The serial detectors traditionally used in Electron Energy Loss Spectroscopy (EELS) have mostly been replaced by parallel detection systems, for reasons of efficiency and ease of use. So far most parallel detection systems have been based on fiber or lens coupled Photo Diode Array (PDA). Although these systems have proven satisfactory they have some limitations: 1) high readout noise, 2) limited correction for gain variations and 3) point spread function or cross talk between channels all of which effects the ultimate sensitivity and resolution. This paper discusses a newly developed parallel detector for EELS based on a CCD and a new type of scintillator and fiber technology. The new detector shows large improvements in all areas of performance.

The sensitivity of a detector is limited by the readout noise at low incident electron dose and the gain correction at high dose. Both these areas have been addressed in the new design. The readout noise is equivalent to approximately 1/2 a primary electron, about 40 times better than a typical PDA.

The electron-optical design and the technical realisation of correctors, monochromators and imaging spectrometers as well as methods of resolution improvement (Focus variation, Holography) have made significant progress in the past few years. For all such devices and methods the mechanical and electrical stability of the microscope are the main challenges.

Such requirements are even more critical in case of Energy Filtering Transmission Electron Microscopes (EFTEMs), where the achievement of highly resolved analytical results requires long detection times (in the range of a few 10s of seconds). Resolution in such case could mean both spatial and energy resolution even at the same time. The stability performance of the combined system is more complex than the sum of the stabilities of the single components. Thus, a specified HT short- and long-term stability of 2 ppm is not sufficient to achieve an energy resolution of 0.2 eV at 100 kV acceleration voltage.

Elemental microanalysis has been important in materials characterization, since the elemental distribution strongly affects the property of various materials. A recently developed post-column energy filter coupled with a slow scan CCD camera makes it possible to carry out elemental mapping with a transmission electron microscope. Here, we develop the elemental mapping technique utilizing the omega filter and imaging plates (3760x3000 pixels). Since the data obtained from the imaging plates consist of a large number of pixels, fine and detailed elemental analysis will be expected.

Energy-filtered images were obtained by a JEM-2010 electron microscope installed with an omega-type energy filter, and they were recorded on imaging plates (FDL-UR-V:25 μm/pixel). The width of an energy-selecting slit was set to be 20 eV. Elemental maps were obtained from the energy-filtered images using the three window technique. Special care was taken to reduce the image shifts among the three filtered images used in the three-window method.

To characterize polymers by transmission electron microscopy it is necessary to use selective staining techniques in order to obtain meaningful results. The combination of a transmission electron microscope with an energy filter system as in the LEO 912 Ω however provides additional analytical information which in particular cases allows imaging without the necessity of staining. Especially a π-π*-resonance at about 6 eV in the low loss electron energy loss spectrum of polymers containing aromatic groups is suitable for imaging. These energy loss electrons are selected by a 2 eV slit in the energy dispersive plane of the spectrometer to form an image. This special type of energy spectroscopic imaging (ESI) can also be referred to as chemical imaging.

This paper presents morphological results on polymer systems containing aromatic components. Micrographs of ultra thin sections were acquired with a 2 eV energy slit at 6 eV electron loss in a LEO 912 π

The superplasticity exhibited by some fine-grained ceramics, notably 3-mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP), offers interesting possibilities to lower manufacturing costs by near net shape forming. Both glassy and crystalline materials have been added to pure 3 Y-TZP to limit grain growth and promote grain boundary sliding during sintering and isostatic pressing. EDS spectrum lines have been used to characterize the distribution and extent of additive phases, but were not able to map some light-element components of additives (e.g., O and B in borosilicate glass). Additionally, quantification of the Si Kα peak was compromised by overlap with the Y L and Z L peaks. To both improve light element sensitivity and investigate potential changes in oxygen bonding at the grain boundaries, undoped 3 Y-TZP and 3 Y-TZP powders processed with 1 wt% barium silicate have been examined with TEM spectrum lines. Additionally, some elemental mapping has been performed. TEM specimens were examined in a 300 kV LaB6 Philips CM30T equipped with a Gatan imaging filter (GIF) at the ORNL SHaRE User Facility.

While transmission and beam damage have been well documented in the TEM at high accelerating potentials, considerably less is known about these phenomena at lower accelerating potentials. Imaging using low accelerating potentials may be attractive as an analytic technique, because chemical degradation associated with electron beam irradiation in organic macromolecules may be a function of accelerating potential (e.g. 1, 2). The present work examines low-energy electron transmission through thin polymer films and beam damage produced by low energy (∼ 0.5-10 keV) electrons.

Thin films of polystyrene were prepared by allowing a ∼ 0.005% solution of G3-5k arborescent polystyrene in toluene to evaporate on a holey carbon film. In solution, this arborescent polystyrene consists of spherical molecules of order 45 nm in diameter (3). When evaporated from dilute solution on a holey carbon film, monolayer thick self-assembled membranes form in some of the holes (figs. 1 and 2). The resulting films are highly reproducible in thickness and have a relatively uniform morphology (fig. 3).

The common experimental method to determine the total inelastic mean free path i by electron energy-loss spectroscopy (EELS) is by the relation : t/λi= ln(It/IO) [1] where t is the specimen thickness, It, is the total integrated intensity, and Io is the intensity of the zero-loss peak. The accuracy of this measurement depends on the thickness determination. Model geometries like cubes, wedges, and spheres enable accurate thickness determination from transmission images.

Spherical polymers with diameters of order 10-200nm can be made from a number of high-Tg polymers by solvent atomization. This research studied atomized spheres of poly(2-vinyl pyridine) [PVP]. A solution of 0.1% PVP in THF was nebulized. After solvent evaporation during free fall within the chamber atmosphere, solid spherical polymer particles with a range of diameters were collected on holey-carbon TEM grids at the bottom of the atomization chamber.

Titanium nitride coatings were grown in a physical vapor deposition system assisted by a direct current reactive magnetron sputtering technique. The vacuum chamber was evacuated with a mechanical pump and cryopump to a base pressure of 10-7 Torr. The Sputtering was performed with a direct current high voltage source (0-1 Kev and 1 A) on a titanium target (99.98% purity). The titanium target was sputtered with a high purity argon -nitrogen mixture. The films were deposited on monocrystalline silicon (mc-Si) (111) substrates at different nitrogen partial pressures from 0.08 mTorr to 1.5 mTorr. Total pressure, power applied to target and substrate temperature were keep constant in all the experiments.

EXEELFS analysis were done using the standard procedure [1,2]. In this case, we are find that the atomic concentration is in good agreement with the respective established stoichioinetry N/T=0.99. Figure 1 shows the window of high energy loss where appears their respective N K-edge and Ti -L23.

Quantitative off-axis electron holography in a transmission electron microscope (TEM) requires careful design of experiment specific to instrumental characteristics. For example, the spatial resolution desired for a particular holography experiment imposes requirements on the spacing of the interference fringes to be recorded. This fringe spacing depends upon the geometric configuration of the TEM/electron biprism system, which is experimentally fixed, but also upon the voltage applied to the biprism wire of the holography unit, which is experimentally adjustable. Hence, knowledge of the holographic interference fringe spacing as a function of applied voltage to the electron biprism is essential to the design of a specific holography experiment. Furthermore, additional instrumental parameters, such as the coherence and virtual size of the electron source, for example, affect the quality of recorded holograms through their effect on the contrast of the holographic fringes.

We simulated in-line holograms as obtained from a point projection microscope (PPM) [1]. The principal of the PPM is shown in Fig.1. A coherent electron wave emitted from a point source illuminates a sample transparent to the wave and travels to an electron detector. Since the wave from a point source is divergent the distance d of two points in the object plane results in a magnified distance D in the detector plane. The magnification of this arrangement is given by M=D/d=(z1+z2)/z1, where z1 and z2 are the tip to sample distance and the sample to detector distance, respectively. As no lenses are used we find a highly defocused shadow image in the detector plane. This arrangement is equivalent to Gabor's in-line holography with a physical source in plane of a lens-focused crossover, and the out-of-focus images are in-line electron holograms.

Microsomal glutathione transferase 1, MGST1, belongs to a recently characterised superfamily named MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) which has evolved divergent functions to protect against reactive lipid intermediates (lipid hydroperoxides and hydroxyalkenals) or indeed harness these molecules for key physiological functions (leukotrienes and prostaglandins). The most thoroughly studied member of this superfamily is MGST1 which is an abundant detoxication enzyme displaying both glutathione transferase and peroxidase activities.

The MGST1 monomer has a molecular mass of 17.5 kD and three to four transmembrane regions can be localized from hydropathy plots (Fig. 1). One transmembrane stretch was shown by trypsin cleavage to be situated between lysine-4 and lysine-41 with the N-terminal on the lumenal side of the membrane. The active site is located on the cytoplasmic side of the endoplasmic reticulum.

Because membrane proteins are localized in a continuous lipid bilayer in the native environment, the situation of membrane proteins in the two-dimensional (2D) crystals is quite similar to the environment in vivo. Thus, electron crystallography using 2D crystals is one of the suitable techniques for structure determination of membrane proteins at atomic or near-atomic resolution. Here we describe the structures of the two membrane proteins, bacteriorhodopsin and aquaporin-1, which were solved by electron crystallography at 2.5 and 4.0 Å resolution, respectively.

Bacteriorhodopsin (bR) is a light-driven proton pump found in Halobacterium salinarium. The atomic model of the protein was first proposed by electron crystallography and we improved the resolution of the structure determination up to 3.0 Å by collecting 366 electron diffraction patterns and 129 images. The resulted map showed not only a bR molecule but also eight surrounding lipids in the asymmetric unit.

Microtubules are involved in many activities within the cell that require highly dynamic activity. For examples, microtubules can grow and shrink as they explore the cell, and the entire microtubule cytoskeleton is restructured as cells prepare for division. Interfering with microtubule dynamics can have serious consequences for the health of the cell, especially in cells that are rapidly dividing. The result of disrupting the normal dynamics is generally an interruption of the cell cycle and consequent induction of apoptosis. This behavior has been exploited with a number of anti-cancer drugs that target tubulin, the main protein in microtubules. Drugs are currently in use that either stabilize or destabilize microtubules. Among the most widely used is Taxol, one of the microtubule stabilizing drugs. Because Taxol produces a range of serious side effects and a substantial fraction of patients treated with Taxol eventually develop resistance to the drug, there is an active search for other drugs that might be more selective and less prone to resistance.

The Na+,K+-ATPase is a transmembrane protein, located in the plasma membrane of virtually all animal cells, which controls Na+ and K+ gradients. It is a member of the P-type ATPase family of ion pumps, a group of enzymes which pump ions against a concentration gradient, forming a phosphorylated intermediate during the pumping cycle. For each mole of ATP hydrolysed, 3 Na + ions are moved out of the cell and 2 K+ ions are moved into the cell. Unlike most other members of this family, which have one subunit, Na+, K+-ATPase is a heterodimer of α and β subunits. The a subunit consists of 1020 amino acids and has been predicted to have 10 membrane-spanning a-helices as well as a large cytoplasmic headpiece which forms the ATP binding and phosphorylation site. The α subunit, 300 amino acids in length, has one membrane spanning helix and has most of its mass located on the extracellular side of the membrane.

The amino acid substitution (B Glu → G6 Val) results in the conversion of Hemoglobin A (HbA) to sickle cell hemoglobin(HbS) which is responsible for sickle cell disease. Under physiological conditions this substitution causes a reduction of the solubility of HbA from about 340 mg/ml to 165 mg/ml (for HbS). One consequence of the reduction in solubility is that HbS polymerizes to form long fiber like structures about 240Å in diameter. The formation of these fibers causes sickle cell disease. The fibers fill the red cell and cause it to assume a characteristic sickle shape. More significantly the fibers cause the red cell to become rigid and, as a result, sickled cells can block blood flow in the capillaries. Understanding the polymerization process in detail is important for understanding the pathophysiology of sickle cell disease and for developing a specific therapy that could be used in its treatment.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50-μum process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We have shown that small segments along the bundle can be treated as single crystals with a unit cell spacing of 144 × 144 × 766 Å. A tomographic reconstruction of the bundle was done from multiple tilt series of images to ∼40 Å resolution. To extend the structural determination of the bundle at a higher resolution, we have used electron crystallographic analysis of single images of the bundles preserved in vitreous ice. Furthermore, we did not employ any helical or crystallographic symmetry (other than PI) in the reconstruction procedure.

Acrosomal bundles from Limulus sperm cells were purified as described earlier. Images of frozen hydrated bundles were taken at 40,000x EM magnification in a JEOL 4000EX electron cryomicroscope using an electron dose of 6 - 10 electrons/Å2.

α-Actinin is an actin crosslinking protein identified in a wide variety of cells. Both muscle and nonmuscle isoforms of α-actinin have been characterized [1]. The molecule consists of two polypeptide chains that form a rod-shaped antiparallel dimer. Each polypeptide is composed of three distinct structural regions: actin-binding domain, four triple helical repeats that form a central rod, and a carboxyl terminal domain that contains two EF hand calcium-binding sites. Here we present the 3D structure of the smooth muscle α-actinin, as determined by cryo electron microscopy at 2.0 nm resolution.

Two dimensional crystals of chicken gizzard α-actinin were formed on positively charged lipid monolayer [2] and preserved frozen hydrated for TEM. The crystal has a unit cell dimension of a = 26.31 nm, b = 20.37 nm, γ= 107.10°, based on internal calibration against TMV.

Gap junctions are composed of a multigene family of proteins called the connexins with a similar sequence and folding topology. Connexons, half-channels made from a particular connexin, will dock and form functional channels with some, but not all, connexons made of other isoforms. This is surprising considering that the primary sequences of the docking domains are conserved and in some cases there are limited amino acid changes in the extracellular loops between compatible connexins. Therefore, some tertiary or quaternary interactions between the extracellular loops of the two docking hemichannels must contribute to the compatibility of a connexon assembled from one connexin for a connexon made from a different connexin. The 3D structures of gap junctions composed of only three isoforms have been investigated with only one case of a gap junction structure at better than 1 nm resolution [1]. The rat liver hemichannel structure is the only one that contains an exposed extracellular surface [2].

α-Actinin is a member of the spectrin superfamily of actin crosslinking proteins. The molecule is an antiparallel homodimer with a polypeptide chain weight of 94-103 kDa. Each chain can be divided into three domains: the N-terminal 250 amino acids forms an actin binding domain, the central domain consisting of four spectrin-like, triple-helical repeats and the Cterminal which contains two EF hand motifs (Baron et al, 1987).

Two models have been proposed for the alignment of the triple helical repeats in the α-actinin structure, an aligned model (Baron et al., 1987) and a staggered model (Taylor and Taylor, 1993). In order to resolve the controversy, we proceeded with the cryo-EM 3D reconstruction from 2D crystal grown on a positively charged lipid monolayer. From rabbit erector spinae aactinin, we obtain better ordered 2D crystals from which we have calculated a 3D reconstruction to ∼15 Å resolution.

Myelin basic protein (MBP) is one of the principal constituents of the mammalian myelin sheath. It is a basic peripheral membrane protein in the major dense line and is believed to play a structural role in maintaining myelin stability through its close association with lipids and other proteins. Immune responses to MBP have been implicated in the pathogenesis of multiple sclerosis (MS), the most common autoimmune disease of the central nervous system in North America and Northern Europe. The correlation between the severity of MS and the deimination of arginyl to citrullinyl residues in MBP was well illustrated in the acute case of MS (Marburg type) that contained MBP with 18 out of 19 arginines citrullinated.

We have studied a recombinant hexahistidine-tagged murine MBP (rmMBP, 18.5 kDa isoform) by electron microscopy of the protein organized as planar arrays on lipid monolayers that consisted of the nickel chelating lipid, l,2-dioleoyl-.yn-glycero-3-[(N(5-amino-l-carboxypentyl)iminodiacetic acid)succinyl] (Nickel salt) (Ni2+-NTADOGS), and the filler lipid, liver phosphatidylinositol (PI).