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Many imaging processes, including both conventional wide-field and confocal fluorescence microscopy, can be described to a good approximation as linear and spatially-invariant. Linearity means that the image of an extended specimen is simply the sum of the images of the parts of the specimen - in the limit the specimen can be regarded as a collection of points, and its image is the sum of the images of the points - i.e. weighted instances of the imaging system's point spread function (psf). In the case of spatially invariant imaging the psf is the same over the entire field of imaging. Mathematically, the image is the convolution of the specimen with the system psf. In principle, this convolution can be reversed to remove degradation introduced by the imaging process, a computational procedure often called deconvolution or, more generally, restoration.

GaN and the related AlN semiconducting materials have recently attracted considerable attention because of their versatile applications for optoelectronics. Large stresses are present in the GaN/AlN thin-film heterostructure and can exceed GPa's. They originate from a large lattice mismatch between the substrate(sapphire)/GaN (14%) and the AlN/GaN interface (-2.7%) in quantum well structures. The strain is expected to induce local piezoelectric fields in these polar materials. It is essential for a further development of GaN based thin films to fully understand the formation and the local strength of these fields. Previous studies show that there are unusual large fluctuations of the patterns in lattice images present across GaN/AlxGa1-xN and GaN/InyGa1-yN quantum wells. They are attributed to compositional fluctuations and have been studied by quantitative high resolution electron microscopy. Differences between the strain profiles and the electrostatic (scattering) potential profiles were observed.

Basic Fibroblast Growth Factor (bFGF) is a multifunctional polypeptide which has been shown to play a pivotal role in the survival and differentiation of nerve cells. Several trophic and non-trophic functions of this protein have been suggested in peripheral nerves. In spite of ample information about the distribution and effects of bFGF in central nervous system, few data are available concerning the localization of this protein in peripheral nerves. In view of the role of bFGF in regulation of trophic and non-trophic functions, we particularly focused on the presence and precise location of bFGF in peripheral nerves at the electron microscope level.Spurr's resin embedded ultrathin sections from adult rats’ sural nerves were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-Zymogenetics) specific for bFGF using two-step immunogold labeling method. Control samples were treated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, VGF, anti-histamine, anti-fibroblast 5B5).

Prosthetic mechanical heart valves (MHVs) made from pyrolytic carbon (PYC) are a highly successful biomaterial application due to their excellent durability and clinically adequate blood compatibility. Their thromboresistance has been attributed to a combination of hemodynamics, material inertness, and surface properties which minimize platelet adhesion and activation. These surface properties include that polished PYC is smooth and that it strongly adsorbs plasma albumin, which is well known to reduce platelet adhesion. However, recent evidence suggests that this explanation is incomplete. Low voltage SEM shows that PYC valve leaflets are actually quite rough at submicron scales and that PYC induces extensive platelet spreading in vitroin the presence of albumin. Surprisingly few studies have examined MHVs explanted from humans or research animals for platelet adhesion and thrombosis using SEM. Moreover, in most studies it appears that even routine sample preparation methods for the preservation of biological samples for SEM,

Few quantitative analysis techniques attempt as large an extrapolation between the compositions of standards and samples than is attempted in electron microbeam x-ray emission analysis. In-situ x-ray microanalysis can be performed for essentially all elements in the periodic table in complex matricies that may contain, in extreme cases, thirty or more detectable elements. Analyses are attempted for the same elements, using the same standards, in various matrices whose average atomic numbers might range from 4 to 94. Unlike most analytical techniques, where suites of standards are synthesized having similar bulk compositions as the samples and bracketing the concentrations of the elements of interest, the standards employed in microbeam analysis are most commonly pure elements, simple oxides, or other binary element compounds. This is true even though matrix effects on electron retardation and scattering, x-ray absorption, and secondary x-ray fluorescence can cause major variations in the differences between relative intensity and relative concentration.

Recently, Inoue et al. succeeded in fabricating ultrahigh-strength Al-based alloys consisting of a nanoscale mixture of α-Al and amorphous phases or a mixture of a-Al, amorphous and icosahedral phases in Al-TM-Ce, Al-TM-Ln (TM: transition metals) and Al-Cr-Co-Ce systems by rapid solidification [1-3]. In order to understand the mechanism of the nanoscale microstructural evolution during the rapid solidification processes in these nanocomposite alloys, we have characterized the microstructures of rapidly solidified Al94.5Cr3Co1.5Ce1and Al96V4Fe2alloys by atom probe field ion microscopy (APFIM) and high resolution transmission electron microscopy (HREM).TEM investigations have revealed that the as-quenched Al94.5Cr3Co1.5Ce1alloy is composed of a nanoscale mixture of amorphous and α-Al. A typical TEM bright field micrograph is shown in Fig. 1. The microdiffraction patterns taken at various locations in the darkly contrasted region have shown that the region consists of a few interconnected α-Al grains and many localized amorphous regions which are trapped within the Al grains.

An O-N-O layer within a DRAM is designed to be an insulator between a single-crystal silicon substrate and a poly-crystalline silicon gate. The nominal structure of the layer is SiO2-Si3N4-SiO2. It has been reported that oxygen and nitrogen diffuse into each other layer while preserving the three layered structure. In order to measure the elemental distribution quantitatively, EELS was used for Si and light elements O and N in this study.The specimen was cut from a 16M-DRAM device and prepared for TEM observation by the crosssection ion milling method. The O-N-O layer was analyzed using a JEM-201 OF FE-(S)TEM equipped with a scanning imaging device including a High-Angle Annular Dark Field(HAADF) detector which is capable of collecting electrons scattered 50 to 110 mrad, and also a PEELS(GATAN model 676). Fig. 1 shows a HAADF image of the O-N-O layer.

An O-N-O layer within a DRAM is designed to be an insulator between a single-crystal silicon substrate and a poly-crystalline silicon gate. The nominal structure of the layer is SiO2-Si3N4-SiO2. A small amount of phosphorus is doped in the poly-crystalline silicon gate to improve the electrical characteristics of the device. Transmission Electron Microscopy (TEM) was first used to view the micro structure in the layer. A bright field image gave clear contrast in the O-N-O layer, however, TEM images do not provide direct information about the elemental distribution. In this study X-ray elemental mapping was thus used to show this distribution.The specimen was cut from a 16M-DRAM device and thinned using the cross-section ion milling method. The O-N-O layer was examined using a JEM-201 OF FE-(S)TEM equipped with a JEOL UTW-EDS detector. Fig. 1 shows a high resolution TEM image of the O-N-O layer.

The speed of automated EBSP orientation gathering has now increased to a point where it is possible to examine large areas of deformed material at a resolution close to the length scale of the dislocation substructures involved (typically <1μm for strains of greater than ∊= l). Investigation of such deformed samples invariably gives rise to diffraction images of low quality and to many double patterns. To assist in a critical selection of the data, it is usual to store a measure of the indexing confidence for each point. Here we describe how deformed samples, which provide the appropriate range of pattern qualities, can be used to calibrate acceptance criteria for unsupervised EBSP measurements on deformation microstructures. An application in the use of such criteria is also described.Two crystals, chosen with orientations that are known to develop a well defined texture spread during deformation, were channel die deformed to a strain of ∊=1.5 (78% reduction).

Accurate characterization of electron diffraction patterns can be tedious, which encourages development of computer assisted tools and methods. We developed a spot measurement tool to characterize rapidly arrays of diffraction spots that are characteristic of a single crystal, and to measure precisely the d-spacing values for individual spots. The spot tool determines these vectors for averaged measurements from many spots in a digital image of the diffraction pattern. Previously we developed automated methods for spot pattern analysis. Why make an operator-assisted tool? This tool is faster than either our automated or entirely manual methods, and it allows assessment of the quality of the data at the beginning of the analysis.It is easy to see regular patterns of spots in a zone axis diffraction pattern (Fig. 1.1). An operator guides the initial calculations by adjusting an array of circles to approximately cover the spots in the pattern to be analyzed.

The near epidemic occurrence of fibropapilloma in green sea turtle (Chelonia my das) (Figure 1) significantly threatens the survival of this species which is protected under the U.S. Endangered Species Act. Although collective evidence suggests a viral etiology, the causative virus of green sea turtle fibropapilloma has not been isolated. To facilitate the isolation and characterization of the causative virus(es), we established 13 cell lines from multiple organs/tissues (tumor, kidney, lung, heart, gall bladder, testis, and skin) of green sea turtles with fibropapilloma. Serial subcultivation of cell lines derived from lungs, testis, eye soft tissues and tumors resulted in the formation of tumor-like aggregates, which attained sizes of 1-2 mm in diameter within two weeks (Figure 2). Media from such cultures, when inoculated onto cells derived from healthy turtle embryos, produced similar tumor-like aggregates, suggesting the presence of a transmissible agent.

The field of immunocytochemistry comprises a large body of methodologies whose purpose is to obtain spatial and temporal information about biological samples with a high degree of chemical specificity. The major differences in immunocytochemical methods are: (1) type of sample preparation (i.e., pre-embedding or post-embedding) and (2) type of detection system (i.e., chromogenic, fluorochromes, or particulate). The history of advances in immunocytochemistry can be understood, to a large extent, through the development of the various detection systems. All of these labeling systems have their appropriate applications and are in use today.The introduction of colloidal gold as a particulate immunoprobe revolutionized immunocytochemistry, particularly at the ultrastructural level. The fact that these probes are discrete particles that are electron dense contributes to their wide acceptance. Due to the particulate nature of these probes, labeling density over a given structure or region of a cell can be determined by simple counting procedures.

Amorphous materials are devoid of periodic long range order, but at the nearest-neighbor level they possess a high degree of short-range order. In amorphous tetrahedral semiconductors, such as Si and Ge, this short-range order arises because each atom attempts to satisfy four bonds arranged as a regular tetrahedron. It is the rotations about each bond, from the second-nearest-neighbor outwards, that result in the loss of long-range order. It is apparent from modeling of amorphous materials, that there is considerable flexibility as to how rapidly the medium-range-order diminishes. The continuous random network (CRN) is a hypothetical tetrahedral extended structure wherein the atoms possess full four-connected coordination, but have minimal medium-range order. However, real amorphous materials infrequently exhibit true CRN-like topologies. Traditionally, diffraction has been used to study short- and medium-range order in amorphous materials. Assuming kinematical scattering, and that every atom has a similar environment, a radial distribution function (RDF) can be extracted which is sensitive only to the averaged atom pair-correlations out to ∼1 nm.

Bulk chemical characterizaton with techniques such as neutron activation analysis (NAA) has been used for many years to identify sources of archaeological artifacts by comparing bulk chemical profiles of archaeological artifacts to profiles determined for samples of raw material for source identification. The chemical approach to determining the provenance of archaeological pottery is complicated by the paste preparation activities of potters and by post-depositional processes. In a ceramic provenance investigation, the investigator typically identifies a series of relatively homogeneous subgroups, which most often are assumed to represent geographically restricted raw material sources. Although the source-specific interpretation is often the most parsimonious explanation of compositional patterning, it is really only the first of several competing hypotheses which, singly or in combination, might account for patterns of similarity and difference in a compositional data set. In this case, two white firing pottery types (SAWH and Ivory) made during the Late and Terminal Formative periods (300 B.C. - 200 A.D.) in southeastern Mesoamerica (Guatemala) were found to be chemically distinct.

Monitoring the performance and capabilities of energy dispersive X-ray spectrometers (EDS) and related x-ray analysis electronics and software is important for maintaining and improving the reliability, sensitivity, and accuracy of the x-ray analysis system. There is growing demand for quality systems through laboratory accreditation, ISO 9000, ISO Guide 25 and related programs that require set quality control procedures for analytical instrumentation. In such cases it is frequently more useful to have one national/international standard. This approach is not only more efficient than having each analyst devise their own system, but the use of the same standard procedures among labs would allow direct intercomparison of results. This intercomparison can help labs and manufacturers determine what are normal versusabnormal results and lead to higher quality instruments and analyses.We are designing a standard procedure to maximize the efficiency of each quality control (QC) measurement so that we spend as little time monitoring the analysis system as is possible.

Conventional scanning electron microscopy permits the use of cryogenic techniques, which has led to the examination of ice-containing samples (eg ice cream) at temperatures of -80°C and below. At these temperatures moderate etching of the ice crystals occurs which helps to identify the crystals in the surrounding glassy matrix. However, imaging at higher temperatures - equivalent to those at which most ice-containing systems are actually utilised - is impossible because of rapid sublimation. We have set out to develop a system which will enable us to examine ice crystals at temperatures of -20°C and below, corresponding much more closely to realistic conditions of use.In the environmental scanning electron microscope (ESEM), a gas can be maintained around the sample. Not only can this be used to maintain the sample in its native state, but the gas is actively involved in the imaging process through its role in signal amplification.

Cobalt disilicide (CoSi2) is an attractive contact material for Si1-xGex devices due to its low resistivity, low Schottky barrier, and good thermal and chemical stability. Previous studies have shown that blanket Co films on Si0.80Ge0.20react at annealing temperatures of 700 °C to form CoSi, CoSi2and a Ge-rich Si1-xGexalloy; no Co-Ge phases were found. The Co preferentially reacts with the Si, which can lead to Ge segregation and island formation. To gain a better understanding of cobalt silicide formation, an electron microscopy investigation of Co/Si1-xGex/Si was performed.Si1-xGex films (x ≤ 0.3) 64 nm to 260-nm thick were grown by molecular beam epitaxy at 550 °C on phosphorus-doped Si <001> substrates. Co films ranging in thickness from 5 nm to 25 nm were electron-beam evaporated (evaporator base pressure < 6.67 x 10-8 Pa) at room temperature on the Si1-xGex films.

Phase segregation is important in determining the properties of many complex polymers, including polyurethanes. Achieving a better understanding of the links between formulation, chemical nature of segregated phases, and physical properties, has the potential to aid development of improved polymers. However, the sub-micron size of segregated features precludes detailed chemical analysis by most existing methods. Zone-plate based, scanning transmission X-ray microscopes (STXM) at NSLS and ALS provide quantitative chemical analysis (speciation) of segregated polymer phases at ∼50 nm spatial resolution. Image sequences acquire much more data with less radiation damage, than spot spectra. After alignment, they provide high quality near edge spectra, and thus quantitative analysis, at full spatial resolution.Fig. 1 shows an image and spectra acquired with the NSLS STXM of a macro-phase segregated TDI polyurethane. Spectral decomposition using model polymer spectra is used to measure the local urea, urethane and polyether content.

Newborn, premature infants have greater susceptibility to infection compared to older newborn infants. Although the reason for greater susceptibility to bacterial infection in premature infants is unknown, there are many reports that focus on polymorphonuclear leukocytes (neutrophils) as the defective cell type in the immature neonatal system. However, we have recently reported that in an rat model of inflammation, neutrophils from adult rats failed to migrate efficiently in neonatal rats. Furthermore, we found that this defect was associated with decreased expression of P-selectin on the surface of neonatal endothelial cells in situ.P-selectin is an adherence molecule that is expressed by activated endothelial cells and serves as the counterligand for CD11/CD18, the β2- integrin that is expressed by activated neutrophils.We hypothesized that endothelial cell P-selectin expression would be less in human umbilical cord veins from prematurely born (preterm) infants compared to normal term infants. To test this hypothesis, we collected umbilical cords from preterm infants (<27 weeks gestation) and term infants (>38 weeks gestation).

We have performed a theoretical and experimental study of the signal composition in the Environmental SEM (ESEM) with the intention of forming a set of general guidelines for optimising the signal to background ratio. In the ElectroScan ESEM, a gas ionisation cascade is used to amplify the secondary electron signals emanating from the specimen surface. The presence of gas in the chamber also gives rise to a pressure dependent background signal derived from ionisation events between gas molecules and high energy primary beam and backscattered electrons, as well as secondary electrons generated by the probe skirt.The signal collected by an environmental secondary detector (ESD) (ElectroScan, 1991) or a gaseous secondary detector (GSED) (ElectroScan, 1994) is an amplified signal which is a composite of at least three contributions. These are the amplified currents arising from the ionisation of the gas by high energy electrons from the primary (Ipe) and backscattered electrons (Ihse).

Recent developments promise the possibility to externally control every aspect of microscopes through a computer interface. In combination with high-resolution cameras and feedback to the microscope, this can be leveraged to create highly automatic routines, e.g., to remotely correct astigmatism. Together with the development of fast computer networks this creates a new branch of microscopy, the so-called “telemicroscopy”. The goal of telemicroscopy is the control of a microscope over a large distance including the transfer of images with an acceptable repetition rate. A big advantage for electron microscopy in particular is the possibility of having access to expensive and well-equipped microscopes. In the field of light microscopy the branch “telemedicine” was created, meaning the “virtual” presence of a colleague or specialist for discussion or diagnosis.Using transmission electron microscopy as an example, the history and special requirements for automation and telemicroscopy will be discussed. In the late 80's the first TEM with a remote control was revealed. Shortly thereafter, first automatic functions for defocus control and astigmatism correction were developed using a video camera as electronic image converter.

A1N is an attractive material because of its mechanical, thermal and electronic properties. However, these properties are often adversely altered by extended defects such as inversion domain boundaries (IDBs), and through impurities associated with the defects, especially oxygen. Thus to improve the properties of the material it is important to understand the structures of the defects. A number of studies have been carried out to characterize these defects using fringe contrast, weak-beam, convergent-beam electron diffraction and conventional high-resolution phase contrast imaging. These techniques all require the use of model structures for data interpretation. In this paper, we report a direct determination of the structures of IDBs and dislocation cores in A1N by highresolution Z-contrast imaging using a 300 kV VG HB603U scanning transmission electron microscope, with a probe size of 1.3 Å.

A dielectric O-N-O multi-layer is formed between a single-crystal silicon substrate and a polycrystalline silicon gate to insulate them. The nominal structure of the layer is SiO2-Si3N4-SiO2. In this study, in order to visualize the elemental distribution, energy filtered mapping technique was used and also the relative concentration was calculated from the intensities of the elemental maps to characterize the structure quantitatively.The specimen was cut from a 16M-DRAM device and made suitable for TEM observation by the cross-section ion milling method. The O-N-O layer was examined using a JEM-2010F FE-TEM equipped with a post column energy filter(GIF 200). Fig. 1 shows energy filtered elemental maps of Si, O and N with an elastic image of the O-N-O layer obtained by the three-window method.As a map is produced after a background intensity extrapolated from two pre-edge images is subtracted from a post-edge image, each pixel of these maps provides energy loss intensity of an element.

The ability to adhere biomaterials to tooth tissue has drastically changed today's dental restorative practice. Modern adhesive techniques enable dentists to restrict operative procedures to the sole removal of diseased tooth tissue without further destruction of the remaining sound tissue. However, adhesive restorations have only a short lifetime of ca. 5 years. Extending the restoration lifetime is therefore one of the primary goals of present research in dental materials science. In practical relevance and direct benefit to that objective are studies that contribute to a better understanding of the interfacial phenomena that take place between restorative material and tooth tissue. Therefore, the resolution of instruments available to elucidate this interfacial interaction is essential. Besides the transmission electron microscope (TEM), a more recent powerful tool to study tooth-biomaterial interaction with high resolution is the atomic force microscope (AFM).

Effective use of the ANL 300kV CFEG/A AEM requires a knowledge of the probe current and size distribution during operation. In most cases this can be calculated once basic parameters of the system are experimentally determined. Central to this is a determination of the source brightness of the Cold Field Emission Gun (CFEG) under standard operating conditions of the instrument. For the last ten years most probe calculations of CFEG's have been done assuming a brightness parameter of β∼×109A/cm2/sterad2at 100 kV, while few if any experimental values have been reported at 300 kV. Using β and the relationship that the operating probe diameter is obtained by adding in quadrature the Gaussian probe size (dg, the CFEG source contribution), and the diameters of least confusion due to spherical abberation (ds) and diffraction limits (dd) one obtains the total probe size dtas :

A BNL-Grace process has been developed to chemically convert in-situ, the chrysotile fibers of sprayed-on fireproofing products to an unregulated glassy material. The effectiveness of this process has been convincingly demonstrated using Analytical Electron Microscopy and X-Ray Diffraction techniques.Asbestos minerals were used in fireproofing materials because of their excellent physical properties including fire resistance, high tensile strength, heat and electrical insulation, and resistance to acids and alkali. But in 1975 the Environmental Protection Agency began regulating materials containing > 1% asbestos.The new in-situ BNL-Grace process, which uses a foamy solution sprayed directly onto asbestos-containing fireproofing chemically digests essentially all the asbestos fibers, transforming them into harmless materials. After treatment, the fireproofing is no longer a regulated material. The process produces essentially no waste.Our problem was to demonstrate that all of the asbestos was converted and that the remaining materials were no longer regulated. Typical analysis methods use conventional optical and Polarized Light Microscopy (PLM) to measure and observe fibers.

Porous silicon has potential applications in the microelectronics industry. It has been investigated as an electroluminescent source1, as a sensing device in chemical sensors and as an antireflective coating for solar cells. The low efficiency of the solar cells is enhanced by the antireflective coating that porous silicon provides, while the cost of fabrication of the cells is decreased. Porous silicon is normally processed by electrochemical anodization of silicon wafers. Since parameters such as current density, anodization time, and surface conditions can affect the microstructure of the films obtained, we varied the current density to study the effect of the microstructure on the resultant reflectance for the purpose of improving it. It has been found that higher current densities result in higher reflectance films than the lower current densities.

The sensory portion of the retina is composed of multiple cell layers with complex functions and interactions vital to ocular homeostasis. Xenobiotics can induce physiological and morphological perturbations of these cell layers, and subsequently compromise vision. We describe an ion channel activator (ICA)-induced retinopathy observed in a preclinical drug safety study in dogs. Purebred beagle dogs received either ICA in vehicle (0.75% Methocel® and 0.05% Tween 80) or vehicle alone (control) for nine months. During the study, visual deficits and fundic changes were observed clinically, leading to premature termination of the study at eight months. The posterior globe of the left eye was fixed by the method of Acland and Aguirre, dehydrated in a series of graded methanol baths, and embedded in epoxy resin. Semi-thin (l-μm thick) sections of the retina were stained with toluidine blue, and examined by light microscopy. Ultrathin (60-90-nm thick) sections of the retina were mounted on copper grids,

This paper addresses the microanalysis techniques used to characterize thermal sprayed Ti coatings on concrete surfaces as anodes in impressed current cathodic protection (ICCP) systems. Thermal sprayed Ti anodes on concrete were electrochemically aged in laboratory studies of anode performance and service life. A thermal sprayed catalyzed Ti anode was installed on the Depoe Bay Bridge on the Oregon Coast in a field demonstration of the ICCP system. Both air and N were used as the atomizing gases during the study. Thermal sprayed Ti coatings are typically applied in an inert atmosphere or vacuum using shrouds or chambers to prevent reactions with the atmosphere. This was not possible with a structure as large as a bridge.Microanalysis of Ti and N together is difficult and the effects of O add to the complexity of the analysis. The ternary Ti-O-N system has not be well studied.

Boron-based thin film materials have numerous uses ranging from application as hard and refractory protective coatings to potential employment in wide-band gap semiconductor electronics. Of particular interest are the boron carbides, nitrides, oxides, and phosphides. These compounds exhibit a broad range of structural and bonding variations. The crystalline form of elemental boron is based on an arrangement of 12-atom boron icosahedra positioned at the vertices of a rhombohedral lattice. Related materials, such as B4C, and B12P, also possess structures closely related to this icosahedrally coordinated prototype. However, the structural coordination of the boron carbides and phosphides will vary with composition. For instance, BP possesses a tetrahedrally coordinated zinc-blende structure, and the carbon-rich boron carbides will form in a graphitic structure. Boron nitride has the added complication that for the same composition (BN) multiple bonding and polytypic variations are possible: both soft, graphitic sp2-rich (e.g., hexagonal and rhombohedral BN) and hard, diamond-like sp3-rich phases (e.g., cubic and wurtzitic BN) can be formed.

Gold particles as markers in immunocytochemical procedures have undergone tremendous changes since their introduction by Faulk and Taylor (1). With the development of both small colloidal gold (<3 nm) and 1.4 nm gold clusters (Nanogold™) for immunocytochemical labeling, steric hindrance has been significantly reduced. In a comparative study using colloidal gold-anti CD3 complexes and particle sizes of 5 nm, 3 nm and less than 3 nm on surface labeling of lymphocytes there was a 1.5 fold increase in labeling density when 3 nm particles were used. There was no increase when smaller particles were used (unpublished data). In another unpublished experiment, labeling densities on an astrocytoma cell line were compared between 3nm colloidal gold-CD44 Fab’ fragments and 1.4 nm gold-CD44 Fab’ fragments. Again there was no detectable differences between particle sizes. The limiting factors remain the size of the IgG or Fab’ fragment, the ability to bind it whether by absorption or by covalent bonding without rendering it inactive and also the accessibility of the target molecule in the tissue.

Introduction: The “real” semiconductor processing world consists of the universe of industrial and government semiconductor laboratories and manufacturing facilities plus a small number of university departments with heavy funding from industry—or a consortium of the above. We are bombarded with reports of how the drive toward faster, denser, lower power consuming and more reliable semiconductor products will accelerate with time. The smallest features of today's devices have already shrunk from view in visible light microscopes! Electron microscopy studies of semiconductor device materials, interconnects, insulators, and device packaging materials play a vital role in these extraordinarily “high tech” and financially important arenas.Shifting semiconductor analytical paradigms: For the most part, the analytical paradigm has shifted: from visible light microscopy to SEM analysis, at low magnification and high accelerating voltage… to much more sophisticated SEM analysis, at high resolution and/or low accelerating voltages… to the many variations of TEM available today… and to new types of scanning probe microscopy.

We are developing a testbed for automated identification of specimens, and acquisition of high resolution images for 3D macromolecular EM. Several software package which have been developed specifically for acquisition of tomographic data sets have illustrated the benefits of automation. These software packages require the user to identify a specimen of interest and then automatically acquire a single tilt series from the identified specimen. The testbed we are developing will provide a generalized system for specimen identification, image acquisition and image quality assessment. The goals of the testbed are to provide a system that will acquire EM images unattended by an operator.The testbed integrates a computer controlled TEM with a distributed real-time image processing environment. Computerized control and image acquisition from the TEM uses the emScope software package (1). This is coupled to existing image processing software packages and new software tools developed specifically for this project.

Today material science requires the use of increasingly powerful tools in material analysis. The last twenty years have witnessed the development of a number of analytical techniques. However, among these techniques, only a few allow observation and analysis of materials at the nanometer level ; one can mention scanning transmission electron microscopy coupled with electron energy loss spectrometry (STEM-EELS), SIMS and atom-probe techniques. In the STEM combined with electron energy loss spectrometry the spatial resolution can be better than one nanometer on a wide scanning area. However the in-depth spatial resolution is limited by the specimen thickness and therefore strongly depends on samples preparation. On the contrary, In SIMS techniques the in-depth spatial resolution can be as good as one nanometer but the lateral resolution is set by the ion beam diameter.

Liquid containing specimens can be stabilized for observation in the Environmental Scanning Electron Microscope (ESEM). It is also possible to examine systems that are primarily liquid, such as colloidal suspensions and gels, or even possess a multi-component liquid microstructure as in emulsions.(l) In order for such investigations to be useful, an understanding of the origins of secondary electron contrast in liquids is necessary. Our aim here is not to produce a complete theory of secondary electron emission in these systems, but to provide general guidelines for discriminating between two liquids based on relative contrast.Secondary electron emission from a substance is determined by three processes: creation, transport through the material, and escape from the surface.(2) Contrast between two regions will be due to differences in one or more of these processes. Initial generation of secondary electrons is primarily a function of the ionization cross-sections of the substance and its density.

Previously, we showed rapid mitochondrial Ca2+transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca2+uptake by the Ca2+uniporter, inhibits mitochondrial Ca2+transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca2+transients.Ca 2+-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM Rhod 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial Rhod 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of Rhod 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.

Solute-atom segregation to grain boundaries has been of interest since the 1930's when it was realized that some steels were susceptible to failure by intergranular fracture when certain impurities were present. Segregation of impurities or intentionally added alloying elements at grain boundaries can greatly affect various grain boundary properties, which in turn affect numerous macroscopic properties. Materials phenomena that have been linked to grain boundary segregation include temper brittleness, fatigue strength, adhesion, precipitation, diffusional creep, intergranular corrosion, and grain boundary diffusivity. Although grain boundary segregation has been extensively studied for many years, the effects of different grain boundary structures on segregation was generally not considered. It has been established both experimentally and theoretically that the level of segregation varies from grain boundary to grain boundary in the same alloy, but there is little direct information on how grain boundary structure influences segregation.

Nanobiotechnology is the fusion of biology and nanofabrication (Hoch, et al. 1996). Nanofabricated devices are increasingly being used in biological studies, and surface modification methods are important for interfacing inorganic devices with tissues.Nanofabricated implants are used to study brain physiology (Najafi and Wise, 1986). These implants can have cross-sectional dimensions as small as 15 μm. However, the implants cause glial scars that inhibit their operation. Figure 1 shows a model implant, with a trapezoidal cross section (base=200 μm, height=130 μm, top=60 μm, length=2 mm), for studying scar formation. Implants were inserted into the brains of rats and tissue was harvested after 1 hr. to 12 wks. Figure 2 shows a thick (100 μm) section of brain prepared 7 days after implantation; the section was immunolabeled for glial fibrillary acidic protein (GFAP) and imaged in a Bio-Rad confocal microscope. The implantation site is surrounded by GFAP positive material indicative of reactive astrocytes.

HRTEM images of α-Al2O3 oriented along [1100] often exhibit a modulation of the basal fringe intensity with a period that corresponds to twice the basal plane spacing. In the example shown in figure 1, note the strong basal fringe contrast in the A12O3 with a period of 4.33 Å. HRTEM image simulations for ideal imaging conditions fail to reproduce this doubling of the lattice fringe period. Instead, simulated images predict that the (0006) fringes (d0006=2.17 Å) should be of equal intensity (e.g., see Figure 3a). Incorrect beam and/or crystal tilt can strongly affect HRTEM image contrast. In particular, anomalous periodicities can arise under beam and crystal tilt conditions for which normally kinematically and dynamically forbidden diffracted beams contribute to the image. More recently, the importance of 3-fold astigmatism on HRTEM imaging has been recognized. As with beam tilt, this lens aberration produces asymmetrical phase shifts in the diffracted beams.

Designing a microscopy facility can be completed in a step by step approach, whether it be a completely new facility or an existing one moving into expanded quarters. The procedure is independent of size. There are several major factors that must be taken into consideration. Check lists can be compiled by the microscopist. One should easily be able to start at any point along the way. The time and effort initially put into the design will pay for itself many times over in convenience and efficiency of the resulting laboratory.Prior to drawing the actual design of the microscopy facility but critical to the design specifics, it is necessary to compile the following information: considerations for determining the size of the facility; general EM lab requirements which affect site determination; activity functional flow diagram; EM room requirements; a list of all the equipment to be placed in the EM facility and exact specifications for each; design features not associated with specific equipment; activities requiring space that are not specific to the equipment; general building requirements not specifically related to microscopy operations; and ergonomic and psychological factors.

Particulate wear debris found around joint arthroplasties has been linked to the premature failure of artificial joints. Cytokines and eicosanoids released by macrophages are known mediators of inflammation and osteolysis. Previous in vitrostudies of wear debris-mediated inflammation have relied upon poorly characterized particulates and peripheral blood cell fractions of mixed cellular composition. The objective of this study was to utilize titanium particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by isolated monocytes.To mimic wear debris that surrounds artificial joints, titanium alloy particulates (1-3 μm in diameter) were obtained. A submicron fraction of these particulates was isolated by differential sedimentation. The morphology, size and concentration of the resulting population was characterized utilizing scanning electron microscopy. Titanium particulate size was estimated from photomicrographs of calibrated latex microspheres mixed with titanium particles (Figure 1). A known volume of titanium particulates in suspension was applied to a carbon planchette and allowed to dry.

A superfamily of actin crosslinking proteins, the calponin-homology domain (CH) family is implicated in directing the assembly and controlling the organization of the cytoskeleton through a highly conserved actin binding domain (ABD). This domain, composed of a tandem pair of CH motifs, is found in actin crosslinking proteins such as fimbrin, alpha-actinin, spectrin, and dystrophin. In addition to its role in crosslinking actin filaments, a putative homologous ABD in intermediate filament-binding proteins, plectin, dystonin, and BPAGlnl, suggests a possible role in crosslinks between desmosomal and vimentin intermediate filaments and actin. Thus, the CH domain could integrate the functions of the actin and IF cytoskeletons by physically linking these structural systems. We present an atomic model for the complex of the CH domain and actin.In crosslinking proteins that organize actin into two and three dimensional networks or gels the ABDs lie at the ends of long, flexible molecules.

Control and optimization of materials’ properties will increasingly depend on the ability to tailor both the structure and chemical composition of the materials at the nanoscale level (100 nm and less). One specific class of “nanostructured” materials is the multilayer film (MLF) structure, which is formed by alternate deposition of two or more different elements or compounds and has a wide range of applications in, for example, the field of data recording and storage. In order to understand MLF structures well enough to improve and optimize their properties, characterization at the atomic level with regard to both structure and chemistry is essential. Interface quality is one of the most important parameters due to the possibility of intermixing between layers. It is thus desirable to obtain information about the interface morphology in these materials at the highest possible resolution.

Digital imaging has made electron microscopy more quantitative, more direct, and more automated. It has vastly changed the way in which microscopy is done today. The conventional photographic emulsion technique for image recording is being replaced by CCD-based digital imaging techniques in virtually all aspects of electron microscopy applications, resulting in increased work efficiency and data accuracy. Many of the microscope's evaluation procedures are traditionally time-consuming and complicated, such as measurements of TEM resolution and spherical aberration coefficient of the objective lens, etc. However, they can be greatly simplified with the use of slow-scan CCD cameras bee and appropriate software.With many superior properties over other image recording media, slow-scan CCD cameras offer possibilities for many new TEM applications, such as TEM autotuning and automated microscopy. On-line image display and analysis have proved to be the key elements in CCD-based digital imaging.

Scanning Capacitance Microscopy (SCM) was first developed in 1985 as a method for sensing tip-to-sample spacing for surface topography profiling in connection with the RCA VideoDisc. Williams and coworkers were the first to use an SCM for obtaining dC/dV doping profiles in semiconductors, albeit with a rather modest resolution of 200 nm. More recently, it has been developed as a 50-nmresolution tool for microscopic doping analysis of semiconductors by measuring the tip-to-sample rf capacitance in an AFM controlled by other means, e.g. by laser beam deflection of a cantilever tip. In this paper we report on the application of SCM to study the 2D doping profiles of InP-based devices, such as multi-quantum well lasers.It is particularly convenient to prepare cross sections of III-V devices, since the material readily cleaves on [110] planes, as compared to silicon where cross sections must be obtained by painstaking polishing.

We report measurements of chemical shifts in the Fe-L x-ray lines of different Fe compounds from xray emission spectra acquired using a microcalorimeter energy dispersive spectrometer (EDS). The observed changes in peak position and relative intensity of the Fe-L x-ray lines are in agreement with measurements obtained using a wavelength dispersive spectrometer (WDS), demonstrating the usefulness of microcalorimeter EDS for high-energy-resolution x-ray microanalysis.Chemical shifts result from changes in electron binding energies with the chemical environment of atoms. In x-ray spectra, chemical shifts lead to changes in x-ray peak positions, relative peak intensities, and peak shapes. These chemical bonding effects can be significant (with x-ray peak shifts on the order of 1 eV), particularly for x-ray lines resulting from transitions involving valence electrons of light elements such as B and C.

To improve the understanding of the mechanical deformation mechanisms associated with friction, wear, and precision machining processes, a detailed set of microploughing experiments were performed on gold single crystal surfaces. Large single crystals that had an edge length of 1.5 mm and a thickness of 1 mm were used for this work. Since the microplough depths ranged from 100 nm to 1600 nm, it was important that the material be damage free at the surface. This condition was verified by alpha particle backscattering. A stand-alone STM head, having a singlecrystal diamond tip, was used in conjunction with a Digital Instruments Nanoscope Ilia controller to generate the 13 μm long microploughs. The vertical ploughing force was measured using a Mettler AE130 semi-microbalance.After generating the microploughs and measuring their geometry with an AFM, some of the microploughs were cross sectioned for TEM observation.

Volcanic eruptions inject tons of silicic material into the atmosphere. Large, dense material falls out within a few hours whereas fine size fractions, < 10 μm radius ash, may remain airborne for weeks to months. Protection of human population centers and aircraft requires predictive atmospheric transport models. Many of these models assume the ash particles are spherical but abundant qualitative studies of ash particles demonstrate irregular shapes ranging from parachute-like bubble-wall shards to polygonal shaped mineral grains. These non-spherical shapes increase atmospheric drag. The accuracy of transport models may be improved with a better understanding of the actual shapes found in fine ash particles. Therefore, the objective of this study is to accurately determine three-dimensional shape measurements via stereoscopic methods using the scanning electron microscope.The scanning electron microscope is an ideal imaging platform for collection of stereo pairs because of its utility in stereoscopic height measurement.

Microanalysis data depe nd strongly on the electron beam energy used in the S E M and the effects are more pronou nced with bulk samples than with thin sections. The situation can be used to advantage but there are also significant problems. The data and any quantitative corrections can be quite different at different beam energies. Th e spectra look different and this can be both helpful and confusing, with low ene rgy x-ra y lines generally more prominent at lower voltages. S p ec tra at several different voltages are particularly informative, both for quantitative analysis and to deco nvo lute three dimensional geometries. There may be no single perfect voltage for an analysis.

Tin dioxide (SnO2) with rutile type structure exhibits unique electronic and optical properties. In applications of this material as gas sensors, a film-type of SnO2provides high ratio of surface area to volume and lead to high sensitivity and fast responses. It has been found that substrate material, deposition conditions, and annealing procedure may directly control the microstructure of thin films, hence control gas-sensing properties. In this paper, we present transmission electron microscopy (TEM) studies of the microstructure and crystal defects of tin oxide thin films on sapphire substrate with subsequent annealing at high temperatures.Tin oxide thin films were deposited on the surface of sapphire by e-beam evaporation of high purity SnO2(99.999%) at 350°C followed by annealing in air at 600°C - 700°C. Microstructures of the films were characterized by X-ray diffraction (XRD) and TEM techniques.