The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

ZrO2:Yb3+ nanocrystalline phosphors with high concentrations of ytterbium ions were prepared using the sol-gel method. X-ray diffraction, high-angle annular-dark-field scanning transmission electron microscopy (HAADF-STEM), energy dispersive X-ray spectroscopy, and high-resolution transmission electron microscopy (HRTEM) were used to characterize the nanocrystalline phosphors annealed at 1000°C. Unit-cell distortion and changes in the crystalline structure of the monoclinic zirconia to tetragonal zirconia, and subsequently cubic zirconia, were observed with increased Yb concentration. Yb ions were randomly distributed into the lattice of the crystalline structure. No segregation of Yb2O3 phase was observed. The substitution of Zr atoms by Yb atoms on different crystalline phases was confirmed by the experimental results and theoretical simulations of HRTEM and HAADF-STEM.

In this work we investigated the chemical composition and structure of (K0.5Na0.5)NbO3 (KNN) single crystals grown by the solid state crystal growth method. The optical, scanning, and transmission electron microscopies were employed for the analysis of the chemical homogeneity and domain structure of the KNN crystal. No compositional inhomogeneities within experimental error were encountered in the KNN single crystals. The domain structure of the KNN single crystal, with a monoclinic unit cell, is composed of large 90° domains of up to 100 μm width, which further consist of smaller 180° domains with widths from 50 to 300 nm.

We examine chemical mapping of reaction phases in a Cu-Al multilayer system using low-loss electron energy loss spectroscopy spectrum imaging and image spectroscopy techniques. The sensitivity of the plasmon peak position and shape to various crystal structures and phases is exploited using postprocessing of spectra into second derivative plasmon maps and line scans. Analytical transmission electron microscopy is complemented by studies of the orientation relationship of the multilayer system using high-resolution electron microscopy of interfaces and selected area diffraction. The techniques have been applied to the Cu-Al multilayer sample and sharply bound epitaxial phases are found, before and after heat treatment.

After a number of replications, human diploid fibroblasts (HDFs) in culture lose the ability to divide, become insensitive to further proliferation and enter a state of replicative senescence (RS). Subcytotoxic doses of several stressful agents such as hydrogen peroxide, tertbutylhydroperoxide or ethanol, are able to cause stress-induced premature senescence (SIPS) in HDFs in vitro. Such senescent cells display many features of RS as growth arrest, senescence associated beta-galactosidase (SA beta-gal), cell enlargement and overexpression of several genes (e.g., p21, TGF beta-1,IGFBP3). During ageing, iron accumulates in several tissues in vivo, and also in senescent HDFs in vitro. Due to its redox-active properties, it promotes hydroxyl radical production (Fenton reaction) and eventually leads to cell injury. Free radical reactions are known to cause the accumulation of intracellular damage resulting in ageing. Iron may thus be able to cause SIPS. The main objective of the present study was to investigate whether the exposure of HDFs to a subcytotoxic concentration of iron is able to cause SIPS.

A detector that looks promising for advanced imaging modalities—such as X-ray absorption contrast imaging, X-ray fluorescence imaging, and diffraction-enhanced imaging—is the controlled-drift detector (CDD). The CDD is a novel two-dimensional X-ray imager with energy resolving capability of spectroscopic quality. It is built on a fully depleted silicon wafer and features fast readout while being operated at or near room temperature. The use of CDDs in the aforementioned applications allows translating these techniques from synchrotron-based experiments to laboratory-size experiments using polychromatic X-ray generators. We have built a dedicated and versatile detection module based on a 36 mm2 CDD chip featuring pixels of 180 × 180 μm2, and we evaluated the system performance in different X-ray imaging applications both with synchrotron-based experiments and in the laboratory environment.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

The differential accumulation of fluorescent molecules in tumorigenic versus normal cells is a well-reported phenomenon and is the basis for photodiagnostic therapy. Through the use of confocal microscopy, the kinetic uptake and accumulation of fusarochromanone (FC101) was determined in two lines of living tumorigenic cells of mesenchymal-epithelial origin and normal fibroblast cells. Like other fluorescent cationic molecules, FC101 showed increased accumulation in tumorigenic cells; however, unlike other molecules, it appeared to be accumulated in a time-dependent manner. Also, unlike traditional fluorescent cationic molecules, FC101, a potent inhibitor of cell growth, showed preferential inhibition of tumorigenic B-16 melanoma cells and MCF7 cells derived from breast cancer adenocarcinoma when compared to normal cardiac fibroblasts. Further analysis of FC101's physicochemical properties using both experimentally obtained and simulated values revealed the likelihood of membrane permeation and oral bioavailability of the compound. These physicochemical properties of FC101 were also used to predict its intracellular localization lending credence to data observed by confocal microscopy.

All clinically-approved and many novel gadolinium (Gd)-based contrast agents used to enhance signal intensity in magnetic resonance imaging (MRI) are optically silent. To verify MRI results, a “gold standard” that can map and quantify Gd down to the parts per million (ppm) levels is required. Nuclear microscopy is a relatively new technique that has this capability and is composed of a combination of three ion beam techniques: scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle induced X-ray emission used in conjunction with a high energy proton microprobe. In this proof-of-concept study, we show that in diseased aortic vessel walls obtained at 2 and 4 h after intravenous injection of the myeloperoxidase-senstitive MRI agent, bis-5-hydroxytryptamide-diethylenetriamine-pentaacetate gadolinium, there was a time-dependant Gd clearance (2 h = 18.86 ppm, 4 h = 8.65 ppm). As expected, the control animal, injected with the clinically-approved conventional agent diethylenetriamine-pentaacetate gadolinium and sacrificed 1 week after injection, revealed no significant residual Gd in the tissue. Similar to known in vivo Gd pharmacokinetics, we found that Gd concentration dropped by a factor of 2 in vessel wall tissue in 1.64 h. Further high-resolution studies revealed that Gd was relatively uniformly distributed, consistent with random agent diffusion. We conclude that nuclear microscopy is potentially very useful for validation studies involving Gd-based magnetic resonance contrast agents.

The successful development of third-order aberration correctors in transmission electron microscopy has seen aberration-corrected electron microscopes evolve from specialist projects, custom built at a small number of sites to common instruments in many modern laboratories. Here we describe some initial results illustrating the two- and three-dimensional (3D) performance of an aberration-corrected scanning transmission electron microscope with a prototype improved aberration corrector designed to also minimize fifth-order aberrations and a new, higher brightness gun. We show that atomic columns separated by 0.63 Å can be resolved and demonstrate detection of single dopant atoms with 3D sensitivity.

The helium ion microscope is a unique imaging instrument. Based on an atomic level imaging system using the principle of field ion microscopy, the helium ion source has been shown to be incredibly stable and reliable, itself a remarkable engineering feat. Here we show that the image contrast is fundamentally different to other microscopes such as the scanning electron microscope (SEM), although showing many operational similarities due to the physical ion interaction mechanisms with the sample. Secondary electron images show enhanced surface contrast due the small surface interaction volume as well as elemental contrast differences, such as for nanowires imaged on a substrate. We present images of nanowires and nanoparticles for comparison with SEM imaging. Applications of Rutherford backscattered ion imaging as a unique and novel imaging mechanism are described. The advantages of the contrast mechanisms offered by this instrument for imaging nanomaterials are clearly apparent due to the high resolution and surface sensitivity afforded in the images. Future developments of the helium ion microscope should yield yet further improvements in imaging and provide a platform for continued advances in microscope science and nanoscale research.

The microstructure of a 55 μm diameter, cold-worked Ni-Ti microwire is investigated by different transmission electron microscopy techniques. The surface consists of a few hundred nanometer thick oxide layer composed of TiO and TiO2 with a small fraction of inhomogeneously distributed Ni. The interior of the wire has a core-shell structure with primarily B2 grains in the 1 μm thick shell, and heavily twinned B19′ martensite in the core. This core-shell structure can be explained by a concentration gradient of the alloying elements resulting in a structure separation due to the strong temperature dependence of the martensitic start temperature. Moreover, in between the B2 part of the metallic core-shell and the oxide layer, a Ni3Ti interfacial layer is detected.

Highly proliferative cancer cells maintain high rates of glycolysis, producing large amounts of acids, mainly lactic acid. Despite this fact, only the interstitial pH of tumours is low, while the intracellular pH of tumours is either normal or higher than that of normal tissues. Monocarboxylate transporters (MCTs) are transmembrane proteins that, by promoting the efflux of the accumulating acids, constitute one of the most important mechanisms in the maintenance of tumour intracellular pH. MCTs play a central role in cellular metabolism and are essential for the transport of metabolically important monocarboxylates, such as lactate, across the plasma membrane.

Nuclear microscopy is a suite of techniques based on a focused beam of MeV protons. These techniques have the unique ability to image density and structural variations in relatively thick tissue sections, map trace elements at the cellular level to the microgram per gram (dry weight) level, and extract quantitative information on these elements. The trace elemental studies can be carried out on unstained freeze-dried tissue sections, thereby minimizing any problems of contamination or redistribution of elements during conventional staining and fixing procedures. The pancreas is a gland with different specialized cells and a complex hormonal activity where trace elements play an important role. For example, zinc has an active role in insulin production, and calcium ions participate in the stimulation and secretion process of insulin. Using nuclear microscopy with a spatial resolution of 1 μm, we have located, using zinc mapping, the islets of Langerhans in freeze-dried normal rabbit tissue sections. The islets of Langerhans contain β-cells responsible for insulin production. Subsequent quantitative analyses have indicated elevations in most elements within the islets of Langerhans, and significantly so for the concentrations of Zn [3,300 compared to 90 μg/g (dry weight)] and Ca [1,100 compared to 390 μg/g (dry weight)].

Spectrum simulation is a useful practical and pedagogical tool. Particularly with complex samples or trace constituents, a simulation can help to understand the limits of the technique and the instrument parameters for the optimal measurement. DTSA-II, software for electron probe microanalysis, provides both easy to use and flexible tools for simulating common and less common sample geometries and materials. Analytical models based on ϕ(ρz) curves provide quick simulations of simple samples. Monte Carlo models based on electron and X-ray transport provide more sophisticated models of arbitrarily complex samples. DTSA-II provides a broad range of simulation tools in a framework with many different interchangeable physical models. In addition, DTSA-II provides tools for visualizing, comparing, manipulating, and quantifying simulated and measured spectra.

The International Technology Roadmap for Semiconductors ranks dopant profiling as one of the most difficult challenges for analysis of semiconductors. Dopant mapping in the scanning electron microscope (SEM) has the potential to provide a solution. This technique has not yet found widespread application, however, mainly due to the lack of a comprehensive theoretical model, uncertain quantification, and its inability to differentiate doping levels in n-type silicon. Although a Monte Carlo model was recently published that closely matched experimental data obtained in p-doped silicon to data obtained from the theoretical model, a large discrepancy between experimental data obtained for n-type silicon was found. Here we present a Monte Carlo model that provides close matches between experimental and calculated data in both n- and p-type silicon, paving the way for a widespread application of SEM dopant contrast.

The cusp epithelium is a specialized branch of the superior epithelium that surrounds the developing teeth of chitons and is responsible for delivering the elements required for the formation of biominerals within the major lateral teeth. These biominerals are deposited within specific regions of the tooth in sequence, making it possible to conduct a row by row examination of cell development in the cusp epithelium as the teeth progress from the unmineralized to the mineralized state. Cusp epithelium from the chiton Acanthopleura hirtosa was prepared using conventional chemical and microwave assisted tissue processing, for observation by light microscopy, conventional transmission electron microscopy (TEM) and energy filtered TEM. The onset of iron mineralization within the teeth, initiated at row 13, is associated with a number of dramatic changes in the ultrastructure of the apical cusp cell epithelium. Specifically, the presence of ferritin containing siderosomes, the position and number of mitochondria, and the structure of the cell microvilli are each linked to aspects of the mineralization process. These changes in tissue development are discussed in context with their influence over the physiological conditions within both the cells and extracellular compartment of the tooth at the onset of iron mineralization.

Richmond, the center of it all! … A wonderful place to visit, live, work, and raise a family is centrally located two hours, by car, from the Atlantic Ocean, Washington DC and the Blue Ridge Mountains and within two hours, by plane, to cities from New York and Atlanta. Central Virginia is home to so much of our nation's heritage; but yet, a contemporary city with a quality of life and cost of living you will quickly appreciate in today's economy. It is the “Gem of the James River”, with many parks and scenic greenways. Thank you, Microscopy Society of America, for choosing Richmond in 2009 as host city for Microscopy and Microanalysis Convention.

We have developed the focused ion beam (FIB) fold-out technique, for transmission electron microscopy (TEM) sample preparation in which there is no fine polishing or dimpling, thus saving turnaround time. It does not require a nanomanipulator yet is still site specific. The sample wafer is cut to shape, polished down, and then placed in a FIB system. A tab containing the area of interest is created by ion milling and then “folded out” from the bulk sample. This method also allows a plan-view of the sample by removing material below the wafer's surface film or device near the polished edge. In the final step, the sample is thinned to electron transparency, ready to be analyzed in the TEM. With both a cross section and plan-view, our technique gives microscopists a powerful tool in analyzing multiple zone axes in one TEM session. The nature of the polished sample edge also includes the ability to sample many areas, allowing the user to examine a very large device or sample. More importantly, this technique could make multiple site-specific e-beam transparent specimens in one polished sample, which is difficult to do when prepared by other methods.