Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Symposia categories include: Advances in Instrumentation and Techniques, Biological Sciences, and Physical Sciences.

The measurement of near-infrared (NIR) cathodoluminescence (CL) with sufficient sensitivity to allow full spectral mapping has been investigated through the application of optimized grating spectrometers that allow the ultraviolet (UV), visible, and NIR CL spectra to be measured simultaneously. Two optical spectrometers have been integrated into an electron microprobe, allowing simultaneous collection of hyperspectral CL (UV-NIR), characteristic X-rays, and electron signals. Combined hyperspectral CL spectra collected from two natural apatite (Ca5[PO4]3[OH,F]) samples from Wilberforce (Ontario, Canada) and Durango (Mexico) were qualitatively analyzed to identify the emission centers and then deconvoluted pixel-by-pixel using least-squares fitting to produce a series of ion-resolved CL intensity maps. Preliminary investigation of apatite has shown strong NIR emissions associated primarily with the rare-earth element Nd. Details of growth and alteration were revealed in the NIR that were not discernable with electron-induced X-ray mapping. Intense emission centers from Nd3+ and Sm3+ were observed in the spectra from both apatites, along with minor emissions from other 3+ rare-earth elements. Quantitative electron probe microanalysis was performed on points within the mapped area of the Durango apatite to produce a calibration line relating cathodoluminescent intensity of the fitted peak centered at 1,073 nm (1.156 eV) to the Nd concentration.

Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350® 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist®; group 3, Listerine®; group 4, Colgate Plax®; LED LCU, group 5, saliva; group 6, Pepsi Twist®; group 7, Listerine®; group 8, Colgate Plax®. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist® groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite.

Tocotrienols are isomers of the vitamin E family, which have been reported to exert cytotoxic effects in various cancer cells. Although there have been some reports on the effects of tocotrienols in leukemic cells, ultrastructural evidence of tocotrienol-induced apoptotic cell death in leukemic cells is lacking. The present study investigated the effects of three isomers of tocotrienols (alpha, delta, and gamma) on a human T lymphoblastic leukemic cell line (CEM-SS). Cell viability assays showed that all three isomers had cytotoxic effects (p < 0.05) on CEM-SS cells with delta-tocotrienol being the most potent. Transmission electron microscopy showed that the cytotoxic effects by delta- and gamma-tocotrienols were through the induction of an apoptotic pathway as demonstrated by the classical ultrastructural apoptotic changes characterized by peripheral nuclear chromatin condensation and nuclear fragmentation. These findings were confirmed biochemically by the demonstration of phosphatidylserine externalization via flow cytometry analysis. This is the first study showing classical ultrastructural apoptotic changes induced by delta- and gamma-tocotrienols in human T lymphoblastic leukemic cells.

Diabetes type 1 is a very common chronic disease that frequently initiates in youth. Patients suffering from this disease have an increased risk of developing complications such as cardiovascular disease or erectile dysfunction. This is partially due to oxidative stress in affected cells, induced by the increasing production of reactive oxygen species (ROS) that, in addition to oxidative macromolecular damage, inactivate nitric oxide (NO). This condition strongly affects blood vessels of corpus cavernosum (CC), decreasing erectile capability of penis. Thus, we hypothesise that treatment with antioxidants present in the green tea in the initial phase of diabetes type 1 may protect CC from structural and molecular changes induced by oxidative damage. In support of this hypothesis a previous study of our group demonstrated that green tea long-term ingestion led to decrease in lipid perivascular deposition and vascular endothelial growth factor (VEGF) and its receptor VEGFR 2 expression in CC of aged rats. VEGF is the main angiogenic factor in tissues that induces proliferation and survival of endothelial cells after binding to VEGFR2. In fact, VEGF/VEGFR2 system is involved in both physiological and pathological angiogenic processes, and most of the molecular changes observed in aged CC derive from oxidative stress coupled to impairment of angiogenic mechanisms. VEGF also engage VEGFR1, nevertheless the exact role of VEGFR1 activation in cavernous tissue is not yet clarified.

The use of transgenic plants for the large scale production of recombinant proteins with commercial and therapeutic value has emerged as an alternative to conventional platforms. Plant based systems, including whole plants and plant cell cultures offer many advantages particularly regarding safety and cost effectiveness. In our laboratory we have been using the model plant Medicago truncatula as a system to express recombinant proteins with a variety of applications.

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The TEAM 0.5 electron microscope is employed to demonstrate atomic resolution phase contrast imaging and focal series reconstruction with acceleration voltages between 20 and 300 kV and a variable dose rate. A monochromator with an energy spread of ≤0.1 eV is used for dose variation by a factor of 1,000 and to provide a beam-limiting aperture. The sub-Ångstrøm performance of the instrument remains uncompromised. Using samples obtained from silicon wafers by chemical etching, the [200] atom dumbbell distance of 1.36 Å can be resolved in single images and reconstructed exit wave functions at 300, 80, and 50 kV. At 20 kV, atomic resolution <2 Å is readily available but limited by residual lens aberrations at large scattering angles. Exit wave functions reconstructed from images recorded under low dose rate conditions show sharper atom peaks as compared to high dose rate. The observed dose rate dependence of the signal is explained by a reduction of beam-induced atom displacements. If a combined sample and instrument instability is considered, the experimental image contrast can be matched quantitatively to simulations. The described development allows for atomic resolution transmission electron microscopy of interfaces between soft and hard materials over a wide range of voltages and electron doses.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

In our study we investigated the relationship between conventional morphometric indicators of nuclear size and shape (area and circularity) and the parameters of gray level co-occurrence matrix texture analysis (entropy, homogeneity, and angular second moment) in cells committed to apoptosis. A total of 432 lymphocyte nuclei images from the spleen germinal center light zones (cells in early stages of apoptosis) were obtained from eight healthy male guinea pigs previously immunized with sheep red blood cells (antigen). For each nucleus, area, circularity, entropy, homogeneity, and angular second moment were determined. All measured parameters of gray level co-occurrence matrix (GLCM) were significantly correlated with morphometric indicators of nuclear size and shape. The strongest correlation was observed between GLCM homogeneity and nuclear area (p < 0.0001, rs = 0.61). Angular second moment values were also highly significantly correlated with nuclear area (rs = 0.39, p < 0.0001). These results indicate that the GLCM method may be a powerful tool in evaluation of ultrastructural nuclear changes during early stages of the apoptotic process.

The microstructure of Ti-35Nb-7.2Zr-5.7Ta (TNZT) and Ti-35Nb-7.2Zr-5.7Ta-0.5B (TNZTB) alloys under different heat treatment conditions has been analyzed. The solution-treated and water-quenched TNZT sample consists mainly of β phase with a very small amount of fine athermal ω precipitate. Precipitation of α can be observed when solution-treated samples are directly aged at 580°C for 8 h. The microstructure of the samples subjected to single-stage aging at 300°C or 400°C consists of ω precipitates in equiaxed β grains. Second stage aging at 580°C for 8 h after first stage of aging at 300°C or 400°C results in the replacement of ω precipitates by secondary α. In all of these samples, the amount of ω or α phase was very small, and therefore they could not be detected by X-ray diffraction studies. However, analysis of selected area diffraction patterns obtained from transmission electron microscopy studies confirms their presence. The addition of boron leads to the formation of dispersed precipitates of TiB in the β matrix of the TNZT alloy and also refines the β grains in the microstructure. However, other microstructural features of the TNZTB alloy are similar to those of the TNZT alloy.

Previously, imaging and analysis with cathodoluminescence (CL) detectors required using high accelerating voltages. Utilization of lower accelerating voltage for microanalysis has the advantages of reduced beam-specimen interaction volume, and thus better spatial resolution, as well as reduction in electron beam induced damage. This article will highlight recent developments in field emission gun–scanning electron microscope technology that have allowed acquisition of high spatial resolution CL images at very low accelerating voltages. The advantages of low kV CL imaging will be shown using examples of a geological specimen (shale) and a specimen of an industrial grade diamond.

Extracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors—platelet derived growth factor-BB and heparin binding-epidermal growth factor—are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formation in vitro and in vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.

The increasing use of environmental transmission electron microscopy (ETEM) in materials science provides exciting new possibilities for investigating chemical reactions and understanding both the interaction of fast electrons with gas molecules and the effect of the presence of gas on high-resolution imaging. A gaseous atmosphere in the pole-piece gap of the objective lens of the microscope alters both the incoming electron wave prior to interaction with the sample and the outgoing wave below the sample. Whereas conventional TEM samples are usually thin (below 100 nm), the gas in the environmental cell fills the entire gap between the pole pieces and is thus not spatially localized. By using an FEI Titan environmental transmission electron microscope equipped with a monochromator and an aberration corrector on the objective lens, we have investigated the effects on imaging and spectroscopy caused by the presence of the gas.

Ionoluminescence (IL) is the emission of light from a material due to excitation by an ion beam. In this work, a helium ion microscope (HIM) has been used in conjunction with a luminescence detection system to characterize IL from materials in an analogous way to how cathodoluminescence (CL) is characterized in a scanning electron microscope (SEM). A survey of the helium ion beam induced IL characteristics, including images and spectra, of a variety of materials known to exhibit CL in an SEM is presented. Direct band-gap semiconductors that luminesce strongly in the SEM are found not do so in the HIM, possibly due to defect-related nonradiative pathways created by the ion beam. Other materials do, however, exhibit IL, including a cerium-doped garnet sample, quantum dots, and rare-earth doped LaPO4 nanocrystals. These emissions are a result of transitions between f electron states or transitions across size dependent band gaps. In all these samples, IL is found to decay with exposure to the beam, fitting well to double exponential functions. In an exploration of the potential of this technique for biological tagging applications, imaging with the IL emitted by rare-earth doped LaPO4 nanocrystals, simultaneously with secondary electron imaging, is demonstrated at a range of magnifications.

A new aberration-corrected scanning transmission electron microscope equipped with an array of Si-drift energy-dispersive X-ray spectrometers has been utilized to acquire spectral image data at atomic resolution. The resulting noisy data were subjected to multivariate statistical analysis to noise filter, remove an unwanted and partially overlapping non-sample-specific X-ray signal, and extract the relevant correlated X-ray signals (e.g., channels with L and K lines). As an example, the Y2Ti2O7 pyrochlore-structured oxide (assumed here to be ideal) was interrogated at the [011] projection. In addition to pure columns of Y and Ti, at this projection, there are also mixed 50-50 at. % Y-Ti columns. An attempt at atomic-resolution quantification is presented. The method proposed here is to subtract the non-column-specific signal from the elemental components and then quantify the data based upon an internally derived k-factor. However, a theoretical basis to predict this non-column-specific signal is needed to make this generally applicable.

Calculi in salivary glands and ducts (sialoliths) can be found in about 1% of the general population. Typically sialolithiasis causes long-term obstruction of salivary secretions, leading to atrophy of the gland with concomitant ceasing of the secretory function and ultimately fibrosis. Although some theories have been put forward regarding the etiology and pathogenesis of salivary calculi, the exact nucleation and growth mechanisms remain elusive. An exhaustive characterization of salivary calculi and systematic evaluation of the existing etiologic theories is therefore required.

In this study, changes produced in aorta and triceps surae muscle of Wistar rats as response to bee venom (BV) envenomation were analyzed by transmission electron microscopy and morphometry. A subchronic treatment of 30 days with daily doses of 700 μg BV/kg and an acute-lethal treatment with a single dose of 62 mg BV/kg were performed. The subchronic treatment resulted in endothelial cell retraction, a thicker subendothelial layer, and thinner elastic laminae and musculoelastic layers in aorta, and thicker endothelium and basal laminae in skeletal muscle. In both tissues polymorphous, swollen mitochondria with disrupted cristae were observed. The acute treatment produced extensive endothelial lesions, breakdown of the collagen layer and migration of muscle cells toward the intima in the aorta, and dilatation of endoplasmic reticulum in the skeletal muscle cells. Mitochondria were almost devoid of cristae or with few circular cristae in the smooth muscle cells while most of the mitochondria presented abnormal circular cristae in the skeletal muscle cells. Degenerative alterations in the aorta were of higher intensity in our experiments—both the intima and media strongly responded to BV, in contrast to those found at the level of the skeletal muscle cells where a moderate degenerative myopathy was recorded.