A new technique for obtaining large platinum-carbon replicas

A problem often encountered in freeze-fracturing is that platinum-carbon replicas roll up or are broken into fragments during tissue digestion and replica washing. In replicas damaged in these ways, tissue orientation and cell identification are difficult. In order to prevent such damage, methods have been introduced of replica strengthening by coating them with plastic films (Steere, 1957; Willison & Rowe, 1980; Stolinski et al., 1983), or with an additional layer of silver or gold (Robards & Umrath, 1978; Robards & Sleytr, 1985). A disadvantage of these methods, however, is that the additional layer must be removed before the replicas can be examined under the electron microscope. Removal of the additional layer results in loss of image quality. A reinforcing plastic film may not be completely digested by the tissue solvent and will then contaminate the replica, while the silver technique requires a silver evaporation source to be present in the vacuum chamber. Instead of replica strengthening methods, some authors recommend putting replicas carrying tissues, which have been partially digested by bleach (Pearce, 1983) or chromic acid (Fetter & Costello, 1986), onto carbon-coated gold grids and then floating them on chromic acid. However, neither this method nor the replica strengthening method assure the production of large unrolled replicas. Moreover, gold grids are rather expensive.     

A replica technique for extracting precipitates from zirconium alloys for transmission electron microscopy analysis

A reliable two-stage carbon replica technique has been developed to extract precipitates from zirconium alloys. Using this technique, all precipitating phases can be extracted from Zircaloy-2, Zr-Cr-Fe, and Zr-Nb-Fe alloys. Precipitate identification using EDS X-ray analysis and convergent beam electron diffraction was greatly facilitated in comparison to thin foils. In addition, the sensitivity for the detection of trace elements in particles was increased using extraction replicas. The chemical compositions of the precipitates as determined from both replica and thin foils were in excellent agreement.     

Automated magnification calibration in transmission electron microscopy using Fourier analysis of replica images

The magnification factor in transmission electron microscopy is not very precise, hampering for instance quantitative analysis of specimens. Calibration of the magnification is usually performed interactively using replica specimens, containing line or grating patterns with known spacing. In the present study, a procedure is described for automated magnification calibration using digital images of a line replica. This procedure is based on analysis of the power spectrum of Fourier transformed replica images, and is compared to interactive measurement in the same images. Images were used with magnification ranging from 1,000 x to 200,000 x. The automated procedure deviated on average 0.10% from interactive measurements. Especially for catalase replicas, the coefficient of variation of automated measurement was considerably smaller (average 0.28%) compared to that of interactive measurement (average 3.5%). In conclusion, calibration of the magnification in digital images from transmission electron microscopy may be performed automatically, using the procedure presented here, with high precision and accuracy.     

A comparative study of replication techniques for use in scanning electron microscopy of orthopaedic implants

Three replication techniques for use in scanning electron microscopy of orthopaedic implants are described and compared. The three consist of one type of silicone replica (Xantopren® Blue) and two types of plastic film replicas (collodion in amyl acetate and Formvar in ethylene dichloride). The clearest image with the highest resolution and contrast is obtained with a Formvar replica. Collodion produces almost as good an image and the replicas are easier to produce. However, neither Formvar nor collodion can be used on implant surfaces which are grossly curved or rough textured since the replicas are not rigid enough to retain the true shape of the surface. Xantopren Blue is considerably easier to use than either of the plastic films, gives a true reproduction of specimen shape and allows replication of rough surfaces. Its only deficiencies relative to the plastic films are resolution and contrast at high magnification. If the sample to be replicated is relatively flat and smooth, and high magnification is desired, plastic film replication materials are recommended.     

Freeze-fracture cytochemistry: a simplified guide and update on developments

A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centred on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemisty works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.     

Seeing is believing? When scanning electron microscopy (SEM) meets clinical dentistry: The replica technique.

In restorative dentistry, the in situ replication of intra‐oral situations, is based on a non‐invasive and non‐destructive scanning electron microscopy (SEM) evaluation method. The technique is suitable for investigation restorative materials and dental hard‐ and soft‐tissues, and its interfaces. Surface characteristics, integrity of interfaces (margins), or fracture analysis (chipping, cracks, etc.) with reliable resolution and under high magnification (from ×50 to ×5,000). Overall the current study aims to share detailed and reproducible information about the replica technique. Specific goals are: (a) to describe detailed each step involved in producing a replica of an intra‐oral situation, (b) to validate an integrated workflow based on a rational sequence from visual examination, to macrophotography and SEM analysis using the replica technique; (c) to present three clinical cases documented using the technique. A compilation of three clinical situations/cases were analyzed here by means the replica technique showing a wide range of possibilities that can be reached and explored with the described technique. This guidance document will contribute to a more accurate use of the replica technique and help researchers and clinicians to understand and identify issues related to restorative procedures under high magnification.     

Replica technique for the inspection and measurement of small internal screw threads

The superplastic behaviour of zinc-22% aluminium alloys can be utilized in the production of replicas of small internal screw threads. Reproduction of size is better than with epoxy resin, moulding rubber or plaster of Paris and the replica has sufficient hardness to enable a contact method of measurement to be used. The replica technique is described and examples of dimensional measurement and of the determination of surface finish parameters are presented.     

Reinforcement and protection with polystyrene of freeze-fracture replicas during thawing and digestion of tissue

An improved method for reinforcing freeze-fracture replicas, which is especially suitable for tough tissues, is described. For this purpose, polystyrene dissolved in chloroform is used to produce a solid protecting layer of plastic on top of the replica, enabling it to withstand the stresses associated with the thawing and digestion of the tissue with strong acids. The method results in production of large clean replicas from tissues such as skin or peripheral nerve which are difficult to process. The method can also be used profitably for reinforcing other softer and more homogeneous tissues.     

A modified cleansing procedure to obtain large freeze-fracture replicas

A method for cleaning freeze-fracture replicas is elaborated which avoids their rolling up and fragmentating during successive steps. Immediately after freeze-fracturing, the replicated tissue is slowly thawed from 77 K to room temperature on solid methanol. Safe passage through the subsequent cleaning steps is facilitated by using solutions of gradually increasing surface tension. After at least 24 h of fixation in methanol, the tissue is digested in half-strength bleach containing 5% ethanol. Following further cleaning in full-strength bleach, the remaining organic material attached to the replica is dissolved overnight at room temperature in 50% saturated sodium hydroxide.     

关于副本定位数据复制新技术的设计

随着计算机网络技术的不断发展,数据网格中副本定位对于网络性能的改进逐渐被人们所认识。本文基于对副本定位数据复制技术的研究,确定了副本创建网格环境后,建立了新技术的域内副本定位机制,提出了动态均衡映射技术域间动态均衡副本定位的方法,完成了系统副本定位模块功能的设计。测试表明,改进的技术数据网格中,域间定位数据元的增加对宿主节点的负载分配没有太大的影响。这一结论对发展大规模网格系统具有重要意义。     

Application of freeze-etch replication for the observation of the cell-extracellular matrix interface of cultured cells

In order to investigate the ultrastructural three-dimensional relationship between extracellular matrices (ECM) and the plasma membranes of cultured cells, a freeze-etch replica method was devised. Bovine corneal endothelial cells were cultured on a Collodion film which covered a hole punched in a plastic coverslip, and were quickly frozen with a slammer with their basal surface facing a liquid nitrogen-cooled copper block. The cells were placed upside-down in a Balzers freeze-fracture machine and freeze-etched, and then platinum-carbon replicas were obtained. The structure of the ECM-plasma membrane interface was observed successfully and so this technique provides a new approach for investigating the ECM-plasma membrane (matrix-receptor) relationship.     

On the measurement of lattice parameters in a collection of nanoparticles by transmission electron diffraction

Improved accuracy in transmission electron diffraction measurements of interplanar spacings can be achieved by adding to the camera constant a term proportional to the square of the diffraction ring radius. A statistical technique using all of the diffraction rings yielded a precision better than 0.05% when measuring the lattice parameter of copper nanoparticles. Gold was used as an internal reference for copper by vapor depositing gold and copper on opposite sides of a thin amorphous carbon film. The source of the squared-radius term is consistent with distortions associated with the magnetic field of the post-objective lenses.     

Electron optical property of a charged foil,observed with a transmitted electron detector device in an SEM

Experimental evidence is presented for the electron optical behaviour of a charged foil area, using the transmitted electron detection device of the scanning electron microscope JSM 50 A (JEOL). The primary electron beam scanning a thin pioloform foil on the one hand produces a charged foil region which on the other hand acts as an electron lens to the primary and scattered electrons. Scanning electron microscopical investigations of air particulates in the submicron size range can be eased by using a transmitted electron detection device both of the bright and dark field operation mode. The image contrast thus may be improved by orders of magnitude, also allowing on line operation of an image analysis system. Using a special preparation technique, depositing the particles on a thin supporting foil which is also used for LAMMA analysis – Wieser et al. 1980, the x-ray spectra of single particles provided by an energy dispersive x-ray spectrometer may be quantitatively interpreted on the basis of the peak-to-background method (Statham and Pawley 1978, Small et al. 1979). Figure 1 shows a schematic of the transmission detector device of the JSM 50 A when operated in the dark field mode. Geometrical dimensions and apertures also are given in Fig. 1. The dark field diaphragm (DFD) on the optical axis of the microscope blocks all electrons (primary electrons and scattered electrons) within an angle of about 10^?2 rad from contributing to the video signal. As long as magnifications above about 350 × are used the primary electron beam hits the DFD thus yielding a transmission scanning electron micrograph in dark field mode. Below this limit or above the corresponding maximal scanning angle (about 7 × 10^?3 rad) of the primary electron beam the rim of the DFD becomes visible in the displayed image as shown in Fig. 2a. At the same magnification Figure 2b shows the sharpened contours of the DFD as obtained by focussing the primary electron beam to the plane of the DFD by lowering the objective lens excitation. By means of the thin bar attached to the DFD (left hand upper corner of Fig. 2b) the DFD may be centered to the optical axis or exchanged to the bright field aperture. Looking to the circular center of Fig. 2a, we recognize the black grid bars and a few black particles whereas the supporting foil looks bright. No video signal can be obtained, because both the grid bars, and to a lesser extent the particles, are not transparent to the primary electrons of 15 keV energy. On the other hand all electrons scattered by the thin foil to an angle of more than 10^?2 rad are seen by the scintillator and hence accumulate a measurable video signal: This is also favoured by the large solid angle outside the DFD, which is about 30 times the solid angle of the DFD itself.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

Techniques for obtaining and observing complementary images with a low-temperature field emission SEM and subsequent comparison of the identical cells in freeze-etch replicas viewed with a TEM

Initial evidence shows that low-temperature (LT) field emission scanning electron microscopy (FESEM) provides high-resolution complementary images of frozen, fractured biological tissues and that the coating or replicas from the tissues can be recovered and viewed in the TEM to compare the identical cellular structures. To observe frozen specimens, a Hitachi S-4000 FESEM was equipped with an Oxford CT 1500 Cryotrans System. The standard Oxford specimen carrier was modified to accommodate a Denton complementary freeze-etch cap that would hold up to 6 hinged 24K gold specimen holders. This combination allowed observation of complementary images of sputter-coated, freeze-etched biological specimens at magnifications up to X30,000. Resolution of sputter-coated images was also compared with that from evaporative coatings. Use of high-vacuum evaporation of Pt/C in conjunction with LT-FESEM provided useful resolutio up to X100,000. In addition, after these specimens were observed in the LT- FESEM, their coating, which consisted of a freeze-etch replica, could be recovered from the frozen tissues and subsequently observed in the TEM at even higher resolutions. Consequently, complementary images of frozen, fractured, fully hydrated cells could be observed in the LT-FESEM and then compared to the complementary images of the identical cells that were present in the replicas, which were recovered from the frozen specimens, and subsequently observed in the TEM. Being able to evaluate, compare, and contrast data from these two different EM imaging techniques as well as from complementary surfaces, not only provides additional information about the ultrastructure of a specimen, but also helps to assess the resolution of coating films, the presence of contaminants and the three-dimensional distortion in replicas.     

An alternative approach to carbon nanotube sample preparation for TEM investigation

A two-stage replication technique (positive replica) is shown to be suitable for transmission electron microscopy (TEM) examination of carbon nanotubes (CNTs) and other one-dimensional nanostructures in their longitudinal direction. This method enables handling the fragile nanostructures, is fast and simple and allows to study the growth mechanism of nanofeatures, including the early stages of their growth. CNTs may also be examined when the growth layers are very thin, and even when only a few nanotubes are on a substrate. Replicas can be taken from various substrate shapes covered with nanostructures and from minute or specifically selected areas of the substrates. CNTs extracted by the replica are not disturbed, and their nanostructures are preserved. It is demonstrated that using positive replicas, HRTEM images from the nanosized carbon forms can also be obtained.     

Systematic inquiry in tests of negative/positive replica combinations for SEM

High resolution replica materials are routinely used in scanning electron microscopy. A systematic evaluating procedure for replica combinations is proposed which details fourteen points to be recorded. These points include quantitative and qualitative information useful when assessing a replica combination for a particular research problem. A case study employing one silicone-based impression material and one epoxy resin is performed as an example of the procedure.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Equipment for deposition of thin metallic films bombarded by fast argon atoms

Deposition of thin metallic films on dielectric substrates using a source of metal atom flow combined with a flow fast argon atoms has been investigated and the investigation results are presented. The fast atoms are produced due to charge-exchange collisions in a vacuum chamber of argon ions, which are accelerated by potential difference between the hollow-cathode glow-discharge plasma and an emissive grid and enter the chamber through the grid. The metal atoms produced due to ion sputtering of a metallic foil placed on the inner surface of the hollow cathode enter the chamber through the same grid. Substrate pretreatment and pulse-periodic bombardment of the growing film by ～1-keV argon atoms both ensure adhesion of copper to glass up to 2 × 10⁷ Pa. The use of a hollow substrate holder, whose inner surface is also covered with the same foil, makes it possible to exclude losses of the depositing metal and allows recommendation of the equipment for beam-assisted deposition of precious metal films.