Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications

Several representative examples are given of the successful application of negative staining across the holes of holey carbon support films using 5% (w/v) ammonium molybdate solution containing trehalose. The inclusion of 0.1% (w/v) trehalose is considered to be most satisfactory, although good data have also been obtained in the presence of 0.01 and 1.0% (w/v) trehalose. The examples given fall into the following groups: protein molecules in the absence of polyethylene glycol (PEG), protein molecules in the presence of PEG (Mr 1000), lipoproteins, lipids and membranes, filaments and tubules, viruses in the absence of PEG, viruses in the presence of PEG, aqueous polymer solutions, and finally for comparison purposes, four unstained samples studied in the presence of trehalose alone. In all these cases, and many others not documented here, successful spreading of the sample across holes has been achieved, with the sample embedded within a thin film of air-dried ammonium molybdate+trehalose. These specimens can be rapidly produced and provide an alternative to negatively stained specimens on carbon support films. Specimen stability in the electron bean is good and such specimens can usually generate superior negatively stained TEM images without flattening and adsorption artefacts. The formation of 2-D arrays/crystals of protein molecules and viruses, suspended across holes in the presence of ammonium molbybdate+trehalose, and trehalose alone, is also demonstrated.     

Computing methods for icosahedral and symmetry-mismatch reconstruction of viruses by cryo-electron microscopy

Three-dimensional(3 D) reconstruction of icosahedral viruses has played a crucial role in the development of cryoelectron microscopy single-particle reconstruction, with many cryo-electron microscopy techniques first established for structural studies of icosahedral viruses, owing to their high symmetry and large mass. This review summarizes the computational methods for icosahedral and symmetry-mismatch reconstruction of viruses, as well as the likely challenges and bottlenecks in virus reconstruction, such as symmetry mismatch reconstruction, contrast transformation function(CTF)correction, and particle distortion.     

Three-dimensional printing of biocompatible scaffolds for tissue engineered constructs

The formation of porous matrix structures (scaffolds) from biocompatible polymers and their composites with finely dispersed hydroxyapatite via air pressure–aided 3D printing and digital light processing (DLP) is studied. A triblock copolymer (polyethylene glycol/poly-ε-caprolactone/polyethylene glycol) and polyethylene glycol diacrylate served as raw polymer materials. The internal structure and surface morphology of the resulting scaffolds and the correspondence of their architectonics to the initial 3D virtual models are analyzed by means of optical and scanning electron microscopy.     

Transmission electron microscopical studies on some haemolymph proteins from the marine polychaete Nereis virens

The hexagonal bilayer haemoglobin molecule from Nereis virens has been investigated in a comparative study using several different negative stain electron microscopical specimen preparations (i.e. by conventional adsorption to continuous carbon support films, by the negative staining-carbon film technique and by negative staining across the holes of holey carbon support films with air-drying and rapid freezing/cryo-negative staining). The benefits and limitations of these different approaches are indicated, with the overall conclusion that negative staining with ammonium molybdate across holes creates the best possibilities for molecular imaging, and also has the potential for the creation of two-dimensional (2D) crystals/arrays at the fluid-air interface. Of the different negative staining procedures presented, cryo-negative staining reveals the greatest details of N. virens haemoglobin. This is exemplified by the direct visualisation of the central linker-assembly within the haemoglobin molecule, a structural feature less clearly defined by the other negative staining techniques. A discoidal lipoprotein molecule (diameter 30-60nm; thickness ca 8nm) has been detected in N. virens, which represents the first documented account of an annelid haemolymph lipoprotein. The biological implications of this lipoprotein for lipid transport remain to be established. The presence of a low concentration of ferritin molecules in N. virens haemolymph is also shown, assisted by the formation of small 2D ferritin arrays in negatively stained specimens prepared across holes.     

Formation and manipulation of regular metallic nanoparticle arrays on bacterial surface layers: an advanced TEM study

The template-directed formation of regular nanoparticle arrays on two-dimensional crystalline protein layers after their treatment with metal salt complexes was studied by transmission electron microscopy. For these investigations, bacterial surface layers (S layers), recrystallized in vitro into sheets and tube-shaped protein crystals with typical dimensions in the micrometer range, were used as the template. As identified by electron holography and scanning force microscopy, the S-layer tubes form alternating double layers when deposited onto a solid substrate surface. Two distinct pathways for the metal particle formation at the templates have been found: the site-specific growth of metal clusters by chemical reduction of the metal salt complexes, and the electron-beam induced growth of nanoparticles in the transmission electron microscope. Both mechanisms lead to regular arrays with particle densities > 6×10¹¹ cm ^-2. Nanoparticle formation by electron exposure takes exclusively place in the flat-lying double-layered protein tubes, where a sufficient amount of metal complexes can be accumulated during sample preparation. Received 6 December 2000     

溶剂对Bi2WO6形貌及其γ射线屏蔽性能的影响

通过简单的溶剂热法,在不添加任何表面活性剂的情况下,系统地研究吡啶、甘露醇、聚乙二醇和乙二醇四种溶剂对Bi2WO6辐射防护材料的影响。并利用XRD,SEM,TEM,HRTEM和DRS等分析技术对Bi2WO6晶体的组成、形貌、比表面积和禁带宽度等进行了表征。实验结果表明,溶剂对防护材料的形貌、光致发光性能和辐射防护性能有较大影响。选用吡啶作为溶剂,有利于材料形成不规则纳米球,提高其结晶度、光致发光强度及射线屏蔽率。该形貌下PS/Bi2WO6材料对155Eu和22Na混合点源的105.310 keV的射线屏蔽率达54.17%,已高于传统防护材料PbWO4。     

A simple synthesis of SnS nanoflakes at ambient conditions

The present work reports a simple route to prepare tin monosulfide semiconductor nanocrystals at ambient conditions. In the presence of complexant L-tartaric acid, SnCl₂·2H₂O was dissolved in ethylene glycol (EG) solution. After adjusting the solution pH value, sodium sulfide dissolved in EG was added into the solution. The solution changed from colorless into brown immediately and phase-pure SnS, which was confirmed by powder X-ray diffraction (XRD) was produced. The transmission electron microscopic (TEM) investigation shows very thin rectangular flake like SnS crystals of 10∼20 nm in size. PACS 81.05.Hd; 81.07.Bc; 81.16.Be; 81.16.Dn; 81.20.Ka     

The use of trehalose in the preparation of specimens for molecular electron microscopy

Biological specimens have to be prepared for imaging in the electron microscope in a way that preserves their native structure. Two-dimensional (2D) protein crystals to be analyzed by electron crystallography are best preserved by sugar embedding. One of the sugars often used to embed 2D crystals is trehalose, a disaccharide used by many organisms for protection against stress conditions. Sugars such as trehalose can also be added to negative staining solutions used to prepare proteins and macromolecular complexes for structural studies by single-particle electron microscopy (EM). In this review, we describe trehalose and its characteristics that make it so well suited for preparation of EM specimens and we review specimen preparation methods with a focus on the use of trehalose.     

Dislocations and mechanical properties of icosahedral quasicrystals

In this article we interpret the mechanical properties of icosahedral quasicrystals with the dislocation theory. After having defined the concept of dislocation in a periodic crystal, we extend this notion to quasicrystals in the 6-dimensional space. We show that perfect dislocations and imperfect dislocations trailing a phason fault can be defined and observed in transmission electron microscopy (TEM). In-situ straining TEM experiments at high temperature show that dislocations move solely by climb, a non-conservative motion-requiring diffusion. This behavior at variance with that of crystals which deform mainly by glide is explained by the atypical nature of the atomic structure of icosahedral quasicrystals.     

Fabrication of tin monosulfide nanosheet arrays using laser ablation

We demonstrate the non-catalytic, low-temperature (∼200°C) synthesis of two-dimensional (2D) tin monosulfide (SnS) nanosheet arrays vertically aligned on the substrate by means of pulsed laser ablation (PLD) process. The prepared nanosheets were characterized using X-ray diffraction, Raman spectroscopy, field-emission electron microscopy, electron probe micro-analysis, and transmission electron microscopy (HRTEM). The growth mechanism of 2D morphological feature, which is widespread but very slim (∼10 nm), is further investigated in detail from the crystallographic point of view by observing highly-resolved lattice images taken from plane-normal and in-plane directions using focused ion-beam manipulation and high-resolution transmission microscopy.     

Controlled fabrication of transparent and superhydrophobic coating on a glass matrix via a Green method

A facile, green method was explored for the organic-inorganic complex coating with superhydrophobic and transparent property on glass matrix. The glass surface was firstly treated with polyethylene glycol (PEG) and SiO₂ organic-inorganic solution and then modified with a layer of 1,1,1,3,3,3-Hexamethyldisilazane (HMDS). The glass samples were characterized by scanning electron microscopy (SEM), water contact angle (CA) measurement, and UV–Vis spectrophotometry. The results showed that the optical transmission over the visible range up to 89 % (in reference to 100 % transmission defined by bare glass substrate), and the water CA of the film reached 168^°. Superhydrophobic coatings with excellent optical transmittance will have potential applications in our daily life.     

Oriented single crystalline TiO2 nano-pillar arrays directly grown on titanium substrate in tetramethylammonium hydroxide solution

Oriented single crystalline titanium dioxide (TiO₂) nano-pillar arrays were directly synthesized on the Ti plate in tetramethylammonium hydroxide (TMAOH) solution by one-pot hydrothermal method. The samples were characterized respectively by means of field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and X-ray diffraction (XRD). Results showed that the TiO₂ nano-pillar with a tetrahydral bipyramidal tip grew vertically on the titanium substrate. HRTEM and Raman results confirmed that the TiO₂ nano-pillar arrays were single crystalline anatase. The controls of morphology, size, and orientation of the nano-pillar could be achieved by varying the solution concentration and hydrothermal temperature. Furthermore, the special morphology of the TiO₂ nano-pillar arrays was caused by the selectively absorption of the tetramethylammonium (TMA) through hydrogen bonds on the lattice planes parallel to (0 0 1) of anatase TiO₂. Less grain boundaries and direct electrical pathway for electron transferring were crucial for the superior photoelectrochemical properties of the single anatase TiO₂ nano-pillar arrays. This approach provides a facile in situ method to synthesize TiO₂ nano-pillar arrays with a special morphology on titanium substrate.     

Formation, TEM study and 3D reconstruction of the human erythrocyte peroxiredoxin-2 dodecahedral higher-order assembly

The production of a higher-order assembly of peroxiredoxin-2 (Prx-2) from human erythrocytes has been achieved during specimen preparation on holey carbon support films, in the presence of ammonium molybdate and polyethylene glycol. TEM study suggested that this assembly is a regular dodecahedron, containing 12 Prx-2 decamers (Mr 2.62 MDa, external diameter approximately 20 nm). This interpretation has been supported by production of a approximately 1.6 nm 3D reconstruction from the negative stain TEM data, with automated docking of the available X-ray data of the Prx-2 decamer. Comparison with other known protein dodecahedral and viral icosahedral structures indicates that this arrangement of protein molecules is one of the fundamental macromolecular higher-order assemblies found in biology. Widespread biotechnological interest in macromolecular "cage" structures is relevant to the production of the Prx-2 dodecahedron.     

Electrochemical fabrication and characterization of lepidocrocite (γ-FeOOH) nanowire arrays

We report on the fabrication of γ-phase iron oxyhydroxide (γ-FeOOH, lepidocrocite) nanowire (nw) arrays within the alumina pores by electrodeposition. An aqueous solution, friendly to alumina matrix, was generated and applied in this study for uniform deposition of γ-FeOOH nw arrays directly through the alumina barrier layer using an alternating current (ac) mode. As-deposited nanowired products were characterized using ⁵⁷Fe Mössbauer spectroscopy (MS), atomic absorption spectrophotometry analysis, field-emission scanning electron microscopy, UV-vis transmission spectroscopy, transmission electron microscopy and X-ray diffraction. The formation of pure lepidocrocite nw arrays in the alumina pores with the average Ø_pore of 45 and 150 nm was verified by transmission MS at cryogenic temperatures.     

Cytological modifications in maize plants infected by barley yellow dwarf virus and maize dwarf mosaic virus

Three inbred lines of maize (33-16, MO17 and B73) differing in their susceptibility to Barley yellow dwarf virus and Maize dwarf mosaic virus were studied to compare the ultrastructural modifications induced by the two viruses in leaf tissues of different age. The results demonstrate that the alterations induced by the two viruses in the different maize lines could depend on the particular line tested.     

Stirring-induced crystallization of polyethylene from Decalin

Stirring-induced solution grown fibrillar crystals of polyethylene have been obtained from solution in Decalin. The crystals were examined by electron microscopy, and their melting and annealing behavior as well as their response to attack by fuming nitric acid were investigated. Crystals formed at lower temperatures exhibit the morphology and properties of conventional shish kebabs. Crystals formed at higher temperatures have a ribbonlike morphology and exhibit different properties from those of conventional shish kebabs.     

Effects of synthesis variables on the magnetic properties of CoFe2O4 nanoparticles

Cobalt ferrite nanoparticles (CoFe₂O₄) have been synthesized using precipitation in water solution with polyethylene glycol as surfactant. Influence of various synthesis variables included pH, reaction time and annealing temperature on the magnetic properties and particle sizes has also been studied. Structural identification of the samples was carried out using Thermogravimetric and Differential thermal analysis, X-ray diffraction, Fourier transform infrared spectroscopy, Scanning electron microscopy, High resolution transmission electron microscopy. Vibrating sample magnetometer was used for the magnetic investigation of the samples. Magnetic properties of nanoparticles show strong dependence on the particle size. The magnetic properties increase with pH of the precipitating medium and annealing temperature while the coercivity goes through a maximum, peaking at around 25 nm.     

On the morphology and structure of poly(p-phenylene terephthalamide) single crystals and spherulites

Poly(p-phenylene terephthalamide) (PPTA) single crystals and negative spherulites obtained from concentrated sulfuric acid solution were studied by polarizing microscopy, transmission electron microscopy, electron diffraction, and infrared spectroscopy techniques. Electron microscopy shows that the single crystals with nearly pyramidal shape are composed of rectangular platelets which are packed in parallel with the same orientation. The long edge of the platelet is parallel to [avec]?, while the macromolecular chains are located in the plane of the platelet and perpendicular to the long edge. The width of the platelet is dependent on the molecular weight. The radius direction of PPTA negative spherulites is [avec]?. Electron diffraction patterns indicate that the unit cell parameters of PPTA single crystals and spherulites are not the same as those of Kevlar fibers. For the former c = 11.5 Å, while for the latter c = 12.9 Å. This indicates that the molecular conformation of PPTA molecules in the single crystals and spherulites is different from the conformation of PPTA molecules in the fibers. Infrared absorption spectra show large shifts of the amide I and amide II bands between the two conformations.     

Icosahedral packing of RNA viral genomes

Many spherelike RNA viruses package a portion of their genome in a manner that mirrors the icosahedral symmetry of the protein container, or capsid. Graph-theoretical constraints forbid exact realization of icosahedral symmetry. This paper explores the consequences of graph-theoretical constraints on quasi-icosahedral genome structures. A key result is the prediction that the genome organization is a Hamiltonian path or cycle and that the associated assembly scenario of such single-stranded spherelike RNA viruses resembles that of cylindrical RNA viruses, such as tobacco mosaic viruses.     

Formation of NiZr2 Binary Metallic Glass： Experimental and Molecular Dynamics Analyses

The local atomic structure of an amorphous NiZr2 alloy is identified by using x-ray diffraction, transmission electron microscopy, and differential scanning calorimeter. Based on the experimental results, molecular dynamics simulation is performed to investigate the glass formation of liquid NiZr2 alloy. Some relevant features of the pair correlation functions are in good agreement with those obtained by experiment. The pair analysis parameters are calculated, suggesting that there exist icosahedral ordering, four-fold symmetrical bipyramid and triangular- faced polyhedral units in the amorphous NiZr2 structure. The result is beneficial to open avenues toward the understanding of fundamental theoretical problems of glass formation of simple binary alloys.