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.     

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.     

Preparation of ultrathin amorphous ice films for cryo-electron microscopy

Electron microscopy of biological macromolecules embedded in vitrified ice films suffers from serious problems caused by excessive inelastic scattering, beam-induced movements of the specimen, deformation of the molecules by adsorption at the water-air interface and insufficient mechanical stability of the films. We have built an environmental chamber to control temperature and humidity independently in order to produce ultrathin water films (< 20 nm) spanning holes with diameters of 20 μm to 1 mm. The surface tension of the water films was reduced by adding lipid monolayers, thus prolonging the usable time for thinning of the film and avoiding adsorption artefacts in the embedded material. After cryofixation in ethane a carbon film was evaporated on each side of the specimen to stabilize the ultrathin ice—lipid film. Mechanical stability and charging effects could successfully be reduced by this preparation method. Collapsing water films could be cryofixed and the shape of the hole was analysed. By the eccentricity of the elliptical holes an estimation could be made of the burst velocity of the rim of the hole and of the cooling rate of the cryofixation process.     

Precise and reproducible deposition of thin and ultrathin carbon films by flash evaporation of carbon yarn in high vacuum

A simple, small device and its use for reproducible flash evaporation of carbon yarn in high vacuum are characterized. Using this flash evaporator, carbon films of any thickness up to 20 nm can be deposited without spark generation under minimized photon radiation, and with an accuracy better than ±0·2 nm. The films have less background structure (imaged in phase contrast) than conventionally rod evaporated films and are therefore suitable for many kinds of thin and ultrathin carbon film applications in electron microscopy, e.g. as backing for formvar films or sections, as isolating carbon layers for autoradiography, as ultrathin films (floated from mica) for support of macromolecules to be metal shadowed and as support and cover for negative staining of various specimens by the sandwich technique.     

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.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.     

On the contraction and dilation of the plastic support membranes under electron-beam irradiation in the transmission electron microscope

Time relation curves are given for Formvar membrane contraction or collodion membrane dilation under electron-beam irradiation. Also, numerical values are given for the contraction or dilation measured in different zones of the Formvar or collodion support membranes.     

TEM-tomography of FAU-zeolite crystals containing Pt-clusters

A method for preparing ultrathin sections (- 20 nm) of inorganic solids has been developed using ultramicrotomy of resin-embedded crystal fragments. Undamaged crystals, oriented along a crystallographic direction, could be imaged with transmission electron microscopy (TEM) at a resolution better than 0.5 nm. The true internal structure of the crystals could be investigated by imaging the second in a series of at least three consecutive ultrathin sections. Such TEM-tomography proved that Pt-ion exchanged FAU zeolite crystals, after reduction and oxidation, are occupied internally and randomly of large platinum clusters mainly in the {111-twin planes. TEM-tomography could be useful in man made nanostructures like semiconductors, epitaxial thin films, hard metal coatings, ceramics, catalysts, and biomaterials.     

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.     

Ion beam-sputtered and magnetron-sputtered thin films on cytoskeletons: A high-resolution tem study

A comparison of ion beam-sputtered and magnetron-sputtered thin platinum (Pt) and tungsten (W) films was made. Cytoskeletons from detergent extracted glioma cells grown on gold grids were coated with Pt or W at thicknesses of 1, 1.5, and 2.5 nm. Transmission electron micrographs were taken at high magnification and the granularity of the metal films was evaluated both on the Formvar film and the filaments of the cytoskeleton. In order to make a comparison between the two deposition methods, the metal deposition rate must be equal when corresponding thicknesses are made. Since ion beam sputtering generally is a slower process than magnetron sputtering, an increased target to specimen distance was necessary with the latter technique. This resulted in a coarser granularity of the W films as compared with the ion beam sputtered. The Pt, however, showed no marked difference between the two techniques at equal deposition rates. The study also demonstrated that varying the deposition rate caused differences in the granularity of the magnetron-sputtered Pt and W films, even if the voltage of the target was kept constant. Decreasing the target to specimen distance which increased the deposition rate resulted in a finer granularity of both the Pt and the W films. At the highest deposition rate the granularity of both the Pt and W films was comparable with the granularity of the ion beam-sputtered films.     

Direct Deposition Autoradiography: Can it Really Be Detecting Radioactivity?

The literature concerning the 'direct deposition method' of electron microscope autoradiography has been examined and certain calculations made. Studies have been made on the interaction of gold chloride with protein films, tissue sections and ultrathin sections. It is concluded that in its present form the direct deposition method does not offer an autoradiographic approach of practical value.     

Electron cryo-microscopy of biological specimens on conductive titanium-silicon metal glass films

Thin films of the metal glass Ti88Si12 were produced by evaporation and characterized by AFM and conductivity measurements. Thin Ti88Si12 support films for electron microscopy were prepared by coating standard EM grids with evaporated films floated off mica, and characterized by electron imaging and electron diffraction. At room temperature, the specific resistance of a thin TiSi film was 10(6) times lower than that of an amorphous carbon film. At 77K, the specific resistance of TiSi films decreased, whereas that of carbon became immeasurably high. The effective scattering cross-section of TiSi and amorphous carbon for 120 kV electrons is roughly equal, but TiSi films for routine use can be approximately 10 times thinner due to their high mechanical strength, so that they would contribute less background noise to the image. Electron diffraction of purple membrane on a TiSi substrate confirmed that the support film was amorphous, and indicated that the high-resolution order of the biological sample was preserved. Electron micrographs of TiSi films tilted by 45 degrees relative to the electron beam recorded at approximately 4 K indicated that the incidence of beam-induced movements was reduced by 50% compared to amorphous carbon film under the same conditions. The success rate of recording high-resolution images of purple membranes on TiSi films was close to 100%. We conclude that TiSi support films are ideal for high-resolution electron cryo-microscopy (cryo-EM) of biological specimens, as they reduce beam-induced movement significantly, due to their high electrical conductivity at low temperature and their favorable mechanical properties.     

Developments in tribological research on ultrathin films

A review of studies of the tribology of ultrathin films is presented, which focuses primarily on the tribological properties of the Langmuir–Blodgett (LB) films, self-assembled monolayers, and the molecular deposition films investigated by the authors and their co-workers. The emergence of the atomic force microscope has helped the development of studies of ultrathin films; particularly LB films which have been studied extensively. Firstly, the results of research into the various factors affecting the tribological properties of LB films and progress in the application of molecular dynamics simulations to study the mechanisms of friction and lubrication are introduced. Then a review of the experimental and theoretical research into self-assembled monolayers is given. Finally, recent advances in the investigation of tribological properties of molecular deposition films on different substrates (Au, Si and silica rock surfaces) are presented and the prospects for the tribological applications of such ultrathin films are addressed.     

Method of platinum-carbon coating of ultrathin sections for transmission and scanning electron microscopy: an application for study of biological composites

Many biological materials are composites containing two or more components with different mechanical properties. This study is concerned with the application of a method of platinum-carbon coating (Pt/C) of ultrathin sections for TEM and SEM studies of the design of natural composite materials. The changes in profile of the ultrathin resin-embedded sections during different stages of the preparation reflect the material properties of the various components: stiffer regions deform less than softer ones. Such changes in the section profile can be visualized by the Pt/C method and used as evidence of specific material properties in particular regions of composite materials. The method increases the relief contrast, improves the 3D-view of structures, and in combination with standard TEM and SEM procedures can provide clear demonstrations of material design. The distribution of chitin crystallites in the insect cuticle and the ultrastructure of the pore canal system specialized for the transport of epidermal secretions to the cuticle surface were studied here as examples.     

Structure analysis of sputter-coated and ion-beam sputter-coated films: a comparative study

Gold, platinum and tungsten films were deposited by low energy input (7 mA, 450 V), or high deposition rate (80 mA, 1500 V), diode sputter coating and by ion beam sputter coating. Film structures on Formvar coated grids and on the surface of coated erythrocytes, resin embedded, sectioned, and recorded at high magnification in a TEM were compared using computer-assisted measurements and analysis of film thickness and grain size. The average grain size of the thinnest gold and platinum films was relatively independent of the mode or rate of deposition but as the film thickness increased, significant differences in grain size and film structure were observed. Thick platinum or gold films deposited by low energy input sputter coating contained large grain size and electron transparent cracks; however, more even films with narrower cracks but larger grain size were produced at high deposition rates. Ion beam sputter coated gold had relatively large grain size in 10 nm thick films, but beyond this thickness the grains coalesced to form a continuous film. Platinum films deposited by ion beam sputter coating were even and free of electron transparent cracks and had a very small grain size (1–2 nm), which was relatively independent of the film thickness. Tungsten deposition either by low energy input or ion beam sputter coating resulted in fine grained even films which were free of electron transparent cracks. Such films remained granular in substructure and had a grain size of about 1 nm which was relatively independent of film thickness. Tungsten films produced at high deposition rates were of poorer quality. We conclude that thick diode sputter coated platinum and gold films are best deposited at high deposition rates provided the specimens are not heat sensitive, the improvement in film structure being more significant than the slight increase in grain size. Thick diode or ion beam sputter coated gold films should be suitable for low resolution SEM, and thin discontinuous gold films for medium resolution SEM. Diode sputter coated platinum should be suitable for medium resolution SEM and ion beam sputter coated platinum for medium and some high resolution SEM. 1–5 nm thick tungsten films, deposited by low energy input or ion beam sputter coating should be suitable for high resolution SEM, particularly where contrast is of less importance than resolution.     

Mass thickness determination by electron energy loss for quantitative X-ray microanalysis in biology

As is well known, electron energy loss spectroscopy can be used to determine the relative sample thickness in the electron microscope. This paper considers how such measurements can be applied to biological samples in order to obtain the mass thickness for quantitative X-ray microanalysis. The important quantity in estimating the mass thickness from an unknown sample is the total inelastic cross section per unit mass. Models for the cross section suggest that this quantity is constant to within ±20% for most biological compounds. This is comparable with the approximation made in the continuum method for measuring mass thickness. The linearity of the energy loss technique is established by some measurements on evaporated films and quantitation is demonstrated by measurements on thin calcium standards. A significant advantage of the method is that the energy loss spectrum can be recorded at very low dose, so that mass thickness determination can be made before even the most sensitive samples suffer damage resulting in mass loss. The energy loss measurements avoid the necessity to correct the continuum measurement for stray radiation produced in the vicinity of the sample holder. Unlike the continuum method the energy loss technique requires uniform mass thickness across the probe area, but this is not usually a problem when small probes (<100 nm diameter) are used.     

Novel nanobubble inflation method for determining the viscoelastic properties of ultrathin polymer films

We describe a novel experimental technique for measuring the viscoelastic properties of ultrathin polymer films. The method is based on the classic bubble inflation technique for measuring the biaxial creep compliance of films, reduced in size to measure films with thicknesses down to at least 13 nm. The method uses the imaging capabilities of the atomic force microscope to determine the time evolution of the geometry of nanobubbles. Using these data, along with the applied pressure, the absolute creep compliance of the films can be determined.     

Direct determination of ultrathin section thickness using latex particles

A method is proposed to determine ultrathin section thickness by means of noncentral sections obtained from latex spheres. The resulting spherical segments provide the parameters necessary to calculate very simply the section thickness without further treatment. The method is illustrated by an example using the grid-sectioning technique and could be generalized to classical microtomy.     

A Method for Evaluating the Thickness of Ultrathin Films

A method for determining the thickness of ultrathin (<10 nm) films using an atomic-force microscope is described. Films were deposited onto a porous glass substrate that has smooth surface areas between the pores when the flow of the evaporated material incident on the substrate is at an angle of 20°–30° with respect to the normal to its surface. In order to obtain pores with sharp edges, the substrate surface was preliminarily sputtered by an oxygen ion beam directed at an angle of 90° to this surface. Images of such films obtained using the atomic-force microscopy technique clearly resolve the position of the pore edge–film boundary, making it possible to evaluate the film thickness by the height of the step between the pore edge and the by surface in the cross section of the surface topography.     

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.