A temperature-jump device for time-resolved cryo-transmission electron microscopy.

We describe a temperature-jump device that permits time-resolved studies of thin cryo-transmission electron microscopy specimens. The specimen is rapidly heated to induce a change in microstructure just prior to cryo-fixation. The apparatus consists of a xenon arc lamp equipped with a shutter controlled by timing circuitry, used in conjunction with an environmental specimen preparation chamber. The specimen is heated by exposure to focused light from the lamp, and then plunged into cryogen. Using a thermocouple constructed from an electron microscope grid, we show that temperature jumps of 30-60 K are achieved with exposure times of 150-450 milliseconds. Micrographs of dimyristoyl phosphatidylcholine (DMPC) vesicles and n-docosane films, subjected to these exposures, show that the specimens are still at least 20-30 K above their initial temperature when they contact the cryogen. This method could be applied to a variety of biological and chemical systems which undergo structural changes activated by a rise in temperature.     

Transmission electron microscopy of mercury metal

Transmission electron microcopy (TEM) analysis of liquid metals, especially mercury (Hg), is difficult to carry out because their specimen preparation poses a daunting task due to the unique surface properties of these metals. This paper reports a cryoTEM study on Hg using a novel specimen preparation technique. Hg metal is mixed with water using sonication and quenched in liquid ethane cryogen. This technique permits research into the morphological, phase and structural properties of Hg at nanoscale dimensions.     

Unexpected property of trehalose as observed by cryo-electron microscopy

Trehalose is an agent useful in maintaining the integrity of many biological systems submitted to various stresses. It is also presumed to improve specimen preparation for electron microscopy and to reduce beam damage. Here we study the effect of trehalose on the preparation and observation by cryo-electron microscopy of thin vitrified films of biological suspensions. We observe that trehalose, as compared to sucrose, can indeed reduce electron beam damage to biological particles, as determined from the dose necessary for the onset of bubbling. Surprisingly, we also find that the contrast of biological particles is higher in a vitrified solution of trehalose than in one of sucrose. This effect can be explained if the water evaporation during the specimen preparation is less in the presence of trehalose than with sucrose, but we do not yet understand the underlying reasons since the evaporation properties of both sugars are similar at a macroscopic level. We conclude that trehalose is truly a remarkable substance and that more investigation is needed in order to fully understand its properties, and that the addition of ca. 3–5% trehalose to biological suspensions is a simple and useful method to reduce commonly arising drying artefacts and water evaporation in the thin film vitrification method.     

Electron diffraction and diffraction contrast imaging of thin organic films

The applications of electron diffraction and diffraction contrast electron microscopy with which to study the structure and dynamics of organic thin films are discussed. The techniques of making thin film deposits on substrates and of forming free-standing thin films over holes on the substrate are described. Selected area electron diffraction and diffraction contrast imaging techniques for thin film studies are elaborated, and examples are given. Methods to reduce radiation damage and environmental protection of the thin film specimen are outlined. The interpretation of electron diffraction and imaging data is given for three cases: (1) The effects of film tilting and molecular tilting (with respect to the film plane) are examined. (2) The detection of phase transition is illustrated. (3) The use of labels to mark film domains is shown together with the measurement of dynamic movement.     

Evaporation during preparation of unsupported thin vitrified aqueous layers for cryo-electron microscopy

Evaporation of water cannot be fully avoided when an unsupported thin vitrified film of an aqueous suspension is prepared for cryo-electron microscopy. This results in increasing concentration of solute which could affect the observed material. We have quantitatively studied this effect by measuring the contrast of polystyrene spheres in a metrizamide solution. The drying effect is generally negligible when specimens are prepared on a hydrophilic perforated support but it is frequently important when hydrophobic films are used instead. A flow of humid air, double blotting with minimal exposure of the thin liquid film to the atmosphere, or an automatic plunger optimizing the blotting conditions are simple methods for reducing drying effects. With this third device acting on a hydrophilic supporting film, the increase of solute concentration is limited to less than 20%.     

Particle-surface interaction in thin vitrified films for cryo-electron microscopy

The concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo-electron microscopy by the classical, single-side blotting method or by blotting both sides of the grid simultaneously. The two-side blotting method gives the most faithful representation of the bulk solution. The single-side blotting method results in particles preferentially adsorbing to the non-blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface seems to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, as for example with lipid vesicles, where the particles are broken as a result of adsorption. Since particles remain adsorbed to the air-liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro-dialysis. A thin film is then formed by blotting the specimen and vitrified by rapid cooling.     

Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging

Cryoelectron microsopy is a widely used technique to observe biological material in an almost physiological, fully hydrated state. The sample is prepared for electron microsopy observation by quickly reducing its temperature to ?180 °C. The high‐speed cooling induces the formation of vitreous water, which preserves the sample conformation. However, the way vitrification occurs is still poorly understood. In order to better understand the phenomenon, we have used a stroboscopic device to visualize the interaction between the electron microscopy grid and the cryogen. By blocking the free fall of the plunger once the grid has penetrated the coolant by half its diameter, we have elucidated the way in which vitrification propagates. The findings were confirmed by numerical simulation. In addition, according to our observations, we now present an alternative way to prepare vitreous specimens. This new method, with the grid parallel to the liquid cryogen surface, decreases evaporation from the sample during its free fall towards the coolant and at the same time achieves a more uniform vitrification over the entire surface of the specimen.     

Validation of convection-limited cooling of samples for freeze-fracture electron microscopy

Rapid freezing is the most important step in sample preparation for freeze-fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) < 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction-limited model, the convection-limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.     

Practical Aspects of Lattice Imaging of Cellulose

The lattice imaging technique for cellulose, a typical electronbeam-sensitive material, was developed by using a conventional 120 kV electron microscope. Routine procedures for specimen preparation and high resolution, low dose electron microscopy are described in detail. A new, simple method was introduced for the preparation of a Formvar micronet to support the thin carbon film. The lattice imaging technique was successfully applied to algal celluloses as well as bacterial cellulose, which is composed of much smaller crystallites than the former. Digital image processing was found to be effective in enhancing the lattice images. The bacterial cellulose ribbon contained crystallites 10–25-nm wide, which is much greater than the basic unit of cellulose fibril extruded from the cell surface. This shows that unit fibrils can fasciate with each other, merging into a single crystallite.     

An improved cryo-jet freezing method

A new cryo-jet freezing apparatus is described that is easy to use and gives good results using a propane-butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.     

Wet STEM: a new development in environmental SEM for imaging nano-objects included in a liquid phase

Environmental scanning electron microscopy (ESEM) enables wet samples to be observed without potentially damaging sample preparation through the use of partial water vapour pressure in the microscope specimen chamber. However, in the case of latices in colloidal state or microorganisms, samples are not only wet, but made of objects totally submerged in a liquid phase. In this case, under classical ESEM imaging conditions only the top surface of the liquid is imaged, with poor contrast, and possible drifting of objects. The present paper describes experiments using a powerful new Scanning Transmission Electron Microscopy (STEM) imaging system, that allows transmission observations of wet samples in an ESEM. A special device, designed to observe all sorts of objects submerged in a liquid under annular dark-field imaging conditions, is described. Specific features of the device enable to avoid drifting of floating objects which occurs in the case of a large amount of water, thus allowing slow-scan high-definition imaging of particles with a diameter down to few tens of nm. The large potential applications of this new technique are then illustrated, including the imaging of different nano-objects in water. The particular case of grafted latex particles is discussed, showing that it is possible to observe details on their surface when submerged in water. All the examples demonstrate that images acquired in wet STEM mode show particularly good resolution and contrast, without adding enhancing contrast objects, and without staining.     

A combined environmental straining specimen holder for high-voltage electron microscopy

A novel specimen holder that enables in situ observation of crack-tip deformation and/or fracture under a controlled environment is developed for a high-voltage electron microscope (HVEM). A window-type environmental cell (EC) that incorporates a uniaxial straining apparatus is built into a side-entry-type single-tilt specimen holder. The gas control in EC, straining apparatus design, limited field of view for crack-tip observation, and specimen preparation for the specimen holder are presented in detail. Experimental results successfully demonstrate that the developed specimen holder is quite useful for the dynamic observation of crack-tip deformation and/or fracture subjected to a hostile environment, such as hydrogen gas.     

An apparatus for freeze-fracturing specimens of dermal collagen in preparation for scanning electron microscopy

A new apparatus is described which facilitates the freeze fracturing of specimens under liquid nitrogen in preparation for scanning electron microscopy. The apparatus is simple and can be made by any competent engineering department.     

Principles of a TEM specimen device to meet highest requirements: Specimen temperature 5–300 K,cryo transfer,condensation protection,specimen tilt,stage stability for highest resolution

A new side-entry specimen device is described which is suitable for high resolution electron microscopy. The specimen can be kept with high stability at any desired temperature between 5 and 300 K. The specimen holder is tiltable around two axes and enclosed in a chamber which can be cooled independently. The specimen is locked into the microscope either at room temperature or at 77 K via a cooling chain. The specimen translating stage is designed according to novel principles which obviate any drift. In particular, the local electron beam heating of a supporting film at very low temperature is considered.     

Natural propane cryogen for frozen-hydrated biological specimens

The properties of natural propane, mixed with 0–4% isopentane, as a cryogen suitable for rapid freezing of this layers of aqueous biological specimen suspensions are discussed. Although natural propane has rather variable properties, its freezing point can be depressed below the temperature of liquid nitrogen by adding a smaller amount of isopentane than is required for depressing the freezing point of pure propane.     

Microtubule structure studied by quick freezing: Cryo-electron microscopy and freeze fracture

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryo-electron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2–3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.     

Phospholipid,Nature's own slide and cover slip for cryo-electron microscopy

Thin films of surface-active compounds, with or without particulate material, can be obtained by immersing and withdrawing a bare specimen grid from a solution/suspension of the compound. Immediately after withdrawing the grid, thinning of the film starts. Thinning is initially powered by gravity and capillary forces and will proceed in thin films (< 100 nm) driven by intermolecular forces until the London-van der Waals attractive forces come to an equilibrium with electrostatic repulsion of similarly charged surfaces of the film. With small unilamellar vesicles prepared from the phospholipid dimyristoyl phosphatidyl choline (DMPC) the draining behaviour of these films was studied by cryo-electron microscopy. Small unilamellar vesicles were observed within the film as well as the coalescence of these vesicles into sheets (‘leaky’ membrane fusion). Sheets dominate the images when films are allowed to drain for longer periods (>3min). Thin films were formed on grids from catalase crystals suspended in a DMPC suspension and vitrified by cooling. High-resolution information was obtained by electron diffraction at low temperature and under low-dose conditions from catalase crystals surrounded by small vesicles as well as from catalase crystals surrounded by sheets of DMPC. In the latter case the water content drops from 99% (DMPC in small vesicles) to less than 30% (DMPC in sheets) during draining. Ferritin was added to a DMPC suspension and thin films were prepared and vitrified. After prolonged draining ferritin molecules were deposited in layers with a stepwise increase in thickness. Draining of thin films has thus a dehydrating effect as well as a sorting and ordering effect. These effects must be considered when using surface-active compounds at air-water interfaces as a slide and cover slip for electron microscopy.     

有机电致发光器件的回顾和发展

综述了有机电致发光薄膜器件 ( OTFEL )的发展过程和 OTFEL的器件制备和结构 ,总结了有机小分子 (包括金属螯合物 ,稀土有机物 ,有机染料 ) ,聚合物 (主要是共轭高聚物 )制成的器件结构 ,发光光谱和亮度。文末展望了OTFEL 的发展前景。     

Profile measurement of a wide-area resist surface using a multi-ball cantilever system

In the semiconductor industry, a device that can measure the surface profile of thin film like photoresist with high accuracy and high speed is needed. Since the surface of photoresist is very smooth and deformable, a device is required that will measure vertically with nanometer resolution and not damage the film during the measurement. We developed an apparatus using a multi-ball cantilever and white light interferometer to measure the surface profile of thin film. However, this system, as assessed with a scanning method, suffers from the presence of a moving stage and systematic sensor errors. Therefore, this paper describes an approach using a multi-ball cantilever as coupled distance sensors together with an autocollimator as an additional angle measuring device, which has the potential for self-calibration of a multi-ball cantilever. Using this method, we constructed an experimental apparatus and made measurements on resist film. The results demonstrated the feasibility of the constructed multi-ball cantilever system with the autocollimator for measuring thin film with high accuracy.     

Ultracryotomy: a technique for cutting ultrathin sections of unfixed frozen biological tissues for electron microscopy

An apparatus is described which facilitates the sectioning of unfixed and unembedded biological tissues for electron microscopy. The specimen is maintained at the temperature of boiling liquid nitrogen; the knife temperature may be controlled between ?50 and ? 150°C. Under the worst operating conditions specimen drift with respect to the knife is about 5 nm/sec, and suggestions are made on how this drift may be further reduced. We propose to call the apparatus an ‘ultracryotome’. Electron micrographs of a variety of unfixed, unembedded and unstained tissues are shown and commented on. Some developments of experimental electron microscopy which may be expected to develop from use of the ultracryotome are listed.