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.     

Cold-stage microscopy system for fast-frozen liquids

The least artifact-laden fixation technique for examining colloidal suspensions, microemulsions, and other microstructured liquids in the electron microscope appears to be thermal fixation, i.e., ultrafast freezing of the liquid specimen. For rapid-enough cooling and for observation in TEM/STEM a thin sample is needed. The need is met by trapping a thin layer ( approximately 100 nm) of liquid between two polyimide films ( approximately 40 nm thickness) mounted on copper grids and immersing the resulting sandwich in liquid nitrogen at its melting point. For liquids containing water, polyimides films are used since this polymer is far less susceptible to the electron beam damage observed for the commonly used polymer films such as Formvar and collodion in contact with ice. Transfer of the frozen sample into the microscope column without deleterious frost deposition and warming is accomplished with a new transfer module for the cooling stage of the JEOL JEM-100CX microscope, which makes a true cold stage out of a device originally intended for cooling specimens inside the column. Sample results obtained with the new fast-freeze, cold-stage microscopy system are given.     

基于椭偏法的火棉胶薄膜光学参数测量

火棉胶醋酸戊脂溶液常被用于医学治疗,在分析材料内部结构时,利用火棉胶醋酸戊脂溶液成膜后表现出的致密性和透光性,将其作为待分析材料的底膜,采用电子衍射的方法获得材料结构。火棉胶薄膜的厚度及材料膜厚对电子衍射结果影响很大,采用光纤光谱仪测量材料膜厚时需要知道火棉胶薄膜的折射率和厚度。为获得不同体积火棉胶醋酸戊脂溶液捞制的火棉胶薄膜的厚度和折射率,利用椭偏原理,采用光谱反射型椭偏仪进行测量,拟合所建模型为Srough/Cauchy/BK7。实验结果表明:该模型能够很好地表征薄膜结构,并得到体积不同的火棉胶醋酸戊脂溶液所捞制的薄膜厚度不同,体积也越大,火棉胶膜厚也越大,Cauchy系数A、B、C系数基本相同。     

Ultrathin formvar support films for transmission electron microscopy

We have found that ultrathin Formvar films are easily and reliably made at an air-water interface by the drop method. By varying the concentration of Formvar in the drop, films of different characteristics can be obtained. Concentrations of 0.25–0.4% in ethylene dichloride produce extremely flat, ultrathin, and stable films that are especially suited for shadowed and negatively stained preparations. Low concentrations (? 0.1%) produce nets consisting of many tiny holes which, after carbon stabilization, are ideal for supporting high-resolution samples. Above 0.5%, films made by the drop method develop bubbles, and this bubble defect makes them unsuitable for section support. For section support, Formvar films made by the stripping method off mica are far superior to those made off glass. The films are more uniform in surface contour and thickness. They are less readily attacked by alcohols. Consequently, they are more resistant to staining procedures involving organic solvents and continue to be strong and uniform for section support.     

Sequential treatment by phosphotungstic acid and uranyl acetate enhances the adherence of lipid membranes and membrane proteins to hydrophobic EM grids

A simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5–10-s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon grids.     

Contrast and quantitation in uniform regions of thin sections using transmission electron microscopy

A direct approach to quantitative measurements of uniform regions in thin sections is described. Accelerating voltages around 80 kV and objective aperture angles of about 9·3 mrad will provide conditions where contrast is directly proportional to specimen mass thickness. An extensive treatment of electron scattering in Formvar films for wide ranges of electron microscopic operating conditions is summarized in a simple, empirical equation. The extent to which Formvar results may be generalized to other materials, both embedding media and structures within the thin section, is treated. Using these results, precise measurements of local section thickness and of specimen density and/or dry mass of regions which penetrate the entire section thickness are possible, with the accuracy dependent upon irradiation effects and specimen makeup.     

Substrate properties affect the mass loss rate in collodion at liquid helium temperature

A previous measurement showed that mass loss from collodion supported by thin carbon films was linear with electron exposure at liquid helium temperature. No other organic solid had shown a linear loss of mass at any temperature. When measurements of collodion were done using titanium supports, the loss of mass proceeded exponentially with exposure at liquid helium temperature. This result suggested that the differing electrical conductivities of these substrates might be the cause of the different mass loss effects. Carbon films, which are typically used at ambient temperatures, have much lower electrical conductivity at very low temperature than titanium films. This suggested that specimen preparation materials and techniques used routinely for room temperature studies may need to be modified when microscopy is done using superconducting objective lenses. For both substrates, the rate of mass loss is slowest at liquid helium temperature.     

Adsorption of DNA molecules to different support films.

Protein-free adsorption of the DNA of the Escherichia coli bacteriophage T7 to carbon, collodion, aluminium-beryllium and aluminium films was studied. It was found that the appearance of DNA strands depended greatly upon the kind of support film used. Direct adsorption of DNA to aluminium-beryllium or aluminium films yielded specimens with 'thin and long' and 'thick and short' regions along the strand. Well extended, uncoiled and unaggregated DNA molecules were obtained only when DNA was adsorbed to carbon, collodion or mica in the presence of intercalating dyes such as ethidium bromide. Adsorption properties of the different films are well correlated with their surface charge. Aluminium-beryllium films carry a strong positive surface charge, aluminium films a weak positive charge and carbon films a weak negative charge. It is suggested that for the preparation of specimens by spontaneous adsorption of well extended and unaggregated strands it is necessary that the DNA molecule is stiffened by a ligand such as an intercalating dye, and that the charge on the surface of the support film is opposite to the charge of the macromolecule.     

Lead and uranium stain artefacts in electron microscopy: a technique for minimizing their occurrence.

A new technique is presented which significantly reduced the occurrence of artefacts by eliminating the air-stain interface when staining thin sections of biological material. It is designed for simultaneous treatment of large numbers of samples. The experiment described deals mainly with the specific staining problems encountered when using grids coated with Formvar.     

A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples

Complementary replicas of samples prepared for electron microscopy by the freeze-fracture/etch technique are extremely valuable in the interpretation of the exposed surfaces, the nature and location of the membrane fracture plane, and as an aid in the recognition of the potential artefacts of this technique. This paper describes a procedure for the preparation of complementary replicas of thin samples sandwiched between copper foil strips and frozen ultra-rapidly in the absence of chemical pretreatments. In this procedure, the copper foil support bearing the replica is floated on the surface of a chromic acid solution, resulting in the controlled dissolution of the copper metal. The replica which remains at the surface of the chromic acid is then stabilized against fragmentation during subsequent cleaning and rinsing steps by placing a 50 mesh gold grid on top of the replica. To minimize agitation of the replica/grid, all cleaning steps are performed in a single depression plate well. The clean replica/grid is picked up from below on a thin Formvar film, dried, and then separated from the extra film. Careful placement of the gold grid on the replicas and low magnification electron micrograph montages of the complementary grids facilitate the location of complementary regions and simplify examination of complementary specimen areas at higher magnification.     

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.     

Thin polymer films as non-charging surfaces for scanning electron microscopy

Films of formvar, 1.2–20.0 nm in thickness, collodion and a 60/40 styrene-vinylpyridine copolymer (precipitated from solution) have been found to provide a noncharging layer on the surface of several types of specimens, including loose, or fibrous specimens. No electron build up was observed in most of the specimens when examined with a scanning beam of up to 30 kv for unlimited periods of time.     

Continuous serial thin sectioning for electron microscopy

The process of serial sectioning for electron microscopy has been refined such that loss of thin sections is kept below 0.1% and the series is continued at will. The method relies on microscopic control of all manipulative steps, Formvar casting on plate glass for coated slot grids, coating of the block with contact cement for reliable ribboning, pickup by a one-step method with grid support in the diamond knife trough, staining in LKB grid holders, gentle treatment of grids in the electron microscope, and a slight modification to the microscope for safe grid withdrawal. The results are particularly applicable to the reconstruction of neuronal microcircuits and larger volumes of neuropil.     

Improved staining of microfilament bundles in plant cells for high voltage electron microscopy

A post-polymerization en bloc staining method for high voltage electron microscopy is described. Embedded specimens were initially trimmed to an area close to the point of interest. Trimmed blocks were stained in 5% uranyl acetate/75% ethanol solutions for 36 h at 333 K. This procedure allowed specimens to be stained without the necessity of exposing Formvar films to damaging solutions. After such staining, both the contrast and fine detail of structures such as microfilament bundles were superior to that seen in material stained with aqueous solutions of uranyl salts.     

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.     

Electron microscopy of critical point dried whole cultured cells

To determine the overall fine structure of whole, unsectioned cells, cells from rat embryos were cultured on Formvar, glutaraldehyde/osmium-fixed, transferred to grids, dehydrated, critical point dried, then examined by transmission electron microscopy at either 80 or 1000 kV. In contrast to air-dried material, critical point dried cells revealed each component clearly and with excellent contrast. All normal cytoplasmic structures (including coated vesicles, polyribosomes, microtubules and other fine components) were readily identifiable. Extensive structures such as microtubules and the endoplasmic reticulum (which appear fragmented in sections) were well displayed. At 1000 kV the beam readily penetrated even the thick nuclear and perinuclear cell regions and produced exceptionally crisp images. The methods described provide a simplified approach to the study of overall cell fine structure.     

Negative staining of freeze-fractured envelopes of Escherichia coli K12.

Envelope fragments of E. coli K12 have been produced by freeze-fracturing "by hand" and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: (i) "grooves" 4-6 nm wide, (ii) spherical particles 6-8 nm in diameter, (iii) "black dots" 3-8 nm in diameter. Treatment of cells with EDTA before freeze-fracturing caused dilation of grooves into holes eventually leading to fragmentation of the outer membrane. A mutant strain deficient in two outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.     

Dynamic hydration effects in an electron microscope cold stage

Water can be a substantial proportion of the residual gas in modern electron microscopes even when frozen hydrated specimens are not used. During measurements of the mass thickness of thin collodion film specimens at low temperatures, it was found that a volatile surface layer (condensed water) modified the apparent rate of mass loss induced by radiation exposure. Mass loss can be enhanced by the presence of water (specimen “etching”), or mass loss can be masked by the dynamic adsorption of water to the specimen surface. The microscope or the grid can be a secondary source of the water; even with cold anticontaminator plates in the vicinity of the specimen, water can be desorbed by x-rays or backscattered electrons. In one typical situation, the mass loss rate appears reduced (due to water adsorption), but the ultimate damage is greater (due to etching). These results illustrate that care must be taken in interpreting mass thickness measurements made in the presence of water and that the lowest stage temperature does not necessarily produce the best observation conditions for all specimens.     

A study of section wrinkling on single-hole coated grids using TEM and SEM

To prevent section wrinkles usually encountered with the use of coated single-hole grids, a simple method was developed. Formvar film resting on a platform with holes (3.5 mm diameter) was heated with a slide warmer (60–65°C). The bottom of a glass petri dish was inverted over the platform to keep the ambient air at the desired temperature. Sections were picked up from the boat of the diamond knife with a single-hole grid and deposited at the orifice of the platform and allowed to dry. The grids were then carefully pushed through the orifice of the platform with the blunt head of a nail (3 mm diameter).     

压电陶瓷微纳米伸缩量测试系统

介绍了一种压电陶瓷的微纳米伸缩量的测试系统。该系统是通过调节加在陶瓷管内外管壁上的电压变化来实现陶瓷管的伸缩,其驱动电压由计算机编程并经过D/A转换后输出,再依次通过低压和高压放大后加在陶瓷管壁上。压电陶瓷的伸缩量则由电感测微仪进行测定。实验结果表明,利用该系统可以很方便地测量压电陶瓷管的伸缩,其伸缩量随着电压的变化而变化,但并非成简单的线性关系,而是呈一条曲线。