Since it was introduced by Faulk and Taylor in 1971, colloidal gold (cAu) has become the predominant marker used for EM cell labeling. Double labeling, using cAu of two distinct sizes, was pioneered a decade later. However, as interest continues to grow of how the myriad proteins, enzymes, receptors, and other macromolecules function with respect to one another in the cellular environment, this method has proven largely inadequate for high resolution, multiple labeling because each cAu label must be of sufficiently distinct size to prevent size overlap and confusion between different labels. As the particle size increases, so does steric hindrance between different labels competing for binding sites, while the spatial resolution of the labeling and overall labeling efficiency decrease. In order to address these concerns and surmount the limitations of current labeling methodologies, we have developed colloidal particles of uniform size from various metals including Ag, Pt, Pd, Rh, Ru, Ni, and Cu, in addition to Au.

Energy filtered transmission electron microscopy has the potential to provide high resolution, spatially resolved, atomic and chemical information. However, aberrations generated by the electron spectrometer blur the energy resolution and limit the atomic or molecular distributions that can be studied. Energy absorptions corresponding to the visible light range fall below an energy loss of 5 eV. The selection of electrons that have lost an amount of energy corresponding to chromophore absorption by the sample thus requires a spectrometer with a high energy resolution over the full image plane. A corrected prism-mirror-prism filter that has a resolution of 1.1 eV, sufficient to select these low energy loss electrons, was developed and installed by us in a Zeiss EM902. Its imaging capability was verified for a number of different chromophores. The chromophore currently under study is that of the green fluorescent protein (GFP).

Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, AU55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved.

Immunocytochemistry generally refers to methods directed toward obtaining information on the in situ distribution of antigens in cells and tissues. Immunocytochemical methods can be applied at either the light or electron microscope levels, or both in concert. The detection of antibody recognition of an antigen (i.e., localization of an antigen) relies upon a reporter system. At the light microscope level, enzymes (e.g., horseradish peroxidase) or fluorochromes are the most widely used reporters in immunocytochemistry. At the electron microscope level, particulate probes (e.g., colloidal gold) are the most widely used reporters. However, enzymes and even fluorochromes can be used at the EM level.

In this abstract, we discuss our use of gold cluster immunoprobes as the reporter system in both light and electron microscope level immuncytochemistry. These gold cluster immunoprobes, are commercially known as NanogoldTM (NG). These probes are very small with a diameter of 1.4-nm.

Immunocytochemistry provides unique information concerning the spatial and temporal distribution of biomolecules in cells and tissues. While techniques associated with immunocytochemical labeling are well developed, there are always improvements that can be achieved. In this abstract, a modification to immunocytochemical methodology that increases the labeling efficiency for certain antigens is described. This modification includes incubation of the sample with sodium dodecyl sulfate (SDS) prior to application of the primary antibody.

Tubulin in microtubules of human neutrophils has been extremely difficult to analyze with antibodies to α-tubulin or β-tubulin using immunocytochemical methods. Using an immunofluorescence assay, we have analyzed several preparative procedures in order to improve the labeling efficiency of these microtubules using anti-tubulin antibodies. We find that treatment of chemically fixed neutrophils with the chaotropic agent SDS improves labeling efficiency for neutrophil microtubules in the immunofluorescence assay.

Since skin basement membrane protein components are labile to conventional chemical fixation and since skin is not amenable to vibratome sectioning, frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slidemounting, usually limit study to the light microscopic level. In the present study, we introduce the use of unfixed, free-floating cryostat sections to characterize immunolocalizations of selected basement membrane protein components at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy.

In cell culture models various and distinct manipulations can be performed in a defined environment. This makes cell cultures very popular for immunohistochemical studies. Such studies are mostly performed at the light microscopic level where fluorescent probes and confocal microscopy provide detailed insights into the distribution and localization of antigens inside cells. In many such cases further studies at the electron microscopic level would give additional information and an often more detailed view. As compared to light microscopy not only is the resolution much higher, but also and even more importantly labeling information is completed with a wealth of cytological details.

This said it is surprising that studies on cell cultures using immunoelectron microscopy are rarely seen. Even more rare are reports in which monolayer cells are fixed and processed in situ without prior trypsinization. Such studies are essential though when cytoarchitecture is of importance or especially when cell-cell adhesion is an issue.

Biochemical studies have documented a role for oxidative stress in complications of diabetes. Our previous cytochemical and immunocytochemical studies demonstrated increased levels of NADH oxidase in retinal endothelial cells and pericytes of the BBZ/Wor diabetic rat which coincided with disruption of the blood retinal barrier. Lipid peroxidation and damage to outer segments was demonstrated in retinas of a streptozotocin-induced rat model of diabetes. Xanthine oxidase has been proposed as a source of superoxide and hydrogen peroxide (H2O2) in oxidative stress mediated complications of diabetes; however, there are no studies which confirm the role and cellular sites of xanthine oxidase in complications of diabetes. In addition, cellular sites of lipid peroxides have not been documented in diabetic retinopathy.

Xanthine oxidase was localized in retinas of BBZ/Wor diabetic rat by cerium based histochemistry using hypoxanthine and NADH as substrates.

In life sciences, the essential part of the functional molecular analysis is the unambiguous identification of biochemical composition of the observed structures. This analysis is expected to create a bridge between functional data pouring from biochemistry and molecular biology laboratories with the molecular architecture data available from ultrastructural images. This goal can be attained by application of various ultrastructural tags (See: Albrecht et al. 1992).

A new promising approach for high fidelity labeling is offered by molecular cloning and expression of molecules containing metal binding sites making them suitable for electron spectroscopic imaging (ESI) (Malecki 1995). A truly enormous potential of ESI relies in its ability for mapping of various elements within the same sample. Interactions of electrons with an atom result in the electrons specific energy loss. Based upon these energy losses distribution of the elements within the sample can be mapped.

Proeutectoid ferrite (bcc α) nucleation on the inclusions and precipitates dispersed in austenite (fee γ), denoted as intragranular ferrite transformation, has been used to refine the microstructure in the heat affected zone to improve the toughness of low and medium carbon steels. It is currently considered that Bl(NaCl)-type complex inclusions/precipitates (e.g. oxide, sulfide, carbide, nitride) are promising ones as ferrite nucleation sites. The present study aims to study the ability of various complex inclusions/precipitates in austenite as nucleation sites in proeutectoid ferrite transformations by standard and analytical electron microscopy.

Table 1 shows the chemical composition of Fe-2Mn-0.2C hypoeutectoid steels with various alloying element additions to change the inclusions/precipitate in austenite. Steel A contains a large density of MnS with the sulfur addition of 470ppm. Steels B and C also contain similar dispersion of MnS as Steel A.

The proeutectoid precipitation of Widmanstatten cementite has recently been shown to occur in two morphologies, either as broad monolithic plates or as aggregates of narrow laths, in a Fe-13 wt% Mn-1.3 wt% C steel, see Figure 1. Each of these morphologies correlates to a specific orientation of the cementite crystal within the austenite matrix; precipitates with a Pitsch orientation relationship adopt the monolithic plate morphology while precipitates with a Farooque-Edmonds orientation relationship develop as laths. In this paper, we examine the morphology and interfacial structure of these Widmanstatten cementite laths.

The typical three-dimensional morphology of Widmanstatten cementite laths can be revealed by etching with a 10% nitric acid solution in methanol. This procedure selectively etches the austenite matrix without affecting the cementite precipitates (see Figure 1). By similarly etching an electropolished thin foil TEM sample, the three-dimensional morphology and internal structure of entire electron transparent cementite precipitates can be examined.

For over five decades considerable attention has been given to magnesium-based alloys, primarily from the physical metallurgy point of view. The driving force for this effort has been the potential applications of Mg alloys in light weight structural components, namely in the automotive and aircraft industries. However, the processing of these alloys through conventional casting technologies have not succeed in reaching a high level of formability and ductility while preserving high specific strength. The combination of rapid solidification and consolidation techniques has arisen as a promising approach to overcome these limitations[l,2]. On the other hand, rapid solidification technologies are opening a wide field for fundamental studies, since different sets of thermodynamic (stability), kinetic and structural conditions are established to control a variety of solid-state phase transformations. The present work reports the applications of electron microscopy techniques to the characterization of the precipitation behavior in the rapidly solidified Mg-based alloys we are investigating.

Coarsening of particles involves a reduction of the total energy of the system. In fluids, the tendency for larger particles to grow at the expense of smaller ones is driven by the reduction of the total surface energy. In solids, the elastic strain energy Eel also needs to be considered. Reduction of Eel gives rise to particle alignment along elastically soft directions and changes of morphology as the particle volume increases. In addition according to several authors, reduction of Eel produces the splitting of particles that reach a critical size. Regular arrays of two, four and eight particles have been observed and explained in terms of splitting. However, splitting is unlikely from a micromechanical standpoint or as a consequence of the elastic interaction between particles. This investigation deals with the determination of translation order domains in coherent γ′ particles.

The introduction of “clean steels” was thought to have cured the problem of temper embrittlement in low alloy steels. However, even these steels exhibit temper embrittlement at services temperature above 400°C. Initial studies of temper embrittlement of clean steels used Auger electron spectrometry of fracture surfaces. The failure was found to occur along the prior austenite grain boundaries (PAGBs) where P segregation was found. It has been reported that the concentration of segregants changes along individual PAGB facets, as well as between the facets of different PAGBs. It was proposed that different elements segregate to different PAGBs, depending on their orientation. Differences in segregation along individual PAGBs were attributed to precipitation during thermal exposure, as identified by AEM. The cause of temper embrittlement is still unknown, as there are doubts about the role of precipitation. This implies that each PAGB may have a different chemistry.

The characterization of the microstructure evolution during welding of nickel base superalloys is required for efficient reuse and reclamation of used and failed components. Previous atom probe analysis of electron-beam and laser-beam welds revealed complex alloying elemental partitioning between the γ and γ phases. Rapid cooling conditions in the weld leads to non-equilibrium partitioning and large amplitude Cr and Co levels in the γ phase. These results indicated that there is a strong relationship between weld cooling rate and the precipitation of γ′ precipitates from the γ phase. To understand and develop predictive models, a systematic investigation of the microstructure evolution in CM247DS alloy under controlled thermomechanical conditions are being performed. This paper describes some recent results on the elemental partitioning between γ and γ′ phases obtained with atom probe microanalysis.

Casting of high temperature austenitic alloys is often used to form components and structures required in the chemical industry. Alloy HP is a Nb-stabilized austenitic alloy for such applications. High carbon levels are selected in order to drive the formation of coarse, intergranular precipitates of various carbides. These precipitates provide resistance to high temperature creep by inhibiting grain boundary sliding. While these precipitates are present in the cast material prior to high temperature exposure, it is the stability of these second phase particles during ageing that determines the long-term creep resistance and lifetime of stressed components. This study deals with the phase distribution in a centrifugally-cast HP component from a steam superheater tube in a styrene furnace, which experienced temperatures from 927°C to 1066°C or more for over 105,000 h.

Materials for high-temperature application, e.g. in gas-turbine compressor discs or steam turbine rotors, are often susceptible to microstructural instabilities during long-term service. Such instabilities reduce the service life due to a deterioration of the mechanical properties. Therefore, it is most important to identify such instabilities, which often take place in sub-micrometer scale. A class of materials widely used in power generating industries are the martensitic 9-12% Cr steels. In the present work STEM-EDX mappings were used to reveal the distribution of alloying elements in 9-12% Cr steels with a martensitic-austenitic microstructure developed recently.

The chemical composition of the material investigated was Fe-10.1Cr-6.2Co-5.2Mn-2.1Ni-1.2Mo-0.47V-0.1 IN (weight-percent). The material was subjected to a two step heat-treatment of normalizing at 1175°C for 1.5h with subsequent quenching and controlled tempering at 600°C for 20h. Due to the high amount of manganese and nickel a duplex microstructure of 30-40vol.% of austenite in a matrix of tempered martensite is formed during tempering.

Inconel 600 is an austenitic Ni-Cr-Fe alloy which is extensively used for tubing in steam generators of pressurized light water reactors (PWR) and CANDU heavy water reactors, because of its excellent mechanical properties and corrosion resistance. However, there have been instances of intergranular stress corrosion cracking of tubes in operating steam generators. The chemistry and the structure of grain boundaries and grain boundary precipitation have emerged as factors of prime importance in understanding stress corrosion cracking and intergranular attack of nickel-base alloys (see e.g. ref. l).

In this study analytical electron microscopy was used to determine the microstructure of grain boundary and matrix precipitates, grain boundary chromium content and dislocation substructure of selected steam generating tubes of CANDU reactors. The results of the in-service materials are compared with as-received material. Two JEOL 2010 STEM instruments were used in this study.

We have determined the structure of the paraelectric phase of BaAl2O4, which is a stuffed tridymite, by different TEM techniques and we will describe the phase transition between the ferroelectric room temperature phase and the paraelectric high temperature phase. We have also obtained HREM images of the higly radiation sensitive acristobalite phase of (Si0,9 Ge0,1)O2 and analysed the extended defects in this material.

The stuffed tridymite BaAl2O4 is ferroelectric at room temperature and undergoes a paraelectric-ferroelectric (PEFE) phase transition. The transition is reversible, takes place over a wide temperature range (400K-670K) and has a dynamical character. BaAl2O4 is easily obtained by solid state reaction of BaCO3, and A12O4,. The stoichiometric amounts of the initial reagents were mixed, grinded in an agate mortar under acetone and pressed into a pellet. The pellet was annealed in alumina crucibles at 1000 °C and 1300 °C for 40 h in air and furnace cooled.

The focus of our study is to demonstrate experimentally how elastic stress effects diffusion behavior and coarsening kinetics in a two-phase binary alloy. This work, based on the theory of Cahn and Kobayashi, focuses on elastic stresses in a thin plate. For the case of phase separation with lattice misfit between solute-rich and solute-poor phases, the diffusion of solute distorts the host lattice and causes elastic stress in the matrix. If the plate is sufficiently thin, the stress can cause the plate to buckle. The buckling stress biases the direction of diffusion, which increases the bending stress even further. Thus, the diffusion and the buckling stress are coupled; each affects the other. The interplay between the two is easiest to observe in a solution in which the solute and solvent have high misfit.