Electron energy-loss spectroscopy (EELS) is now a standard tool to investigate the local chemistry and bonding of defects in solids. to first order, the energy thresholds of the ionization edges in EELS spectra are determined by the identity of the element [1], while small shifts are induced by different bonding coordination and charge states [2]. The shapes of ionization edges in EELS spectra reflect the local bonding environments. We present first-principles calculations that incorporate electron-hole interactions and are in excellent agreement with experimental data obtained with X-ray absorption spectroscopy (XAS) and EELS. The superior energy resolution in XAS spectra and the new calculations make a compelling case that core-hole effects dominate core-excitation edges in all of the materials investigated: Si, SiO2, MgO, SrTiO3 and SiC. These materials differ widely in their dielectric constants leading to the conclusion that core-hole effects dominate all core-electron excitation spectra in semiconductors and insulators.

The promise of advanced technological applications in optical and electronic devices has led to a significant recent research effort in the structure-property relationships of defects in GaN. in particular, the major scientific issues arise from the high density of threading dislocations induced during thin film growth by film-substrate lattice mismatch. There is still debate as to the exact effect of these dislocations on the overall properties; they may or may not be electrically active and are thought to decrease the lifetime of devices. As such, a fundamental understanding of the electronic properties of these defects will facilitate the development of new and improved devices.

The analysis of the electronic structure of dislocations in GaN is performed here by a combination of atomic resolution electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) and multiple scattering (MS) simulations. Experimentally, a Z-contrast image of the dislocation core is obtained first1 and used to position the probe for EELS.

Electron loss near edge structure (ELNES) reflects transitions of inner shell electrons to site- and symmetry-projected unoccupied electronic states, which in a solid lie above the Fermi level and may be calculated using advanced electronic structure calculations. The direction of momentum transfer, q, imparted during the inelastic scattering process is dependent on the angle over which the scattered electrons are collected. It is usual to resolve the momentum transfer, into components parallel and perpendicular to the incident beam direction (q∥and q⊥). in a specimen region which is crystallographically anisotropic, the exact direction of the momentum transfer and hence energy states available to the excited electron will depend on the specimen orientation with respect to the incident electron beam. Practically, this can present problems where quantitative comparisons and/or modelling of electronic structure between different specimen regions at different orientations, or within a polycrystalline material, or even between different samples are required.

Introduction: Atomically resolved chemical analysis by means of electron energy (core-) loss spectroscopy has been postulated since the technical availability of highly focused sub-nm STEM beams. We study the feasibility of this approach, as compared to the alternative of spatial difference spectra, where interfacial resolution is achieved by subtracting boxes at and apart from the interface. A nanobeam EELS signal is generated by the dynamical propagation of a conical wave through the specimen. This wave acts as 3D-excitation envelope for inelastic scattering and is needed to link the ab-initio calculated atom- and angular-momentum-projected density-of-state (DOS) functions to measurable EELS signals.

Projected DOS: For the rhombohedral twin grain boundary in sapphire (FIG 1) as model system, DOS for p states in oxygen have been calculated 4 as described in , for the most stable twin structure obtained by ab-initio calculations.

In the last 6-7 years there has been considerable experimental and theoretical interest in GaN and its alloys for use in light emitting applications. GaN is a direct gap semiconductor with a bandgap at ∼ 3.4 eV. When alloyed with in and/or Al the bandgap can be tuned to any wavelength ranging from UV to red. to further the development of GaN based devices, reliable methods are required to measure and predict the optical and electronic properties. EELS is one of the few techniques that can study these properties with high spatial resolution (< 1 nm) if a small electron probe is used, such as that in a scanning transmission electron microscope (STEM). High spatial resolution is necessary as, to improve efficiency, devices are normally based on quantum well structures.

Gd3+ doped Ce oxides are very promising candidates as electrolytes for solid oxide fuel cells operating at ∼ 500 °C. For their successful commercial implementation, a full understanding of the defect chemistry in the bulk and at grain boundaries is essential. in particular, the contribution of the grain boundaries to the total ionic conductivity through such effects as the segregation of impurities, dopants and vacancies is of crucial importance. Here the effect of the atomic structure on the local electronic properties, i.e. oxygen coordination and cation valence at grain boundaries of the fluorite structured Gd0.2Ce0.8O2-x ceramic electrolyte is investigated by a combination of Z-contrast imaging and electron energy loss spectroscopy (EELS) in the JEOL 201 OF STEM (operating at 200keV, and aligned for a probe size ∼ 0.2 nm).

Preliminary O K- and Ce M45-edges were acquired from points in the grains (A and B) and grain boundary shown in Figure 1.

LiCoO2 is currently the preferred cathode material in secondary Li-ion batteries. Stoichiometric LiCoO2 has rhombohedral symmetry and belongs to the R3m space group. This layered structure has Li and Co ions ordered along (111) planes to form alternating cation layers. The Co3+ ion, located in octahedral sites, forms a strong bond with neighboring oxygen atoms to produce O-Co-O sheets. Li layers are sandwiched between these sheets of CoO2.

It has traditionally been accepted that in lithium metal oxides (LiMO2) the valence of the transition metal compensates for the charge on the intercalated Li. The theoretical boundaries for Li concentration in an electrode of LixCoO2 are 0≤x≤l. Using a simple ionic charge transfer model, the fully-lithiated material has ions with charges of Li+, Co3+, and O2-. in the CoO2 end member the ions have charges of Co4+ and O2- This simple model incorrectly leads to the conclusion that the oxygen valence is virtually unaffected by Li intercation.

Introduction

Fluid transport systems of organisms [1] in general and the blood vascular system in particular are considered to be optimally designed. From Murray's laws [2] it is concluded that an arterial bifurcation where the diameter of the parent vessel (d0) relates to the larger (d1) and the smaller daughter vessel (d2) according to d03 = d13 + d23 is optimal. Interestingly, existing data predominantly refer to arterial branchings of the fully developed circulatory system of mammals. to the best of our knowledge there are no data available on arterial bifurcations and venous mergings in an initially growing but then regressing tubular system of blood vessels as it is found in the gill filter apparatus of the anuran tadpole where the highly complex three-dimensional vascular network totally disappears at the end of metamorphosis.

Microwave-assisted processing of biological samples began with Mayers. Subsequently, many investigators reported using microwave ovens for various aspects of biological sample processing for light microscopy (LM) and/or transmission electron microscopy (TEM.) The use of ice-encased fixation and water-immersion resin polymerization marked the beginning of rapid, reproducible microwave-assisted processing techniques.

Mcrowave protocols are now often used for LM and TEM processing. Examples include paraffin embedding for LM, in situ hybridization, decalcification, immunological staining plus clinical studies and research projects for TEM.

Recently my lab has begun to microwave process biological samples for scanning electron microscopy (SEM.). We fix, dehydrate and dry with hexamethyldisilazane in less than 1 ½ hours. The only part of the process not utilizing microwave assist is the final 15 minute drying step in a conventional oven.

The history of microwave-assisted processing has been dominated by the idea that microwave heating was an integral part of the equation. The separation of a microwave component from the heating effects of the radiation during sample processing has been experimentally difficult. Combined with this difficulty has been the closed cavity design of microwave ovens. This design is typical of laboratory and household ovens and results in the formation of “hot” and “cold” spots within the chamber. These spots produce regions in close proximity to each other which have widely varying heating effects on samples.

A second factor to consider with microwave heating is the effect wattage output has on rate and extent of microwave induced heating. Peak wattage outputs of all laboratory and most household microwave ovens are in excess of 650W. As a result the vast majority of all microwave-assisted protocols are based on heating parameters associated with high wattage processing.

The investigation of connective tissue ultrastructure has historically relied on chemical fixation to stabilize micro architecture. These fixatives are not effective in retaining many matrix molecules, including proteoglycans, and allow precipitation of many other components in subsequent processing steps. The result is a preponderance of open space between matrix components. Cells shrink considerably and a precipitation of molecules in highly concentrated regions leads to artifacts including the lamina lucida and lamina densa of basement membranes. Cryo-fixation at high pressure followed by freeze substitution seeks to immobilize tissue components within vitreous (non-crystalline) ice, which is later substituted with acetone at temperatures below the recrystalization temperature of water. Formation of large ice crystals during any part of the preparation protocol significantly distorts structure and can be recognized in the compound microscope. Recognition of smaller freezing artifacts, including microcrystalline ice, is imperative for interpretation of resulting images.

The nematode C. elegans is a simple model for genetic studies of cell and tissue development. There is a need to improve the preservation of embryonic and early larval stages, during which nematode tissues elaborate and separate. However, these stages are particularly resistant to fixation and embedment due an impenetrable eggshell and larval cuticle. Their small size at these ages precludes mechanical cutting, which has been used successfully for immersion fixation of older stages. Here we compare the quality of preservation under three rather different regimes: using laserholes to permeabilize the eggshell during the primary fixation step, using microwave energy to enhance the first fixation step, or using fast freezing and freeze substitution to circumvent the standard immersion procedure. Vancoppenolle et al (2000) have recently demonstrated very good results through enzymatic weakening of the eggshell prior to immersion fixation. Their data are comparable to what we achieve by either the laserhole or microwave methods.

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the procedure necessary to detect the target structure of one technique may negatively affect the target of the other. Structural impairments may also limit the application of the two techniques. to overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on Chromosome spreads, intact cells and semi-thin sections of methyl methacrylate-embedded tissue. Microwavestimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for all techniques, yielding high sensitivity and excellent morphological preservation compared to conventional processing techniques. Co-localization of in situ hybridization and immunohistochemistry probes with high morphological resolution was achieved using computerized image reconstruction. The latter may allow for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level.

In efforts to better understand the cell biology of fungi and their pathogenic interactions with plants, we have pursued the use of cryo-preparative techniques for specimen preservation and immunocytochemistry at the light and electron microscope level. The chemically diverse composition of plant and fungal walls has proven very challenging for immunofluorescence using confocal microscopy. Typically, to allow probe access in these organisms, immunolabeling has required sectioning or enzymatic digestion of cell walls. The ideal method would preserve the spatial fidelity and antigenicity of the target molecule as well as maintain the integrity of the surrounding tissue. Our early work using methacrylate de-embedment, as described for sectioned plant material, was very effective for freeze-substituted fungi. Further improvements, using a combination of slow freezing and freeze-substitution followed by methacrylate de-embedment, permitted whole mount fluorescence antibody labeling of a variety of phylogenetically diverse fungi without enzymatic digestion or sectioning (Fig.l).

There are many strategies for documenting the distribution of gene products or other molecules within cells or tissues. Depending on the required information, techniques can range from cell fractionation to methods that employ electron microscopy: some preclude examination of living cells, and thus can not provide dynamic temporal information. Time sequence data are paramount when gene expression is transient, as is often the case during fungal-pathogen plant-host interactions. Fluorescent dyes and protein tags (e.g., green fluorescent protein, GFP) allow monitoring of specific molecules, organelles or cells over time, for simultaneous recording of both spatial and temporal information. in addition, fluorescent markers are amenable to powerful 3-D analysis using confocal or multi-photon imaging methods. We have achieved good expression of fluorescent proteins in the fungal blast pathogen Magnaporthe grisea, and have generated a functional fusion protein between GFP and P-tubulin (Figs. 1,2).

Resolving the true fine structure and behavior of soft bodied organisms such as arthropod larvae, acari, nematodes, annelids and pupae by conventional scanning electron microscopy has been difficult. Classical preparation techniques, involving immersion fixation, dehydration and critical point drying, cause significant deleterious changes in specimen structure due to effects of surface tension, osmotic stress and mechanical damage. The intact exoskeleton, cuticle or other limiting membranes of these organisms, creates a formidable obstacle to the penetration of fixatives and solvents, which are only effective after a long period of time (hours). During this time, behavioral postures and much of the structural integrity are lost and most organisms detach from their hosts.

In previous studies, our laboratory utilized methods in low temperature freeze stabilization, to preserve and observe the undisturbed fine structure of biological samples. Plunge freezing, although more rapid, resulted in many parasitic organisms dislodging from hosts and being lost.

Shadowing is widely used for biological macromolecules and their complexes structure determination . It has been shown that tungsten shadowing produces fine granularity and good structural detail resolution. We have developed a working prototype of an electron beam evaporator (“electron gun“) based on the principle described by V. Vasiliev. The electron gun has characteristics that allow us to deposit high quality films of carbon or even pure tungsten. in addition we have constructed a prototype sample handling apparatus for freeze drying and shadowing. The prototype allows tilting of +/- 90° in 2° increments, rotation (if desired) at 6 rpm, controlled heating at a rate of 1-2° per min, and cooling using a liquid nitrogen trap attached to the device. The system is vacuumed by 480 1/sec maglev turbomolecular pump with scroll pump at the first stage. It produces oil-free vacuum in 2-5*10-7 torr range, which we believe, is sufficient for good quality shadowing.

Classic studies of gill vasculature indicate the gross anatomy of elasmobranch gills is similar in most species, and in general, resembles those of teleosts. However, studies of fine gill structure examining arterio-arterial pathways in several species of batoid elasmobranchs (Order: Rajiiformes, Suborder: Myliobatoidei) have reported some structural differences. These differences include the presence, in urolophids (Urolophus jamaicensis, U. mucosus, U. paucimaculatus), of 1) a tip channel, possibly an extension of the afferent filament artery that runs along the top of the filament corpus cavernosum, and 2) a vascular arcade, a vessel which connects the afferent filament arteries of a hemibranch near the tip of each filament (FIG. I). Until recently these structures were believed to be unique to the urolophids as they were not previously found in either rajids (Raja erinacea, R. clavata) or the shark Scyliorhinus canicula. Animals of mixed species and sexes were collected, perfused, and acrylic casts made of the circulatory system.

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.

Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

In the early 1960s, concerns about artifacts in preparing biological material for electron microscopy led to a new technique whereby samples are rapidly frozen, fractured under high vacuum, the fractured surfaces shadowed and replicated with a thin metal-carbon coat, and the cleaned replica examined in a transmission electron microscope. Pioneered by Moor and Muhlethaler subcellular structures are revealed with extraordinary three-dimensional clarity at near-molecular resolution. Furthermore, it was observed and proven at model systems as well as biological membranes that lipid bilayers split along their hydrophobic interior during freeze-fracture procedure. Therefore, freeze-fracture electron microscopy (FFEM) has the unique advantage of accessing the hydrophobic interior of biological (FIG.l, 5 and 6) as well as artificial bilayers (FIG. 2, 4, 5, and 6). Here it permits study of pattern generated by intrinsic proteins as well as lipids (FIG. 1 and 2).