisted molecular beam epitaxy were designed to form NWs in the range of 0.20<x<0.40 with different lengths and growth temperatures. The NWs exhibit a well-defined core-shell radial structure with a complex chemical distribution along and across the growth direction that finally affects the NW morphology. All the wires have an initial stage with a maximum Al content in the core slightly above the GaN/AlxGa1-xN interface, which initially decreases exponentially with the NW height depending on the nominal Al content and the growth temperature. In longer NWs, this trend changes and evolves increasing both the Al/Ga ratio and the core diameter as well as sharpening the shell. Adatom surface kinetic differences and the geometrical shadow effect during the growth are the probable drivers of this behavior.

In GaAs-based pseudomorphic high-electron mobility transistor device structures, strain and composition of the InxGa1−xAs channel layer are very important as they influence the electronic properties of these devices. In this context, transmission electron microscopy techniques such as (002) dark-field imaging, high-resolution transmission electron microscopy (HRTEM) imaging, scanning transmission electron microscopy-high angle annular dark field (STEM-HAADF) imaging and selected area diffraction, are useful. A quantitative comparative study using these techniques is relevant for assessing the merits and limitations of the respective techniques. In this article, we have investigated strain and composition of the InxGa1−xAs layer with the mentioned techniques and compared the results. The HRTEM images were investigated with strain state analysis. The indium content in this layer was quantified by HAADF imaging and correlated with STEM simulations. The studies showed that the InxGa1−xAs channel layer was pseudomorphically grown leading to tetragonal strain along the [001] growth direction and that the average indium content (x) in the epilayer is ~0.12. We found consistency in the results obtained using various methods of analysis.

Silicon oxide used as an intermetal dielectric (IMD) incorporates oxide impurities during both its formation and subsequent processing to create vias in the IMD. Without a sufficient degassing of the IMD, oxide impurities released from the IMD during the physical vapor deposition (PVD) of the glue layer of the vias had led to an oxidation of the glue layer and eventual increase of the via resistances, which correlated with the O-to-Si atomic ratio of the IMD being ~10% excessive as verified by transmission electron microscopy (TEM) analysis. A vacuum bake of the IMD was subsequently implemented to enhance outgassing of the oxide impurities in the IMD before the glue layer deposition. The implementation successfully reduced the via resistances to an acceptable level.

Samples of Zn-21Al-2Cu alloy (Zinalco) that will be heavily deformed were prepared using five different manual mechanical metallographic methods. Samples were analyzed before tensile testing using the orientation imaging microscopy-electron backscatter diffraction (OIM-EBSD) technique. The effect of type and particle size during the final polishing stages for this material were studied in order to identify a method that produces a flat, damage free surface with a roughness of about 50 nm and clean from oxide layers, thereby producing diffraction patterns with high image quality (IQ) and adequate confidence indexes (CI). Our results show that final polishing with alumina and silica, as was previously suggested by other research groups for alloys that are difficult to prepare or alloys with low melting point, are not suitable for manual metallographic preparation of this alloy. Indexes of IQ and CI can be used to evaluate methods of metallographic preparation of samples studied using the OIM-EBSD technique.

Meso-scale structure in polymeric foams determines the mechanical properties of the material. Density variations, even more than variations in the anisotropic void structure, can greatly vary the compressive and tensile response of the material. With their diverse use as both a structural material and space filler, polyurethane (PU) foams are widely studied. In this manuscript, quantitative measures of the density and anisotropic structure are provided by using micro X-ray computed tomography (microCT) to better understand the results of mechanical testing. MicroCT illustrates the variation in the density, cell morphology, size, shape, and orientation in different regions in blown foam due to the velocity profile near the casting surface. “Interrupted” in situ imaging of the material during compression of these sub-regions indicates the pathways of the structural response to the mechanical load and the changes in cell morphology as a result. It is found that molded PU foam has a 6 mm thick “skin” of higher density and highly eccentric morphological structure that leads to wide variations in mechanical performance depending upon sampling location. This comparison is necessary to understand the mechanical performance of the anisotropic structure.

The orientation of fibers in assemblies such as nonwovens has a major influence on the anisotropy of properties of the bulk structure and is strongly influenced by the processes used to manufacture the fabric. To build a detailed understanding of a fabric’s geometry and architecture it is important that fiber orientation in three dimensions is evaluated since out-of-plane orientations may also contribute to the physical properties of the fabric. In this study, a technique for measuring fiber segment orientation as proposed by Eberhardt and Clarke is implemented and experimentally studied based on analysis of X-ray computed microtomographic data. Fiber segment orientation distributions were extracted from volumetric X-ray microtomography data sets of hydroentangled nonwoven fabrics manufactured from parallel-laid, cross-laid, and air-laid webs. Spherical coordinates represented the orientation of individual fibers. Physical testing of the samples by means of zero-span tensile testing and z-directional tensile testing was employed to compare with the computed results.

High quality fixation often inactivates epitopes and gentler fixation can fail to preserve biological structure at the required resolution. For studies of male reproduction, immunofluorescence techniques using paraformaldehyde fixation associated with paraffin as an embedding medium gives good epitope preservation, although the cell becomes morphologically compromised. On the other hand, glutaraldehyde associated with a plastic resin has been used with success to recognize and distinguish each spermatogonial cell subtype, but the antigenic sites become inaccessible to antibodies. Here we describe a new method that provides excellent morphological details of testicular cells while preserving the binding capacity of epitopes. Using a combination of glutaraldehyde and paraformaldehyde as a fixative and LR White resin for embedding, we show that it is possible to clearly recognize spermatogonial subtypes (Aund, A–A4, In and B spermatogonia) on 1-μm thick-sections and to label epitopes such as bromodeoxyuridine, a marker used for cellular cycle studies in the testis. The information gained from this procedure can be critical for understanding spermatogonial process of self-renewal and differentiation.

Since semiconductor devices are being scaled down to dimensions of several nanometers there is a growing need for techniques capable of quantitative analysis of dopant concentrations at the nanometer scale in all three dimensions. Imaging dopant contrast by scanning electron microscopy (SEM) is a very promising method, but many unresolved issues hinder its routine application for device analysis, especially in cases of buried layers where site-specific sample preparation is challenging. Here, we report on optimization of site-specific sample preparation by the focused Ga ion beam (FIB) technique that provides improved dopant contrast in SEM. Similar to FIB lamella preparation for transmission electron microscopy, a polishing sequence with decreasing ion energy is necessary to minimize the thickness of the electronically dead layer. We have achieved contrast values comparable to the cleaved sample, being able to detect dopant concentrations down to 1×1016 cm−3. A theoretical model shows that the electronically dead layer corresponds to an amorphized Si layer formed during ion beam polishing. Our results also demonstrate that contamination issues are significantly suppressed for FIB-treated samples compared with cleaved ones.

Closed form analytical equations used to calculate the collection solid angle of six common geometries of solid-state X-ray detectors in scanning and scanning/transmission analytical electron microscopy are presented. Using these formulae one can make realistic comparisons of the merits of the different detector geometries in modern electron column instruments. This work updates earlier formulations and adds new detector configurations.