Sputter shadowing improved by using a tungsten target

This work builds upon a previous paper (W. Colquhoun, 1984, J. Ultrastruct. Res. 87, 97) in which a sputter shadowing device was briefly described. The device allowed TEM specimens to be shadowed in a conventional sputter coater. Images obtained by sputter shadowing with a standard Au/Pd target were of good quality but were slightly inferior to the best that could be obtained by e--beam evaporation of tungsten. Here we show that construction and use of a tungsten target greatly improves the quality of the sputter shadowed deposit. Images of DNA and ribosomal subunits contrasted by sputter shadowing with tungsten are shown. The DNA images indicate that sputter shadowing with tungsten is a gentle contrasting technique. The sputter shadowed images of the 30 S ribosomal subunits show the major features of the particle revealed by evaporation shadowing using the most sophisticated of methods in that technology. Advantages of sputter shadowing are discussed and a rationale for the improved grain obtained by sputtering tungsten is suggested.     

Interfacial Growth of Activated Carbon on WO3 Nanoplates for Enhanced Photocatalytic Activity by Surface Plasmon Resonance

Plasmonics - A highly efficient, visible-light-driven and hybrid-activated carbon-doped pure tungsten trioxide (WO3) composite photocatalyst, with greatest stability has been synthesized by using...     

Single bacteriorhodopsin molecules revealed on both surfaces of freeze- dried and heavy metal-decorated purple membranes

The flat sheets of the purple membrane from Halobacterium halobium contain only a single protein (bacteriorhodopsin) arranged in a hexagonal lattice. After freeze-drying at -80 degrees C (a method that is superior to air-drying), shadowing with tantalum/tungsten, and image processing, structural details on both surfaces are portrayed in the range of 2 nm. One surface is rough and lattice lines are clearly visible, whereas the other is smooth and the hexagonal order seems to be absent. The optical diffraction patterns, however, indicate a hexagonal lattice for both surfaces. In addition, these diffraction patterns are characteristic and easily distinguished. The orientation of the two surfaces was identified by silver decoration: partial condensation of silver on purple membranes enabled the smooth surface to be identified as the plasmatic and the rough surface as the exoplasmic surface. After image processing, the exoplasmic surface shows a triplet structure which exactly fits the projected structure determined by Unwin and Henderson (1975. Nature(Lond.). 257:28-32) at molecular resolution, whereas, on the plasmatic surface, four image details per unit cell are visible. Three of them match the arrangement of bacteriorhodopsin, whereas the fourth must be located over a lipidic array. Summarizing these results, it is possible to show the part of each single bacteriorhodopsin protein that is present in the surfaces of the purple membrane. By "shadowing" the membranes perpendicularly, we prove that these components of the surfaces are mainly portrayed by a decoration effect of the tantalum/tungsten condensate.     

The mechanism of action of molybdenum and tungsten upon collagen structures in vivo

The effect of in vivo administration of molybdenum (as sodium molybdate) and tungsten (as sodium tungstate) was investigated in the skin of laboratory rats. It was proved that the amount of both bound molybdenum and tungsten in collagen is relatively small being 0.05 and 0.06 moles per mole respectively. Besides the fraction of firmly bound molybdenum and tungsten a much higher extractable pool of both these metals was found. It was also demonstrated that in vivo shadowing of collagen is caused by the fraction of loosely bound metals. On the other hand pronounced changes were shown in the mechanical properties of connective tissue after molybdate and tungstate administration. Surprisingly, the change in mechanical properties indicated a lower level of cross-linking after the administration of the investigated metals. It is therefore concluded that bitopical binding of molybdenum and tungsten in the collagen structure is unlikely. It also appears that the biological effect of these metals is due to the competition with copper and the interference with the physiological cross-linking reactions based on the partial blockade of lysyloxidase.     

Identification of Spermatozoa in Criminological Investigations

Materials used for study were viral smears or ultra-thin sections containing viral cell inclusions. They were stained with the Feulgen reaction and other cytochemical procedures. Stained preparations were dried and then shadow-cast with metallic chromium for 30 seconds in a bell jar with a vacuum of at least 0.1 µ (10^?4mm.) of mercury, and placed at a shadowing angle of 10–12°. Shadow-cast preparations were cleared with xylene and mounted in Canada balsam. Dried smears or deparaffinized sections without staining were suited to this method also. A virus which stained indistinctly with cytochemical procedures alone could be adapted to visible light microscopy by shadowing, and in addition, used for observations on its chemical composition.     

Sputter shadowing

A device has been constructed which allows specimens to be shadowed in a conventional sputter water. This process of sputter shadowing lends to specimens a contrast suitable for imaging in the transmission electron microscope (TEM). The process has the practical advantages over metal evaporation shadowing of lower instrumentation costs, less user training, and less time expenditure per shadowing operation. It provides on a single grid a spectrum of shadowing contrasts from which optimal imaging for a particular specimen can be chosen. The process minimizes radiant and metal deposition heating of the specimen and, thereby, may better preserve its structure during the contrasting procedure. The grain resulting from sputter shadowing differs significantly from that obtained by metal evaporation shadowing and the possibility for using this difference to improve resolution in shadowed preparations is discussed.     

Hochauflösende Gefrierätzung

Zusammenfassung Die Anwendungsmöglichkeit der Gefrierätztechnik auf molekularbiologische und cytochemische Probleme ist vielfach durch das zu geringe Auflösungsvermögen der derzeit üblichen Simultanbeschattung mit Platin-Kohle begrenzt. Zwar lassen sich durch Beschatten mit höchstschmelzenden Metallen Abdrucke mit höherer Auflösung erzielen, jedoch scheinen bisher alle Versuche, diese Methode in der Gefrierätzung anzuwenden, fehlgeschlagen zu sein, da sie zu Präparatveränderungen führten. Außer hoher Auflösung müssen nämlich Beschattungsmethoden, um für die Gefrierätzung geeignet zu sein, auch folgende Bedingungen erfüllen: kurze Aufdampfzeit, geringe thermische Präparatbelastung und große chemische Stabilität des Abdruckes. Durch die Verwendung von Tantal-Wolfram als Aufdampfmaterial und die Konstruktion eines geeigneten Elektronenstrahl-Verdampfers, der eine Reduzierung der Wärmestrahlung und ein Ausschalten des Ionenbeschusses ermöglicht, ist es nunmehr gelungen, obige Bedingungen zu erfüllen und die Beschattung mit höchstschmelzenden Metallen auch in der Gefrierätzung erfolgreich einzusetzen. Die mit Tantal-Wolfram beschatteten Präparate sind entsprechenden Platin-Kohle-Abdrucken an Auflösung deutlich überlegen.Da die Wärmebelastung des Präparates in der Gefrierätztechnik von entscheidender Bedeutung ist, wurde sie für Ta/W und Pt/C-Beschattungen untersucht. Bei Ta/W-Be-dampfung liegt der für unsere Anordnung berechnete Wärmefluß bei 16 mW·cm^–2, der gemessene beträgt 23 mW·cm^–2. Bei Pt/C-Verdampfung ist die berechnete Wärmebelastung gleich hoch wie bei Ta/W. Der an einem üblichen Verdampfer gemessene Wärmefluß beträgt aber 41 mW·cm^–2. Rechnung und Experiment stimmen darin überein, daß die Versuchsbedingungen, wie Aufdampfrate, Verdampfergröße und -abstand auf die thermische Präparatbelastung einen stärkeren Einfluß haben, als die Art des aufgedampften Materials.

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Improved resolution in freeze-etching

Summary Resolution in freeze-etching is primarily limited by the need for shadowing the replica. In addition to giving high resolution, any technique suitable for freeze-etching must allow short shadowing times, a low thermal load for the specimen, and must provide a final film capable of surviving the drastic chemical procedures used for cleaning the replica.Simultaneous evaporation of platinum and carbon is at present the standard shadowing method in freeze etching. Replicas of higher resolution can in principle be obtained by using very high melting metals such as tungsten or tantalum. Extensive specimen damage caused by long evaporation times and excessive thermal load have however prevented the successful application of such ultrashadowing methods to freeze etching.Since neither long shadowing times nor a high thermal load are properties intrinsic to ultrashadowing, a suitable electron beam evaporator for high melting metals was built and thus ultrashadowing successfully applied to freeze-etching. All parts of the gun are water cooled and can be outgased by electron bombardment. The source can thus be operated reproducibly at high rates and without affecting the vacuum. The actual source (3 mm Ø) is the only hot part of the gun not shielded from the specimen. The ions which are generated during evaporation, and can cause considerable specimen damage, are deflected from the specimen by an electric field. The evaporation is done in 7–10 seconds, the source-specimen distance is 200 mm. For the shadowing material we use a tantalum-tungsten alloy. The resultant films are stable in 70% sulfure acid.At lower magnification freeze-etched specimen which are shadowed with Ta/W look just like Pt/C replicas which indicates that no additional artification took place. High magnification micrographs of ultrashadowed objects show a resolution considerably higher than those of platinum-carbon replicas published in the literature.Since heat damage is a crucial problem in freeze-etching the thermal load for tantalum-tungsten-and platinum-carbon-shadowing was calculated. For both methods a theoretical value of approximately 16 mW·cm^–2 was obtained provided the above shadowing conditions are observed and the ions are deflected from the specimen. Furthermore, the calculations show that the load caused by thermal radiation can be reduced drastically, without increasing the shadowing time, if the gun is operated at a higher rate thus allowing the use of either a smaller source or a longer source-specimen distance.Measurements of the thermal load agreed basically with the calculations. The values measured using the tantalum-tungsten gun were about half the ones obtained during platinum-carbon shadowing with a commercial evaporator.

     

Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.     

Inactivation of Escherichia coli glutamine synthetase by thiourea trioxide

Incubation of Escherichia coli glutamine synthetase with thiourea trioxide resulted in partial inactivation of the enzyme. This reagent specifically modifies lysine residues to form homoarginine. By amino acid analysis 2.3 ± 0.3 residues of homoarginine are produced per enzyme subunit after treatment with thiourea trioxide. Previously we determined that thiourea dioxide totally inactivated glutamine synthetase and modified both lysine and histidine residues (J. Colanduoni and J. J. Villafranca (1985) J. Biol. Chem. 260, 15,042–15,050). Thiourea trioxide reacted with the same lysine residues of glutamine synthetase as thiourea dioxide. The K_m values for the thiourea trioxide modified enzyme were determined and are 210 ± 30 μm and 10 ± 1 mm for ATP and glutamate, respectively. Both values are about threefold higher than for native enzyme assayed under the same conditions. Fluorescence titrations of native and thiourea trioxide labeled enzyme showed that ATP binding was virtually unchanged by the modification while glutamate and methionine sulfoximine bound about twofold more weakly to the modified enzyme.     

Effect of zooming on texture features of ultrasonic images

Background

The effect of age on common carotid artery diameter is unclear for varying atherosclerosis risk levels.

Methods

Cross-sectional data from the Atherosclerosis Risk in Communities Limited Access Data set were used to estimate the association of age with B-mode ultrasound common carotid artery diameter for three atherosclerosis risk levels. Based on information from clinical examinations, B-mode ultrasounds, questionnaires, blood and other tests, participants were categorized into three groups: pre-existing disease (prevalent stroke and/or coronary heart disease), high risk group (no pre-existing disease, but prevalent diabetes, hypertension, plaques/shadowing, body mass index >= 30, current smoking, or hyperlipidemia), and a low risk group (no pre-existing disease, no plaques/shadowing, and no major elevated risk factors). Multivariable linear regression analyses modeled the common carotid artery diameter relationship with age.

Results

Age was positively and significantly associated with common carotid artery diameter after risk factor adjustment in the overall sample, but age had a larger effect among persons with evidence of atherosclerosis (interaction p < 0.05). Each year of older age was associated with 0.03 mm larger diameter/year among persons with pre-existing disease, with 0.027 mm larger diameter/year in the high risk group, but only 0.017 mm/year among the low risk group. Results were qualitatively similar using plaques/shadowing status to indicate atherosclerosis severity.

Conclusion

The significant impact of age on common carotid artery diameter among low risk, middle-aged, black and white men and women suggests arterial remodelling may occur in the absence of identified risk factors. The significantly larger impact of age among persons with, compared to persons without identified atherosclerosis or its risk factors, suggests that arterial remodelling may be an indicator of exposure duration.     

Evaluation of the technical performance of three different commercial digital breast tomosynthesis systems in the clinical environment

The aim of this work was to research and evaluate the performance of three different digital breast tomosynthesis (DBT) systems in the clinical environment (Siemens Mammomat Inspiration, Hologic Selenia Dimensions, and Fujifilm Amulet Innovality). The characterization included the study of the detector, the automatic exposure control, and the resolution of DBT projections and reconstructed planes.The modulation transfer function (MTF) of the DBT projections was measured with a 1 mm thick steel edge, showing a strong anisotropy (30–40% lower MTF_0.5 frequencies in the tube travel direction). The in-plane MTF_0.5, measured with a 25 μm tungsten wire, ranges from 1.3 to 1.8 lp/mm in the tube-travel direction and between 2.4 and 3.7 lp/mm in the chest wall–nipple. In the latter direction, the MTF peak shift is more emphasized for large angular range systems (2.0 versus 1.0 lp/mm). In-depth resolution of the planes, via the full width at half maximum (FWHM) from the point spread function of a 25 μm tungsten wire, is not only influenced by angular range and yields 1.3–4.6 mm among systems. The artifact spread function from 1 mm diameter tungsten beads depends mainly on angular range, yielding two tendencies whether large (FWHM is 4.5 mm) or small (FWHM is 10 mm) angular range is used. DBT delivers per scan a mean glandular dose between 1.4 and 2.7 mGy for a 45 mm thick polymethyl methacrylate (PMMA) block.In conclusion, we have identified and analysed specific metrics that can be used for quality assurance of DBT systems.     

Tungsten particle-induced nicking of supercoiled plasmid DNA

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.     

An initial test of a method for the estimation of real mean particle size from shadowed samples

During shadowing, a "cap" of metal develops on small particles. This cap increases apparent particle with (measured normal to the shadowing direction) by an extent which cannot be predetermined. The extent of this increase in particle size (here defined as the "cap," X) is estimated in the present method by using opposite (180 degrees sample rotation) bidirectional shadowing. It is argued that the bidirectional cap is the sum of the two unidirectional caps, and therefore that X = 2A - (B + C), where A is the mean bidirectionally shadowed particle size, and B and C are the two mean unidirectionally shadowed particle sizes. As a validation of the method, the mean diameter of air-dried ferritin was estimated and the results appear to confirm the hypothesis (mean diameter by present method, 10.7 +/- 0.2 nm; mean diameter by previous methods, 10.89 nm).     

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.     

Arsenic trioxide induces Hsp70 expression via reactive oxygen species and JNK pathway in MDA231 cells

In the present study, we determined the molecular pathways that induce the heat shock proteins (Hsps) after treatment of cells with arsenic trioxide. Administration of arsenic trioxide to MDA231 cells leads to induce Hsp70, which is accompanied by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). We showed that arsenic trioxide-induced Hsp70 expression was caused by activation of ROS and prevented by the antioxidant N-Acetyl-Cysteine (NAC). SP600125 and dominant-negative SEK suppressed Hsp70 promoter-driven reporter gene expression, suggesting that JNK would be preferentially associated with the protective heat shock response against arsenic trioxide stress. In addition, SP600125, a specific JNK inhibitor, significantly reduced the amount of phosphorylated HSF1 upon administration of arsenic trioxide. It is likely that Hsp70 expression against arsenic trioxide exposure protects cells from oxidative injury and apoptotic cell death by means of JNK activity.     

An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus

An electron microscopic analysis of human and bovine vitreous humor after rotary shadowing showed the presence of both collagen fibrils and an extensive loose network of hyaluronan molecules. No interaction between the collagen fibrils and the hyaluronan molecules was observed under the conditions used for rotary shadowing. Periodic "struts" were present on the surface of the collagen fibrils. These struts showed an organization the same as that previously observed for type IX collagen on the surface of collagen fibrils from chicken cartilage and vitreous. However, the knob of the noncollagenous NC4 domain of cartilage type IX collagen was not observed at the ends of the struts in a manner identical to that of chicken vitreous humor. Zonular fibrils were dissected out from bovine eyes and shown by rotary shadowing to contain a beaded fibril which is similar in morphology to the "elastin-associated" microfibrils of many connective tissues. Experiments in which the zonular fibrils were stretched and fixed prior to rotary shadowing showed that the distance between each bead is variable and can be accounted for by the bowing out of overlapping filaments which connect each bead.     

The tungsten metallome of Pyrococcus furiosus

The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope (187)W (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 μM, of which ca. 30% appears to be free tungsten, probably in the form of tungstate or polytungstates. The remaining 70% is bound by five different tungsten enzymes: formaldehyde ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase and the tungsten-containing oxidoreductases WOR4 and WOR5. The membrane proteome of P. furiosus is devoid of tungsten. The differential expression, as measured by the tungsten level, of the five soluble tungsten enzymes when the cells are subjected to a cold-shock shows a strong correlation with previously published DNA microarray analyses.     

A simple method for freeze-drying of macromolecules and macromolecular complexes

We present a simple approach for effective freeze-drying and rotary shadowing of large molecules, molecular assemblies, and cell organelles. Simply, a suspension of specimen is adsorped to a glass coverslip, stabilized, and rinsed with 30% methanol. A second coverslip is "sandwiched" on top, and excess methanol is withdrawn from the edges then frozen by plunging into liquid nitrogen and split. Following either rotary or unidirectional shadowing and replication, the coverslip is dissolved in hydrofluoric acid. In addition to avoiding the problems encountered with air-drying specimens for rotary shadowing, the technique also reproducibly provides the thin layer of solution necessary for proper freeze-drying, regardless of how hydrophobic the sample is. The "glass sandwich" technique allows modification of the glass substrate (making it hydrophobic with carbon or hydrophilic by soaking it in alcian blue) which clearly alters the shape of macromolecular assemblies such as myosin filaments and decorated thin filaments.