Objective: To demonstrate the quality of a new model which gives a close representation of the in situ gland for studying functional characteristics of lacrimal acinar cells. A method has been long sought which would serve as a good in-situ model for studying functional attributes of lacrimal acinar cells. Previous methods used cell plates coated with Matrigel as a growth interface for lacrimal acinar cells.

Two problems associated with this method are: cell cultures seeded this way provided a restrictive, planar growth interface, and the inability to maintain cultures beyond seven days, due in part to a lack of proliferation. A modification of recent methods (Guo et al., 1998) was implemented. The cell preparations were coated with a Hepato Stim® Medium (supplemented with EGF, DHT) and Matrigel. The medium is an extracellular matrix enriched solubilized preparation developed from the basement membranes of the Englebreth-Holm-Swarm mouse sarcoma line.

Mollusk nacre, the opalescent lining of shells such as abalone, has a fracture toughness about 3000 times greater than that of aragonite (CaCO3), the mineral of which it is almost entirely composed. Since it owes its fracture strength and toughness to its composite organic/inorganic microstructure, details of nacre formation provide clues for materials scientists hoping to mimic nacre's microstructure in fracture-resistant synthetic materials. TEM and SEM provide detailed structural information, which contributes to the understanding of nacre.

An abalone shell was fractured to expose the stacked-plate morphology for imaging in a Philips XL30 FEG-SEM. Also, an ion-milled, cross sectional sample was prepared for imaging in a Philips CM200 FEG-TEM. While SEM shows tablets of aragonite with a highly uniform thickness of 0.5 μm, stacked in interdigitated columns (Fig. 1), cross-sectional TEM reveals organic layers, ∼10 nm thick, which separate the aragonite lamella (Fig. 2), consistent with previous studies.

Mechanical properties of the structural elements of mollusk hard tissues were determined using nanoindentation techniques. Red abalone (Haliotis refuscens) has a dual laminate ceramic/polymer biocomposite, evolved for protection against impact. The nacre (mother-of-pearl) section of the shell has excellent bending strength (180 MPa) and fracture toughness (12 MPa m1/2), orders of magnitude stronger and tougher than monolithic CaCO3, it's primary component. The outer prismatic structure is comprised of columnar calcite (rhombohedral CaCO3) crystallites oriented normal to the shell surface and the inner nacreous structure consists of layers of pseudo-hexagonal shaped aragonite (orthorhombic CaCO3) platelets arranged in a brick and mortar microarchitecture (Fig. 1). The platelets (0.25 μm thick and 5 μm edge length) are surrounded by a thin layer (10-25 nm) of organic phase within and between the individual layers. This sub-um layered structure results in excellent bulk properties, not present in man-made composites of similar phase composition.

The goal of this project was to analyze the cellular localization of Z-RNA, within middle fibers (MF) of the adult dog ocular lens (1.5 yr) (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. B-DNA can adopt the left-handed Z-DNA conformation in vitro (1). Right-handed A-RNA can be transformed into left-handed Z-RNA (2). Z-RNA has been studied in cultured cells (3). Evidence supports the presence of Z-DNA in vivo (1). Removal of DNA binding proteins by fixatives can initiate supercoiling which stabilizes Z-DNA sequences (1).

Anti-Z-RNA polyclonal antibody probes were developed in rabbits immunized with multiple injections of Z-RNA: Br-poly[ribosomal(G-C)]. Regarding immunohistochemistry, lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (2.5 μm) (Fig. 2). Computerized image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.

The detection and localization of iodine in biological tissues have traditionally presented problems associated with sample preparation of soluble elements. Standard sample preparation for biological tissue involves methods which solubilize or extract iodine.Furthermore, analytical methods for detection of elements typically have been studied in smooth flat materials. We describe two methods using frozen pig skin for detecting iodine in tissue, creating digital color maps of iodine concentrations in cross sections of pig skin, and visualizing flow properties of an iodine-releasing coating at skin surfaces.

Biological samples were analyzed with the Philips XL30 SEM integrated EDAX systems using peak identification and digital color mapping software packages. Iodine-releasing coatings were applied to pig skin explants and processed by freeze-drying. The stratum corneum and hair follicle regions of pig skin remained undistorted and identifiable after dissection and processing. Iodine was detected in the coating, in the stratum corneum, and in the tissue surrounding hair follicles (Fig. 1).

Microbiologically influenced corrosion (MIC) is of wide concern in marine and non-marine environments. Biofilms and corrosion products associated with microorganisms cause numerous problems in aqueous environments, such as increased fluid frictional resistance, reduced heat transfer, and many types of corrosion, all of which can lead to failure of materials. Corrosion of metals has been extensively examined using TEM, but examination of MIC with TEM has only just begun (Blunn, 1986; Chio, 1996). Previous studies examining microbial colonization of copper surfaces and distribution throughout corrosion products demonstrate copper immobilization by bacterial biofilms (Blunn, 1987). In the current study, Pseudomonasputida attachment to corroding iron particles was examined in a sealed environmental cell in a JEOL 3010 scanning transmission electron microscope (STEM).

Iron filings were produced from carbon steel (C1010) using 600 grit sandpaper, collected with a teflon coated magnet, degreased in acetone and sterilized in ethanol. Filings were incubated in distilled water until corrosion was visible under a dissecting microscope.

Because of their amphiphilic nature, lipids tend to self-assemble into membranes and other topologically complex structures that can serve a number of functions in both natural and synthetic environments. The structure of lipid assemblies often varies on length scales of 2-100 nm, and morphological studies of such structures often require electron-optical methods. Image contrast in an electron microscope is usually generated by large defocus for unstained samples or by positive/negative staining methodologies. We currently are developing alternate approaches to generate image contrast based on spatially resolved Electron Energy Loss Spectroscopy (EELS) [1]. This has allowed us to distinguish between different lipid species as well as between lipids and protein. Low-loss spectra taken from cholesterol, ceramide and protein (BSA) show that these materials have characteristic spectroscopic fingerprints due to both π and σ valence-electron excitations that are sufficiently different to distinguish between them.

The elemental composition of polyphosphate bodies (PPB's) and other components of the cell have previously been determined by using energy dispersive X-ray spectrometers(EDX). In this present study we perform a quantitative analysis of normally grown cells and cells that were grown in the overplus phenomenon.

Cells of Gloeocapsa alpicola were grown in modified Fitzgerald's media and harvested after a logarithmic growth phase of 14 days. The overplus cells were grown, as has been previously described. The cells were exposed to 20ppm of Al, Cd, Cu, Mn, Ni, Pb, and Zn, as well as a control, for one hour. The cells were then air dried on formvar coated grids or fixed and embedded in EPON according to Luft's procedure.

For Xn-ay analysis cells were first located using the TEM mode, and then the microscope was switched to the STEM mode. Analyses of cell components were carried out using the spot mode (75Kv).

Epidemiological studies conducted first in the USA and later in the UK, suggest that a relationship exists between increased cardio-respiratory hospital admissions, morbidity and mortality rates and increases in PM10 concentrations. In urban environments, ultrafine diesel exhaust particles (DEP), accounts for 20-80 % by mass of the airborne PM10 arising from vehicular activities. In previous work, we used well characterised DEP as a surrogate for PM10 and examined its bioreactivity in vivo by assessing lung permeability, inflammation and epithelial cell markers in lavage fluid. Delivery of a single instillate of l mg DEP into the rat lung was not found to cause progressive damage but did produce a transient change in lung permeability. In the experiment described here, we instilled two different doses (control [NaCl], 0.25 and 1.25 mg) of DEP into the rat lung and assessed the responses using the methods described above with the addition of a new technique known as gene expression profiling.

According to the findings by the National Resource Defense Council (NRDC), dust, soot, and many particles in polluted air over the nation's 239 major cities cause 64,000 of premature heart and lungrelated deaths each year. In light of the new reports, the Environmental Protection Agency (EPA) has announced the plan to impose tougher restrictions to include fine particulate less than 2.5 microns (PM2.5). The new regulations could cost more than $2.5 billion a year to implement in a city like Chicago alone. Urban trees as a component of urban ecosystem can provide cost effective phyto-mitigation and phyto-remediation strategy for improving urban environment and quality of life. Previous studies have indicated that trees in Chicago removed 234 tons of particulate matter less than 10 microns (PM10) annually. To the extend that trees can control particulate pollution there is potential for improved air quality and substantial cost savings.

People spend the majority of their time indoors, mostly in the domestic environment, where there may be significant air pollution sources. Indoor air pollutants can be derived from the ingress of contaminated air from outdoors (e.g., heavily trafficked roads, presence of industry, allergens) and from specific indoor sources such as combustion appliances, building materials, and consumer products. The activities and behaviour of individuals, for example, cooking, heating, cleaning, smoking, pets, can also influence the production and levels of indoor air pollutants. There is a body of literature concerning indoor concentrations of pollutants such as CO, NO2, formaldehyde, volatile organic compounds, house dust mites, fungi, bacteria and their health effects. However, there have been few, if any studies, conducted in the UK to date to compare and contrast indoor and outdoor particulate matter (PM). Epidemiological studies consistently suggest that increases in outdoor PMIO (particulate matter with an aerodynamic diameter of less than l0μm) concentrations are linked to health effects (morbidity and mortality in persons with cardiopulmonary diseases).

Because of the session at this Microscopy and Microanalysis 2000 meeting concerned with the microanalysis of irregular surfaces, it seems appropriate to briefly review the methods used for the characterization of rough surfaces. This includes both mathematical tools for the concise description of surface roughness, and instruments used to acquire the necessary data. These methods are widely used in industry to characterize and specify the roughness of surfaces prepared by various machining, grinding, polishing, chemical etching, and physical and chemical deposition techniques, and to correlate the surface roughness with performance.

Historically, surface roughness has been measured by performing a linear traverse with a mechanical stylus that is sensitive to vertical displacements of nm but with a lateral resolution on the order of pm, which is quite similar to the dimensions of the region analyzed by X-ray microanalysis. Recently, more comprehensive characterizations have been obtained using a raster scan over surface areas.

Some of the key aspects affecting analysis of a rough surface are described in Fig. 1. If a hypothetical sample consisting of 96% Si, 1% O, 1% Na, 1% Al and 1% Fe is analysed at 20kV at constant beam current with an x-ray detector elevated at 30deg from horizontal then the measured intensities of the various characteristic lines will vary with tilt. While absorption of low energy x-rays is reduced as the sample tilts towards the detector, there is also a gradual loss of intensity due to increased backscatter. At the lower beam voltage of 5kV, absorption of x-rays at zero tilt is already small and Fig.2 shows that variation in intensity with positive tilts is substantially reduced, although absorption still becomes significant if the sample is tilted away from the detector.

The classical schemes to convert the x-ray intensity into concentration, using the ZAF or the ϕ (ρz ) methods, are valid for specimens having homogeneous composition and flat surfaces . Quantitative schemes have also been developed for x-ray microanalysis of multi-layered specimens. More recently, a quantitative method has been proposed for the microanalysis of spherical inclusions embedded in a matrix as well as Monte Carlo simulations of x-ray emission from porous materials.

For the case of specimens having a non-planar surface, a quantitative method based on the peak to background ratio, using photons of the same energy, has been proposed1. However, this method has some pitfalls. First, this method is based on the assumption that the peak to background ratio is independent of the specimen roughness which is not strictly correct because the ionization cross sections and the bremstrallung cross sections are not the same. Therefore, the shapes of the ϕ (p z ) curves are not the same for characteristic and continuum photon of the same energy resulting in different absorption corrections. The result is that the peak to background ratio will vary with beam position on a rough surface.

From its inception, electron microprobe analysis was almost exclusively done under conditions of normal electron probe incidence. The radial symmetry of this geometry greatly simplified the development of quantitative equations, and these equations where further refined based on large amounts of data also collected at normal incidence. However, as x-ray detection systems where added to scanning electron microscopes (SEMs), samples were often viewed under conditions of non-normal incidence and attempts were made to modify the various correction procedures to give acceptable quantitative results. Little justification for these methods has ever been published and so the current study was undertaken to compare theoretically calculated x-ray emission from a well characterized sample, in this case NiAl (.685 wt. % Ni, .315 wt. %A1) with experimentally measured results collected as a function of tilt angle. The theoretical calculation where done using a Monte Carlo (MC) program developed by Gauvin and Lifshin.

The energy of the electron beam, in conventional electron probe microanalysis, is generally in the range of 15-25 keV which provides the necessary overvoltage to excite efficiently the K and L x-ray lines for elements with atomic numbers in the range of about 5-83. One of the primary microanalytical methods for obtaining compositional information on particles is X-ray analysis in the electron probe and these same voltage criteria have been applied to the procedures developed for this purpose. The main difference in analytical procedures for bulk samples and particles is that corrections have to be applied to the particle k-ratios or calculated compositions to compensate for: 1) the penetration or scattering of electrons out of the particle volume and 2) variations in the absorption due to particle geometry of x-rays less than about 3 keV. In general, particle corrections improve the accuracy and reduce the relative uncertainty estimates from several tens of percent for uncorrected data to about 10% for corrected data.

In the recent years the Monte-Carlo simulation has been used successfully to exploit and understand fully the capabilities of electron microscopes. In this paper, we propose a new parametrization of Mott scattering cross-section for the calculation of the total cross-section as well as the polar angle of collision. This parametrization gives better results than Rutherford cross-section for Monte Carlo simulation at low beam energy without the numerous data files needed to use the exact Mott cross-section.

The calculation of elastic scattering cross-section can be performed with the Rutherford equation using the screening parameter, δ, the energy of the incident electron, and the electron wavelength, differential cross-section is given by:

The vast majority of applications of electron probe x-ray microanalysis takes place on specimens which deviate significantly from the ideal configuration. Classic quantitative x-ray microanalysis makes the tacit assumption that the only reason the unknown differs in emitted x-ray intensity from standards is that there is a difference in composition between them. While this seems trivial, it forces the analyst to eliminate a major non-compositional source of possible intensity differences, namely the geometric effects associated with surface roughness. An important early paper by Yakowitz demonstrated that deviations from an ideal flat surface due to surface topography could seriously degrade the accuracy of analysis. Yakowitz interrupted the grinding and polishing procedure on pure elements and alloys at various stages and measured the variance in the x-ray intensity as the probe was scanned across the surface as compared to the predicted distribution based on counting statistics.

In many areas of the world slags pose potential environmental threats to their surroundings. Heavy metals, toxic and radioactive elements contained in some slags may leach into drinking water and contaminate agricultural areas. Leaching experiments can estimate short-term alteration rates, but such tests cannot reproduce long-term weathering processes. Such studies are particularly important in the case of U- and Th-bearing slags which may remain hazardous for 103 104 years. These radioactive slags are of concern to decisions regarding the termination of a nuclear materials license and the subsequent allowances for unrestricted public use of the smelting sites and slag piles. To thoroughly evaluate leaching from slags, one must understand the distribution of elements among the slag phases and the stability of each phase. Questions regarding the distribution of elements among multiple phases are best answered by detailed electron microprobe (EMP) studies.

Slags are often mineralogically and compositionally complex, and may have complicated exposure histories.

Plutonium (Pu), with an atomic number of 94, is the highest atomic number naturally occurring element on Earth. It is formed when naturally occurring Uranium 238 captures a neutron that can be created by spontaneous fission and alphaneutron reactions. The natural abundance of terrestrial Pu is very small; its concentration is about one part in 10 of the uranium present in naturally occurring uranium ores. The first man-made plutonium was produced at the University of California cyclotron and identified on February 23, 1941. Just a few months after the first Pu was produced it became clear how metallurgically complex this material is. Plutonium is known to have six allotropes (crystalline forms) at atmospheric pressure, between room temperature and 640°C, the melting point of the pure metal. This is the largest number of allotropes known for any element.