It is well established that the response of devices based on the giant magnetoresistance (GMR) effect depends critically on film microstructure, with parameters such as interfacial abruptness, the roughness and waviness of the layers, and grain size being crucial. Such devices have applications in information storage systems, and are therefore of great technological interest as well as being of fundamental scientific interest. The layers must be studied at high spatial resolution if the microstructural parameters are to be characterized with sufficient detail to enable the effects of fabrication conditions on properties to be understood, and the techniques of high resolution electron microscopy, transmission electron microscopy chemical mapping, and atom probe microanalysis are ideally suited. This article describes the application of these techniques to a range of materials including spin valves, spin tunnel junctions, and GMR multilayers.

A technique is proposed for reducing unwanted diffraction contrast when imaging second phases in crystalline materials using transmission electron microscopy. With the suggested name of plasmon-ratio imaging, the technique uses an energy-filtered imaging system to record and determine a ratio for two images taken at energies in the low loss region. Unlike core-loss imaging, the use of very thin specimens is not required. It is concluded that it is often possible to create a ratio image in which the contrast is dominated by energy loss, that is, chemical differences, rather than by diffraction effects.

The protozoan parasite Toxoplasma gondii is a representative of apicomplexan parasites that invades host cells through an unconventional motility mechanism. During host cell invasion it forms a specialized membrane-surrounded compartment that is called the parasitophorous vacuole. The interactions between the host cell and parasite membranes are complex and recent studies have revealed in more detail that both the host cell and the parasite membrane contribute to the formation of the parasitophorous vacuole. By using our a new specimen preparation technique that allows three-dimensional imaging of thick-sectioned internal cell structures with high-resolution, low-voltage field emission scanning electron microscopy, we were able to visualize continuous structural interactions of the host cell membrane with the parasite within the parasitophorous vacuole. Fibrous and tubular material extends from the host cell membrane and is connected to parasite membrane components. Shorter protrusions are also elaborated from the parasite. Several of these shorter fine protrusions connect to the fibrous material of the host cell membrane. The elaborate network may be used for modifications of the parasitophorous vacuole membrane that will allow utilization of nutrients from the host cell by the parisite while it is being protected from host cell attacks. The structural interactions between parasite and host cells undergo time-dependent changes, and a fission pore is the most prominent structure left connecting the parasite with the host cell. The fission pore is anchored in the host cell by thick structural components of unknown nature. The new information gained with this technique includes structural details of fibrous and tubular material that is continuous between the parasite and host cell and can be imaged in three dimensions. We present this technique as a tool to investigate more fully the complex structural interactions of the host cell and the parasite residing in the parasitophorous vacuole.

Cryptosporidium parvum (Apicomplexa, formerly Sporozoa) is the causative agent of cryptosporidiosis, an enteric disease of substantial medical and veterinary importance. C. parvum shows a number of unique features that differ from the rest of the class of coccidea in which it is currently grouped taxonomically. Differences occur in the overall structure of the transmission form and the invasive stages of the parasite, its intracellular location, the presence of recently described additional extracellular stages, the host range and target cell tropism, the ability to autoinfection, the nonresponsiveness to anticoccidial drugs, the immune response of the host, and immunochemical and genetic characteristics. These differences have an important impact on the infectivity, the epidemiology, the therapy, and the taxonomy of the parasite. The present article describes the structural analysis of the parasite using light and electron microscopy with an emphasis on structural details unique to C. parvum.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 μm wide and up to 10 μm long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.Abbreviations: EM: electron microscope; L/P: lactate/pyruvate; QT: QuickTime; CCCP: carbonyl cyanide 3-chloro-phenylhydrazone; CMXRos, chloromethyl-X-rosamine; Rh123, Rhodamine 123; RTM, real time microscope

Along with rapidly developing nanotechnology, new types of analytical instruments and techniques are needed. Here we report an alternative procedure for electrical measurements on semiconductor nanowhiskers, allowing precise selection and visual control at close to atomic resolution. We use a combination of two powerful microscope techniques, scanning tunneling microscopy (STM) and simultaneous viewing in a transmission electron microscope (TEM). The STM is mounted in the sample holder for the TEM. We describe here a method for creating an ohmic contact between the STM tip and the nanowhisker. We examine three different types of STM tips and present a technique for cleaning the STM tip in situ. Measurements on 1-μm-tall and 40-nm-thick epitaxially grown InAs nanowhiskers show an ohmic contact and a resistance of down to 7 kΩ.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Extended abstract of a paper presented at the Pre-Meeting Congress: Materials Research in an Aberration-Free Environment, at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, July 31 and August 1, 2004.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

The first dedicated local electrode atom probes (LEAP [a trademark of Imago Scientific Instruments Corporation]) have been built and tested as commercial prototypes. Several key performance parameters have been markedly improved relative to conventional three-dimensional atom probe (3DAP) designs. The Imago LEAP can operate at a sustained data collection rate of 1 million atoms/minute. This is some 600 times faster than the next fastest atom probe and large images can be collected in less than 1 h that otherwise would take many days. The field of view of the Imago LEAP is about 40 times larger than conventional 3DAPs. This makes it possible to analyze regions that are about 100 nm diameter by 100 nm deep containing on the order of 50 to 100 million atoms with this instrument. Several example applications that illustrate the advantages of the LEAP for materials analysis are presented.

The influence of a lack of sufficient electrical conductivity on the results of quantitative electron probe microanalysis has been investigated on a number of oxides. The effect of surface charging and the way it alters the emitted X-ray signals has been studied. It is shown that the presence of conducting coatings, such as carbon or copper, will affect the interelement X-ray intensity ratios, whatever the thickness of the coating may be. Although the effects for heavier elements may be acceptable, they cannot be ignored for a light element such as oxygen, where strong variations with coating thickness were observed. Quantitative analyses of oxygen, on uncoated well-conducting oxide specimens, using uncoated well-conducting hematite (Fe2O3) as a standard yielded excellent results in the range between 4 and 40 kV with the φ(ρz) software used. As soon as coated nonconducting specimens were examined, using the same hematite standard, coated under exactly the same conditions, widely scattering and noncoherent results were obtained. These discrepancies can only be attributed to a lack of conductivity.

Committee Members

2004 Local Arrangements Committee

Energy dispersive X-ray spectrometry of uncoated insulators performed at low beam energy (incident energy ≤ 5 keV) and in the variable pressure scanning electron microscope and the environmental scanning electron microscope is subject to spectral artifacts. Charging decelerates the incident beam electrons and reduces the impact energy, lowering the available overvoltage to excite characteristic X-ray peaks. The Duane–Hunt limit of the X-ray bremsstrahlung continuum is commonly used as a diagnostic of charging. Dynamic charging effects can hide the true impact of charging on the X-ray spectrum. Careful examination of the behavior of the X-ray spectrum with time and other variables is needed to avoid spectral artifacts, particularly on relative X-ray intensities.

Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.

A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.7–3.9 Å are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7 Å. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0 Å. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

A study of screw dislocations in hydride-vapor-phase-epitaxy (HVPE) template and molecular-beam-epitaxy (MBE) overlayers was performed using transmission electron microscopy (TEM) in plan view and in cross section. It was observed that screw dislocations in the HVPE layers were decorated by small voids arranged along the screw axis. However, no voids were observed along screw dislocations in MBE overlayers. This was true both for MBE samples grown under Ga-lean and Ga-rich conditions. Dislocation core structures have been studied in these samples in the plan-view configuration. These experiments were supported by image simulation using the most recent models. A direct reconstruction of the phase and amplitude of the scattered electron wave from a focal series of high-resolution images was applied. It was shown that the core structures of screw dislocations in the studied materials were filled. The filed dislocation cores in an MBE samples were stoichiometric. However, in HVPE materials, single atomic columns show substantial differences in intensities and might indicate the possibility of higher Ga concentration in the core than in the matrix. A much lower intensity of the atomic column at the tip of the void was observed. This might suggest presence of lighter elements, such as oxygen, responsible for their formation.

Extended abstract of a paper presented at the Pre-Meeting Congress: Materials Research in an Aberration-Free Environment, at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, July 31 and August 1, 2004.

A numerical model has been developed to simulate images obtained from the three-dimensional atom probe. This model was used to simulate the artefacts commonly observed in two-phase materials. This model takes into account the dynamic evolution of the atomic-scale shape of the specimen during field evaporation. This article reviews the model and its applications to some specific cases. Local magnification effects were studied as a function of the size, the shape, and the orientation of precipitated phases embedded in the matrix. Small precipitates produce large aberrations in good agreement with experiments. The magnification from such precipitates, as measured from the simulation, is only found to match the theoretical value for mesoscopic scale precipitates (size similar to the specimen size). Orientation effects are also observed in excellent agreement with experiments. The measured thickness of a grain-boundary-segregated film in the simulation is found to decrease with the angle between the normal to the grain boundary and the tip axis. Depth scaling artefacts caused by variation in the evaporation field of atoms in multilayer structures were successfully simulated and again showed good agreement with effects observed experimentally.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.