There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.

Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

High resolution, quantitative cell labeling analysis for electron microscopy is typically accomplished using colloidal gold (cAu) particles which have a uniform diameter such that each particle conjugates no more than one ligand with monovalent specificity, such as an Fab antibody fragment. in this way, one particle labels exactly one target molecule. The corresponding particle size which satisfies this requirement, in the case of the Fab fragment, is 3-5nm. Under these constraints, multiple labeling cannot be performed using the standard cAu technology in which particles of various diameters are used such that each size corresponds to a unique label. Particles larger than 5nm may conjugate multiple Fab fragments, and thus a single particle may lable multiple target sites. Conversely, several particles smaller than 3nm may bind to a single Fab fragment so that several particles may label a single target molecule. Recent work in our lab has focused on the development of a labeling system which maintains the advantages of the standard cAu labeling technology, namely the use of electron-dense particles with excellent secondary and backscattered electron emission properties which make them readily detectable by both TEM and SEM, while at the same time offering greater versatility by permitting multiple labeling for high resolution, quantitative analysis. This novel labeling system makes use of colloidal particles which have unique and distinctive shapes, and composed of a variety of different metals including Au, Ag, Pt, Pd, Rh, and Ru. These elements all form stable aqueous colloidal suspensions, and the particles have surface charge properties similar to those of Au, permitting the adsorption of proteins and other ligands to them by the same conjugation techniques used with cAu (fig. 1).

Current computer chip technology is based on lithographic methods that limit components to ∼0.3 microns in size, due to the wavelength of light, and the photoresist/coating/etching processes. The size directly determines computer speed, complexity and cost, and advances in computers over the years have mostly been due to reduction in component size. It is here proposed to construct nanowires that are approximately 2 nm in diameter, or 150 times smaller than currently available. For 2 dimensions, this translates into a 1502 = 22,500-fold computational advantage. Additionally, 3 dimensional construction is proposed, bringing the potential improvement factor to 3,375,000. While it is probably unrealistic that this factor of packing density can be fully achieved, even several orders of magnitude improvement over current technology would be significant.

A wire width 2 nm may be achieved by placing gold quantum dots along a DNA template. Ends of the DNA-nanowire may be designed with sequences to attach by hybridization to complementary sequences on target connection pads, so that the two ends will seek and automatically wire correctly in solution. This strategy is easily adaptable to 3-dimensional wiring. Conduction between gold quantum dots may be studied as a function of spacing, size and coatings.

Molecular immunolabeling allows us to recognize location of molecules within complex biomolecular assemblies, cells, and organs. This technology helps us to facilitate correlations of the data available from biochemistry, molecular biology, pathology, and molecular imaging laboratories concerned with the gene expression as well as location and functions of its products. in this sense, molecular imaging serves as an integrative factor for life sciences endeavors (Albrecht et al. 1992, Bazett-Jones et al. 1996, Hainfeld and Powell 2000, Malecki et al. 1998, Robinson et al. 2000, Malecki 2000). Nevertheless, the essential requirement for pursuit of those projects is availability of molecular markers, which are detectable with various imaging instruments (Malecki 1996). Correlations between positron emission tomography (PET) providing metabolic information, magnetic resonance imaging (MRI) delineating precise anatomical location, and energy filtering transmission electron microscopy (EFTEM) expanding insights into the molecular level are particularly powerful. We have designed three main strategies for pursuit of this project: (a) expression of fusion proteins containing amplified metal binding sites (Malecki et al. 1998), (b) expression of recombinant single chain variable fragment (scFv) antibodies containing elemental recipient (Malecki et al. 2000), and (c) covalent coupling of IgG and Fab with organometallic clusters (Malecki 1996). Herewith, we report the results obtained through imaging of organometallic clusters inserted into the recombinant single chain variable fragment (scFv) antibodies.

A promising aspect of ESI is its application in the detection of elemental labels introduced into biomolecules for cell and molecular biological techniques. Even though colloidal gold labeling for electron microscopy (EM) is highly developed, availability of alternative labels, especially for double or triple labeling applications would be helpful because of difficulties with gold concerning i) detection (gold diameters ≤1nm), ii) discrimination due to gold particle size variations in one size class, and iii) different labeling efficiencies depending on gold granule size. An alternative labeling molecule should contain a high concentration of a specific chemical element which is not or in minor concentrations present in the system under surveillance, and has to have the potential to be discriminated from “biological” elements by ESI.

With respect to ESI, one candidate for elemental labeling is boron. It meets the criteria described above and substantial experience in the synthesis of labeling compounds exists. From the chemical point of view, the preferred labeling structure is a so called dendrimer, a highly branched regular three-dimensional monodisperse macromolecule. Dendritic structures offer a large variety of functionalities to incorporate an element detectable by energy filtering transmission electron microscopy (EFTEM).

Human CD46 (membrane cofactor protein) is expressed on all nucleated human cells and is a cellsurface regulator of activated complement as well as a receptor for both the measles and herpes 6 viruses. CD46 is also expressed in particularly high levels on some tumor cell lines . Complement leads to the recognition of foreign material, targeting it for removal and immune cytolysis. The primary role of CD46 is to bind to one of the complement components, C3b, thereby protecting host cells from autologous complement attack. in addition to its role as a complement regulatory factor, CD46 acts as a measles virus (MV) receptor. Despite the development and use of an effective MV vaccine, at least one million deaths occur each year. MV infection leads to downregulation of cell surface CD46 and immunosupression. MV (and probably also C3b) is internalized shortly after complex formation with CD46, yet, despite much work in this area, precisely how MV enters cells is largely unclear.

The purpose of this research project was to characterize the distribution of left-handed Z-RNA sequences within the epithelial cells of the adult noncataractous crystalline dog lens: the meridional rows (MR). This was achieved by using anti-Z-RNA IgG polyclonal antibody probes. Both light microscopy (LM) and electron microscopy (EM) were used to analyze the tissue binding of the anti- Z-RNA antibodies. Nucleic acids can adopt many different helical conformations (1), such as Z-DNA (Fig. 1) and Z-RNA (2,3). The lens is made up of a single cell type, which is a monolayer of undifferentiated epithelial cells covering its anterior surface (Fig. 2). At the equator of the lens, these cells elongate and form concentric layers of the secondary (nucleated) fiber cells, which undergo cell death-terminal differentiation (denucleation). The epithelial monolayer consists of several cell types, each of which has unique characteristics.

The production of anti-Z-RNA polyclonal antibody probes was achieved using four

The ultimate goal of the immunocytochemistry is to get the best signal with the lowest background while preserving ultrastructure. Unfortunaly too often the experimentator faces a compromise between density of the signal and ultrastructure preservation (1). with the advent of hight resolution confocal light microscopy, two photon imaging and other new technologies there is a need to provide the best immunocytochemical complementary results with the high resolution of the electron microscope. But often for exemple the high level of labelling observed by immunofluorescence microscopy is not matched by immunocytochemistry (2). One approach to obtain comparable levels of labelling is to use comparable protocols. We propose here such a new immunogold method. to illustrate our point we used a very convenient biological material, the adherent cells grown on 35 mm plastic culture dishes. The following procedure was applied : Confluent endothelial cells were fixed in situ for 3 hours with the following freshly prepared and filtered solution : 1 % 1-lysine, 4% paraformaldehyde, 0.04% glutaraldehyde, 0.25% sodium metaperiodate in 0.04M sodium cacodylate buffer, pH 7.4.

To obtain high resolution backscatter electron (BSE) images in field emission SEM (FESEM), one must consider selection of accelerating voltage, beam current, working distance between the specimen and the backscatter detector (in-lens or below-the-lens position for the specimen), the type of BSE detector, and the type of metal used to coat the specimen to improve conductivity and signal collection [1]. A new generation of below-the-lens FESEM have been tested for BSE imaging on biological samples, but no information exists on whether or not high resolution imaging is possible. Here we report the comparison of detection of a colloidal gold standard (6, 12, 18 nm) by high resolution BSE imaging using Autrata-modified YAG detectors in an in-lens FESEM and in a below-the-lens FESEM.

Standards were prepared by mixing colloidal gold particles of 6 nm, 12 nm, and 18 nm. The gold particles were attached via poly-l-lysine to glass chips and coated with <1 nm Pt by ion beam sputtering.

In 1999 Thermo NORAN completed licensing agreements with NIST for the microcalorimeter x-ray spectrometer technology developed by Martinis, Irwin, Hilton and Wollman.Steady progress has been made to commercialize this technology.

Briefly, the NIST microcalorimeter consists of a Transition Edge Sensor (TES) which measures the temperature rise in an absorber due to an incident photon. The sensor must be operated at temperatures below 100 milliKelvin, which is provided by an Adiabatic Demagnetization Refrigerator (ADR), coupled with a traditional liquid helium cryostat. Additional enabling technology includes a Superconducting Quantum Interference Device (SQUID) and a SQUID Array for device readout and low temperature signal amplification. All of this technology has been widely used in cryogenic research facilities for many years, but is not readily available for commercial purchase and use.

Initial work focused on adapting the NIST design to a modern FESEM. The available ports in an FESEM required scaling the heat shields, and cold extension diameters by 68%.

The high performance of single-pixel microcalorimeter EDS (μ,cal EDS) has been shown to be very useful for a variety of microanalysis cases. The primary advantage of jxcal EDS over conventional EDS is the factor of 25 improvement in energy resolution (∽3 eV in real-time). This level of energy resolution is particularly important for applications such as nanoscale contaminant analysis where it is necessary to resolve peak overlaps at low x-ray energies. Because μcal EDS offers practical solutions to many microanalysis problems, several companies are proceeding with commercialization of single-pixel μal EDS technology. Two drawbacks limiting the application of uxal EDS are its low count rate (∽500 s−1) and small area (∽0.04 mm for a bare single pixel, ∽5 mm2 with a polycapillary optic). We are developing a 32x32 pixel array with a total area of 40 mm2 and with a total count rate between 105 s−1 and 106 s−1.

The concept utilized in charge coupled devices (CCD’s) for detection and imaging of light signals involving lateral movement of charges and extremely low capacitance of the detector and readout electronics has spawned a variety of new ideas in the design of nuclear detectors. Initially, silicon drift detectors (SDD’s) were developed for high energy physics applications. More recently, a vigorous effort to develop new structures for x-ray spectroscopy and light detection has started. Drift structures have been designed in a variety of topologies and materials (such as Si, CdZnTe, and HgI2) to satisfy the requirements of many different applications. The most interesting features that can be achieved with drift structures include: a) Large active area devices with low capacitance and low electronic noise, b) Very high signal throughput, c) Operation at or near room temperature, d) High sensitivity over the large entrance electrode to low energy xrays and short wavelength light, f) Single carrier charge collection allowing for elimination of hole contribution to the spectral broadening in compound semiconductor detectors such as HgI2, CdTe, and CdZnTe, f) 2D resolution of few tens of micrometer in both directions over few cm2 active areas, and g) Possibility of using more sophisticated schemes of charge collection by switching between integration and drift mode.

The analysis of samples with a scanning electron microscope often requires element distribution images. Until recently, element mapping was strongly limited by the count rate that could be processed with conventional EDS systems using Si(Li) or Ge crystal diode detectors. The enhancement of secondary and backscattered SEM images can now be achieved through the addition of rapid color imaging. Using a new methodology for color enhancement, features as small as 1 micrometer can be colored and easily distinguished. The addition of color to SEM images enables easy visualization of element distributions and allows the microstructural features and compositional variations of different materials to be more readily identified. Persons unfamiliar with electron microscopy can more readily interpret color images than black and white or gray scale images. This new technology for imaging elements has great potential to revolutionize electron microscopy.

The detector technology behind this new methodology is the silicon drift diode (SDD).

As scientists look to determine elemental composition of smaller and smaller particles using a scanning electron microscope, the usefulness of conventional EDS detectors (Si(Li)) becomes limited. to obtain analytical information from the SEM on extremely small particles the analyst can reduce the excitation energy of the electron beam; excitation energies of around 3kV are regularly used for the analysis of these particles. with only low energies (up 3keV) to work with, the scientists have to use L, or in some cases M, spectra to determine the chemical composition of the particle. A standard Si (Li) with a resolution of ≈100eV @3keV (130eV at 6keV) will be unable to adequately resolve spectra for confident characterization work to be carried out for these low energy lines. A new generation of EDS detectors for microanalysis is required to fulfill the scientists requirements for fast, simultaneous chemical determination of these small particles.

Low temperature heterogeneous catalytic routes for polymers are of considerable interest in the chemical sciences and technology because they are economical and environmentally beneficial. However such routes have been difficult because of an incomplete understanding of process control and low yields. Currently, hydrogenation of aliphatic dintriles in solvents is used in the chemical industry to manufacture the corresponding diamenes which are subsequently reacted with adipic acid solutions and polymerized to produce the polyamide, nylon (6,6).

Here we report an alternative, low temperature heterogeneous catalytic process for the polymerization reactions using novel environmental-HRTEM (EHREM) in liquid environments. EHREM under simulated reaction conditions provides direct, in situ real-time information on the dynamic structural and chemical changes and reaction modes of operation. We prepared high surface area heterogeneous catalysts including cobalt-ruthenium nanoclusters supported on rutile titania using a single step sol-gel technique, (shown in Fig. 1).

The crystallographic orientation and atomic termination layer of oxide catalysts are known to influence their reactivity. The objective of this work was to measure how the relative photochemical reactivities of two ternary titanates vary with surface orientation and composition. The surface structure property relationships derived from these observations can be used to define optimized photocatalyst microstructures.

To measure the relative reactivity of surfaces with different orientations, crystallites (∽ 20 μm in diameter) in a polycrystalline ceramic were examined individually. First, polycrystalline specimens of BaTiO3 and SrTiO3 were synthesized using conventional ceramic processing. Surfaces for analysis were prepared by polishing and thermal etching in air. The surface orientations of selected grains in the sample were then determined from backscattered electron diffraction patterns recorded in a scanning electron microscope. to make a local measurement of the photochemical activity of each grain, we used a well established probe reaction (the reduction of aqueous Ag+ to Ag0) that deposits metallic silver on the surface as a reaction product.

Combustion of natural gas for power generation leads to NOx formation due to the high temperatures encountered. Catalytic combustion allows the entire combustion process to be completed at temperatures where NOx formation can be avoided. The catalyst of choice is supported PdO. As temperature is increased, PdO decomposes to Pd metal with profound effects on catalyst reactivity. Persistent hysteresis in reaction rates have been related to the decomposition of PdO into Pd and its reformation.

Understanding and controlling the phase transformations, and the resulting activity variations, is of enormous importance for high temperature combustion catalysts where predictable catalyst activity is necessary for stable operation. Farrauto et al. studied the phase transformations of PdO to Pd using thermogravimetric analysis (TGA). They concluded that while PdO decomposes to Pd at high temperatures during the heating cycle, upon cooling the Pd does not transform to PdO till the temperature drops by several hundred degrees.

New EPA regulations limiting the emissions of NOx from diesel engines will require a reduction by 70% in the amount of NOx leaving the tailpipe of diesel powered vehicles by the year 2004. Some form of exhaust aftertreatment will need to be applied to diesel engines to meet the new stricter requirements. One option is to develop a “lean-NOx” catalyst that uses a hydrocarbon reductant (e.g. diesel fuel) to convert NOx into N2 and O2. Two of many constraints on such a catalyst are that it achieve a high degree of conversion over a fairly broad temperature range and that the amount of reductant necessary to reach the 70% goal is sufficiently small as to be cost effective. A number of promising systems comprise either platinum group metals or base metals on oxide supports. Such catalyst systems typically involve near-atomic dispersions, or ultra-fine clusters, of the heavy metal species on the oxide support.

Recently a number of reports have been made in which direct methods (DM) were used to solve bulk inorganic crystal structures with electron diffraction (ED) data, either ab initio or in a combined approach using phase information from images. This paper reports ab initio application of DM to experimental ED data from the zeolite mordenite. Data from mordenite [001] were taken using a JEOL 3010 TEM at 300 kV. For collecting data, a small condenser and spot size were used to obtain a defocused probe 50 nm in size positioned over the edge of a thin area. Patterns were recorded with a CCD camera. Fig. 1 compares experiment with modeled kinematical data based on a published structure. Phase determination by DM was performed with the minimum relative entropy approach. As a test, ab initio DM was performed on modeled kinematical data.The result, shown in Fig. 2, is not distinguishable from a picture of the projected potential.

Heterogeneous catalysts are extremely important to the chemical industry. The goal of much current research is to develop predictive models for real world catalysts in order to optimize and exploit new processes. The first step in any predictive modeling is to understand the systems that are currently in use. A key question regarding these materials is the distribution of atoms within the catalyst particle, i.e., are the materials equally distributed within the particle or is there clustering or shelling of the constituents. This is a more general question than just catalytic systems and has applicability to many small cluster problems where normally immiscible systems can be forced to mix or order when confined to small volumes.

EXAFS analysis has provided useful information for these systems; due to the small scattering cross-section and large beam sizes of x-rays, the systems have only been studied as ensemble averages.