Siemens Elmiskop Research Articles

The chronicles of coronaviruses: the electron microscope, the doughnut, and the spike

The advancement of medicine owes in large measure to a German engineer Ernst Ruska, whose invention of transmission electron microscope in 1931 won him the 1986 Nobel Prize in Physics, when it comes to infectious diseases. Encouraged by his physician brother Helmut Ruska to use the prototype instrument for the study of viruses, the course of virology was shifted to a different and unprecedented level. Virus could then be seen, identified and imaged. The University of Maryland happened to acquire an American model of transmission EM, the RCA EMU, using which the first structural study was done for the first known coronavirus (then was simply known as infectious bronchitis virus) in 1948. The virus was described as rounded bodies with filamentous projections. The magnification was not great and the resolution was poor. The study was followed by a series of studies using improved techniques and better EM spanning the next decade. An upgraded version RCA-EMU2A gave better images in 1957 and the virus was described as doughnut-like structure. Using Siemens Elmiskop, D.M. Berry and collaborators made the first high-resolution pictures in 1964. The thick envelope which gave doughnut-like appearance and filamentous projections reported before could be discerned as discrete pear-shaped projections called the spikes. These spikes form a corona-like halo around the virus, which were also seen in novel human viruses (B814 and 229E) that caused common colds. The discoverer of B814, David Tyrrell and his aid June Almeida, a magnificent electron microscopist, established that IBV, B814 and 229E were of the same kind of virus in 1967, which prompted to create the name coronavirus in 1968. This article further highlights the detail structural organisation of coronaviruses emanating from these pioneering research.

The ultrastructure of the muscle coat of human gastro-oesophageal junction, with special reference to "interstitial cells of Cajal".

The muscle coat of the human lower oesophageal sphincter and stomach was studied 5 cm above and 4 cm below the gastro-oesophageal junction. Four subjects were operated on for motility disorders of the esophagus, two for a hypertensive lower oesophageal sphincter and two for an epiphrenic diverticulum; six subjects were operated on for oesophageal or gastric carcinomas. Specimens were fixed in phosphate-buffered OsO4, embedded in Epon, contrasted with uranyl acetate and lead citrate and observed under a Siemens Elmiskop Ia electron microscope. Both the oesophageal and gastric muscle cells, which showed features typical of this cell type, were innervated by multiple varicosities that were rich in synaptic vesicles; these varicosities were generally rarely encountered at distances less than 1000 Å from muscle cells. Only a very few, close neuromuscular junctions were detected. Special cells, which correspond to the “interstitial cells of Cajal” as reported by other authors, were discerned at the periphery of muscle cell bundles. These cells were characterized by an elongated cell body with many thin branches and an oval, sometimes indented nucleus. Some pinocytotic vesicles were located at the cell periphery. These cells were surrounded by a discontinuous basal lamina and were seen in close contact with each other and with muscle cells; the close contact areas were often very wide. The cytoplasm contained variable amounts of mitochondria, a well-developed smooth endoplasmic reticulum and a Golgi complex. As a characteristic feature, bundles of thin filaments were located at the cell periphery and were attached to electron-dense areas of the cell membrane. Morphologically, these filaments resembled myofilaments; they were present in variable amounts and were sometimes very numerous. The observation that the cytoplasmic organelles and filaments varied in number, is probably related to the different functional properties of these cells. Interstitial cells were richly innervated by varicose nerve fibers that were densely packed with synaptic vesicles; many close junctions to nerve endings were also detected. These morphological data lead us to assume that the interstitial cells demonstrated by the electron microscope do not correspond to the cells initially identified by Cajal and cannot even be considered connective tissue cells. We propose that they are specialized smooth muscle cells that are involved in generating spontaneous, myogenic electrical activity in the gastrointestinal tract.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

Active and Passive Mechanisms Drive Secretory Granule Biogenesis during Differentiation of the Intestinal Parasite Giardia lamblia

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.

Methylation of the Imidazole Side Chains of the Alzheimer Disease Amyloid-β Peptide Results in Abolition of Superoxide Dismutase-like Structures and Inhibition of Neurotoxicity

The toxicity of the amyloid-beta peptide (Abeta) is thought to be responsible for the neurodegeneration associated with Alzheimer disease. Generation of hydrogen peroxide has been implicated as a key step in the toxic pathway. Abeta coordinates the redox active metal ion Cu2+ to catalytically generate H2O2. Structural studies on the interaction of Abeta with Cu have suggested that the coordination sphere about the Cu2+ resembles the active site of superoxide dismutase 1. To investigate the potential role for such structures in the toxicity of Abeta, two novel Abeta40 peptides, Abeta40(HistauMe) and Abeta40(HispiMe), have been prepared, in which the histidine residues 6, 13, and 14 have been substituted with modified histidines where either the pi- or tau-nitrogen of the imidazole side chain is methylated to prevent the formation of bridging histidine moieties. These modifications did not inhibit the ability of these peptides to form fibrils. However, the modified peptides were four times more effective at generating H2O2 than the native sequence. Despite the ability to generate more H2O2, these peptides were not neurotoxic. Whereas the modifications to the peptide altered the metal binding properties, they also inhibited the interaction between the peptides and cell surface membranes. This is consistent with the notion that Abeta-membrane interactions are important for neurotoxicity and that inhibiting these interactions has therapeutic potential.

Staining terminal cisternae proteins in rat skeletal muscle triads: Selectivity of a new silver technique

A selective silver staining of proteins within the lateral components of triads and diads in fixed striated muscle cells and in isolated sarcoplasmic reticulum vesicles using a mercuric acetate postflxation has been demonstrated by us.In the present work we modify the technique in order to study the mechanism of the reaction . First, the mercuric postfixation step was omitted and replaced by a treatment with diluted ammonia, a procedure thought to wash out the excess of the reducing aldehyde fixative. Second, a treatment with mercuric acetate was employed on the ammonia treated muscles. The rat gastrocnemius muscle was fixed by immersion (12-24 h) in 3% glutaraldehyde, washed briefly with water and placed (12-24 h) into an ammonia:distilled water (1:4 v/v) solution. After washing with water, 1% acetic acid and water, muscles were treated overnight with 50% (v/v) ammoniacal silver nitrate at 37°C in darkness, washed with three changes of 5% (w/v) sodium sulphite and water, embedded in Poly B, sectioned and examined unstained with a Siemens Elmiskop I electron microscope.

Characterization of germ cell nuclei in freeze-fracture replicas using seminiferous tubules isolated by transillumination

The present investigation was under taken, combining the identification of specific germ cell types present in a particular stage of the seminiferous wave with freeze-fracture techniques to follow the changing pattern of their nuclear pore distribution during spermatogenesis. Male albino adult rats were used. Segments of seminiferous tubules were separated (stages VII-VIII; IX-XI; XII-XIV) by a technique described by Parvinen and Vanha-Perttula where the stages of the cycle were recognized and isolated by transillumination under a dissecting microscope. The tubular segments were immediately fixed in glutaraldehyde, immersed in glycerol, freeze-fractured in a Balzers BAF 301 apparatus at-110°C and examined under a Siemens Elmiskop I EM. The only spermatogonia located in the studied stages (type A) are ovalcells in contact with the basal lamina. Pore distribution is non-random with a relatively in conspicuous aggregation. Preleptotene spermatocytes (stages VII-VIII) are easily identified by being numerous, their location over the basal lamina and the irrelatively small size. They exhibit a striking clustering of pores, in groups of 15-30 separated by pore-free areas (fig.1). Leptotenes (fig.2, 3) (stages IX-XI) and zygotenes (fig.4) (XII-XIII) change the pattern to a more even distributional though clearly maintaining some degree of clustering.

Diffusion of TEM grid material into amorphous silicon films

While investigating the silicide formation reaction between thin bilayers of amorphous Si (a-Si) and Cr, an unintended interaction occurred between the specimens and the supporting TEM grids. In an attempt to understand this phenomenon, the interaction between a single film of a-Si and TEM grids made of Cu, Ni, Au and Mo were investigated by in situannealing in a Siemens Elmiskop 101. The 60 nm films, prepared by e-beam evaporation of pure Si onto glass substrates, covered with an acetone-soluble release agent, were amorphous.In Fig. 1 the interaction between the a-Si film and a Cu TEM grid is shown. Areas I, II, III and IV in (a) show different stages of the interaction, area IV being closest to the Cu grid bar, and area I being pure a-Si. The reaction started at about 600°C by crystallisation of the a-Si film at different spots in contact with the grid bars, and fanned out from these nucleation sites (area II).

Study of dislocation loops in Arsenic-Rich as-grown GaAs

Grown-in dislocations in GaAs have been a major obstacle in utilizing this material for the potential electronic devices. Although it has been proposed in many reports that supersaturation of point defects can generate dislocation loops in growing crystals and can be a main formation mechanism of grown-in dislocations, there are very few reports on either the observation or the structural analysis of the stoichiometry-generated loops. In this work, dislocation loops in an arsenic-rich GaAs crystal have been studied by transmission electron microscopy.The single crystal with high arsenic concentration was grown using the Horizontal Bridgman method. The arsenic source temperature during the crystal growth was about 630°C whereas 617±1°C is normally believed to be optimum one to grow a stoichiometric compound. Samples with various orientations were prepared either by chemical thinning or ion milling and examined in both a JEOL JEM 200CX and a Siemens Elmiskop 102.

Rapid freezing, cryosectioning, and x‐ray microanalysis on cardiac muscle preparations in defined functional states

AbstractElectrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosections are cut with a LKB cryoultramicrotome at temperatures of −130 to −140°C, freeze‐dried at 10−6 Torr vacuum and carbon‐coated before analysis. The freeze‐dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMol/kg d.w.) of Na, Mg, P, S, Cl, K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x‐ray microanalysis can be successfully applied to heart physiology.

Optimized Energy Dispersive X‐Ray Microanalysis With A Siemens Elmiskop 102 Electron Microscope

SUMMARYThis paper describes modifications made to the objective lens of a Siemens Elmiskop 102 electron microscope which enable a Kevex Si(Li) detector to be brought to within 20 mm of the optic axis, and which give rise to a relatively‘clean’ X‐ray microanalysis system. The modifications do not affect the performance of high resolution, double‐tilt capability of the instrument.

Counting superhelical turns in electron micrographs of large plasmids

Dark-field electron microscopy allows high resolution imaging of super- coiled DNA. Plasmid molecules isolated from Halobacterium halobium were spread by basic monolayer techniques with cytochrome c. They were subsequently mounted onto thin indirectly deposited carbon films and positively stained with ethanolic uranyl salts. A Siemens Elmiskop 102 was operated at 10 000 and 40 000 times magnification using tilted and hollow cone dark-field illumination respectively. Micrographs obtained (Fig. 1) allowed contour length determination by tracing molecules and direct counting superhelical turns. This led to calculation of the superhelical density of large plasmids. The contour length of supercoiled and of relaxed circular molecules which were contained within the same spreading solution and thus photographed from the same grid were evaluated by projection of micrographs and tracing with a digitizer connected to a Wang 2200 calculator. The values obtained are (49.1 ± 2.5) μn and (52,7 ± 2.2) μm for superhelical and relaxed circles respectively.

Quantitative transmission electron microscopy of small agglomerates in HF-O

High amounts of gases or carbon are soluble on interstitial sites of the hexagonal lattice of hafnium. The gas atoms influence nearly all physical properties; e.g., the critical resolved shear stress τ (CRSS) is drastically increased with an increasing content of gas atoms. Different authors measured the temperature dependence of τ for different 0 concentrations (cO ≲ 1.5 at%). The CRSS increases strongly with decreasing measuring temperature T. Ruano and Elssner concluded intuitively from the non continious shape of the τ-T curve, that besides the interstitially solved single gas atoms also agglomerates of gas atoms should be present in the specimens. These agglomerates act as strong barriers against plastic deformation. - A superposition of the 2 types of barriers (single interstitial gas atoms and agglomerates) could explain the shape of the τ-T curve. TEM studies were performed to prove this assumption of the existence of agglomerates. Hafnium specimens with different 0 content (0.13, 0.65 and 1.17 at%0) were electrolytically thinned and investigated with a Siemens ELMISKOP 102 operated at 125 kV.

Transmission electron microscopy of SI-AL-O-N alloys

It is expected that Si-Al-O-N alloys are important high temperature construction materials. The phase diagrams for Si-Al-O-N alloys were studied systematically mainly by X-ray diffraction work (for a summary see). Different stable phases were found. For the understanding of the physical and mechanical properties it is of great interest to know for the different stable phases the microstructure and the morphology, which can be obtained by TEM observations. Results of some TEM studies are reported here utilizing not only the conventional TEM but also the lattice fringe imaging technique.Specimens of the different phases were produced as described in They were prepared for TEM observations. For high resolution work a Siemens ELMISKOP 102 (operating voltage 125 kV) was used fitted with a double tilting stage (± 45°), for conventional TEM studies the specimens were examined in an AEI EM7 high voltage EM operated at 1 MeV.

Transmission electron microscope studies in special ceramics

The use of high temperature special ceramics which are usually complex materials based on oxides, nitrides, carbides and borides of silicon and aluminum, is critically dependent on their thermomechanical and other physical properties. The investigations of the phase diagrams, crystal structures and microstructural features are essential for better understanding of the macro-properties. Phase diagrams and crystal structures have been studied mainly by X-ray diffraction (XRD). Transmission electron microscopy (TEM) has contributed to this field to a very limited extent; it has been used more extensively in the study of microstructure, phase transformations and lattice defects. Often only TEM can give solutions to numerous problems in the above fields, since the various phases exist in extremely fine grains and subgrain structures; single crystals of appreciable size are often not available. Examples with some of our experimental results from two multicomponent systems are presented here. The standard ion thinning technique was used for the preparation of thin foil samples, which were then investigated with JEOL 200A and Siemens ELMISKOP 102 (for the lattice resolution work) electron microscopes.

Ultrastructural Description of three Different Epithelial Cell Types in Rat Mammary Gland.

The mammary gland is composed of epithelial and myoepithelial cells. In the present work, we show that the epithelial component is composed of three types of cells differing in their ultrastructural profiles.Sprague-Dawley virgin female rats, 60 to 100 days old, were sacrificed during estrus. The mammary gland was fixed in Karnovsky's fluid (1), embedded in Epon-Araldite (2), thin-sectioned, stained with uranyl acetate and lead citrate, and examined in an Hitachi HU-11A or a Siemens Elmiskop 1A. To see whether ultrastructural differences correspond to size differences, the nuclear and cytoplasmic areas of 1000 cells were determined from photographs at 10,000X, using a compensating polar planimeter.The population of epithelial cells in the mammary gland is comprised of three different cell types (Fig. 1): a) light cells, b) dark cells, and c) intermediate cells.

Magnification in electron-shadow microscopy

An accurate and reproducible determination of the magnification in point-projection or electron-shadow microscopy is obtainable by using as a reference the objective lens current required for infinite magnification. The magnification at any other objective lens current is then determined as a function of the difference between that current and the current at infinite magnification. It is generally a simple matter to choose a reproducible current difference. A replica of a crossed grating with line spacing of 0.463 mu m was used as a test specimen in experiments with a converted Siemens Elmiskop 1. The relationship, magnification=8720/n, where 12.8 n mA is the current difference, was found to hold to better than 3%.

A temperature controlled hydration or environmental stage for the Siemens Elmiskop 1A

A hydration stage is designed for the Siemens Elmiskop 1A with a minimal modification to the microscope. The stage is temperature controlled in the range of -10 degrees C to 50 degrees C, saturated hydration condition being automatically obtained at all temperatures. The stage is easy to operate. It is suitable for temperature and hydration dependent experiments on both biological and non-biological materials and for dynamically observing other mixed phase physical or chemical interactions.

Inner Shell Ionization by 60 keV Electrons, Application to Microanalysis

By using a Castaing-Henry filtering device adapted on a Siemens Elmiskop I electron microscope, we have directly observed plasmon and inner shell excitations by 60 keV electrons on electron energy loss spectra. Inner shell excitation edges have been detected up to 1900 eV.By comparing L and K inner shell excitation profiles in the case of Magnesium, Aluminium and Silicon, it is concluded that the optical selection rules, △1 = ±1, explain the shape of the spectrum after the edge in a first approximation.

Observation of Domains and Phase Separation in Hydrated Lipid Bilayers

The domain structure and phase separation in lipid single bilayers were observed directly by selective area electron diffraction (SAED) and selected reflection dark field electron microscopy (SRDFEM), using an environmental stage for a Siemens Elmiskop IA. The specimens were viewed in a fully hydrated state in the temperature range between 0°C and 50°C. Below the transition temperature, three orders of a hexagonal pattern were recorded, whereas diffuse rings only were seen above the transition temperature.Selective area electron diffraction were done by restricting the illumination area to few micrometers in diameter, using a pointed filament and a 10 μm second condenser aperture. Below the transition temperature of dipalmitoylphosphatidylchoiine bilayers, differentially oriented diffraction patterns were distinguishable between adjacent membranes areas (domains) five micrometers apart. Occasionally, two or more superimposed patterns were recorded from the same area, indicating the observation area covered several domains.