The use of a simple method to avoid cell shrinkage during SEM preparation

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO₄-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO₄, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.     

Lock-in technique applied to cathodoluminescence of biomedical specimens in the SEM

Very low signals or disturbances by unwanted, foreign signals often lead to a restriction in the application of the cathodoluminescence (CL) method in the scanning electron microscope (SEM). This is even true if one uses an optimal CL detection system. We, therefore, introduced the lock-in-amplification technique, which has proved very successful in investigations of semiconductor materials into the biomedical field. After attaching the lock-in system to our SEM which has a special CL equipment, we found that this technique could remove the disturbance caused by the light emitted from the heated filament, which can be reflected into the CL detector. Specimens on polished Al-stubs or on Au-coated glass slides could be imaged with improved contrast. The same was true if we measured the wavelengths of the CL. A general improvement of the signal-to-noise ratio in all specimens could not be detected. However, the beam current could often be reduced when using the lock-in technique without a decrease in the quality of the CL image. A disadvantage of the commercially available lock-in amplifier is that pictures need a longer exposure time than without lock-in amplification.     

A container for handling small specimens during preparation and examination in the scanning electron microscope (SEM).

The difficulties of handling small specimens of less than 1 mm3 are considerably reduced when the specimens are placed in a small container designed to be compatible with the dimensions of the preparatory instrumentation and of the microscope. The container described is unaffected by solvents used in the preparation of specimens for scanning electron microscopy.     

Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicillium nalgiovense cultures

Penicillium nalgiovense cultures, which are used in the food industry, were found to be collapsed when prepared by standard procedures for scanning electron microscopy. Neither freeze-drying nor critical point-drying preserved the structure of cultures grown on agar media. Cryofixation and preparation of frozen hydrated samples using the Hexland Cryotrans CT 1000 attachment in conjunction with an AMR 1000A scanning electron microscope yielded micrographs of uncollapsed structures which could be used for morphological characterization. Several additional steps had to be used in sample preparation to achieve satisfactory results. Samples were held in a humid chamber prior to freezing; growth substrate was trimmed as thinly as possible (less than 1 mm above the support); the sides of samples were painted with a conductive cement to their upper edge; and frozen samples were coated intermittently with gold sputtered in several 2-min bursts.     

Some methods for more efficient processing of SEM specimens

Three techniques are described for increasing the quantity of specimens which may be processed for SEM while maintaining the quality of the final product. They are: a simple stainless steel holder for safe manipulation of coverslips during incubation and fixation; a coverslip carrier which permits four or more coverslips to be dehydrated and critically point dried at the same time; and a simple pattern for making baskets to hold relatively large but delicate specimens during processing for SEM. Processing soft tissues for scanning electron microscopy (SEM) presents many problems depending on the size, shape and type of specimen. Cohen (1974) presented an overall view of this problem. Methods for handling cells in suspension have been suggested by Baker & Princen (1975), Newell & Roath (1975) and Rostgaard & Christensen (1975). Tissue culture specimens are also difficult to process for SEM. These may be long-term cultures such as fibroblasts or kidney cells, or short-term preparations such as the collecting of white blood cells or bacteria on coverslips. Nemanic (1972) described a method using Tygon tubing with slits to hold the coverslips after incubation. Perecko et al. (1973) gave details of a holder for processing six small (6 mm) coverslips after incubation. Rice et al. (1976) designed several complex multipurpose carriers for 9 × 22 mm coverslips and for cells on silver or cellulose membrane filters. Broers et al. (1975) used a mesh receptacle for bacteria attached to silicon dioxide slivers. Many investigators simply process pieces of plastic cut from the tissue culture dish itself. Using the plastic directly avoids the problems of incubating cells on glass coverslips. Coverslips are fragile and difficult to remove from the culture dish or flask. Each coverslip must be processed individually, increasing the risk of breakage and limiting the number which can be done at one time. For processing larger tissues there are commercial baskets available. Cohen (1974) illustrated mesh baskets for odd sizes and shapes of soft tissues. We have devised a simple holder which facilitates the use of 12 mm 0 grade coverslips in several types of culture dishes. We have developed a multipurpose carrier which increases the number of coverslips which can be processed for SEM and reduces the number of manipulations needed for each individual coverslip. This carrier can be used for histological and histochemical studies as well as SEM processing. We have modified Cohen's technique (1974) for mesh baskets for larger soft tissue specimens.     

Cryo-electron microscopy of vitrified specimens: An approach to the study of bulk specimens

We are using and developing cryo-electron microscopy of vitrified specimens. Our main interests concern the structure of muscle and muscular components. Micrographs which generally contain periodic features are analyzed by numerical image processing methods. To detect artifacts induced by the electron microscopy techniques, we correlate our results to those obtained by X-ray diffraction. In this paper, we describe our approach to the study of bulk specimens. Vitrification of such specimens is assessed by cryo-X-ray diffraction. Microscopy is done on cryo-substituted specimens.     

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.     

Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

An attempt to model distributions of machined component dimensions in production

In this study, normal, log-normal, triangular, uniform, Weibull, Erlang and unit beta probability density functions are tried to represent the behaviour of frequency distributions of workpiece dimensions collected from various manufacturing firms. Among the distribution functions, the unit beta distribution function is found to be the best fit using the chi-square test of fit. An attempt is made for the adoption of the unit beta model to x-bar charts of quality control in manufacturing. In this direction, upper and lower control limits (UCL and LCL) of x-bar control charts of dimension measurements are estimated for the beta model, and the observed differences between the beta and normal model control limits are discussed for the measurement sets.     

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.     

The use of Er:YAG laser for cavity preparation: an SEM evaluation

OBJECTIVE: The purpose of this study was to evaluate morphological changes in cavities prepared by the Er:YAG laser (2.94 mum) at different parameters of irradiation and by a diamond bur. EXPERIMENTAL DESIGN: Cavities were prepared on 27 human molars (n = 3): G1, 15 Hz/160 mJ enamel/6 Hz/200 mJ dentin; G2, 15 Hz/180 mJ enamel/6 Hz/200 mJ dentin; G3, 15 Hz/160 mJ enamel/6 Hz/250 mJ dentin; G4, 15 Hz/180 mJ enamel/6 Hz/250 mJ dentin; G5, 15 Hz/180 mJ enamel/10 Hz/180 mJ dentin; G6, 15 Hz/160 mJ enamel/10 Hz/180 mJ dentin; G7, 15 Hz/160 mJ enamel/10 Hz/160 mJ dentin; G8, 15 Hz/180 mJ enamel/10 Hz/160 mJ dentin; G9, diamond bur. For SEM analysis, samples were fixed (2.5% glutaraldheyde, 12 h, 4 degrees C), dehydrated (25-100% ethanol), dried, and sputter-coated with gold. RESULTS: Despite the changes on energy and repetition-rate settings, all laser-treated samples showed no evidence of thermal damage or signs of burning and melting. Er:YAG laser ablated dental hard tissues showed exposed enamel prisms, dentin surface without smear layer, and opened dentinal tubules. CONCLUSION: Different Er:YAG laser parameters were effective for ablation of hard tissues, creating an irregular and microretentive morphological pattern without hard tissue damage.     

The preparation of field-ion-microscope specimens containing grain boundaries

A back polishing technique has been devised which increases the efficiency of a field-ion-microscope study of grain boundaries. The rate of back polishing is predicted from the relationship d_md = 2·5 × 10⁴ nm²/msec. This relationship is shown to be valid experimentally and theoretically. Its validity implies the persistence of constant current density conditions during electropolishing of sharp, needle-shaped specimens down to diameters of 100 nm.     

An attempt to quantify the limits of failure detection by ferrography

An attempt is made to determine whether ferrography is likely to be able to detect the levels of damage that would lead to catastrophic failure in mining gear transmissions.

A level of maximum sustainable damage before catastrophic failure would occur is set. The level is necessarily very approximate, but it provides an estimate of the amount of wear debris that would be released into the oil. This amount is considered against the background level of wear debris likely in underground equipment to give the increases produced by failures.

The increases in the direct reading Ferrograph values produced by given increases in ferrous wear debris are derived from experimental data and are related back to the levels of maximum sustainable damage in mining gear boxes.

It is concluded that for some mining gear transmissions ferrography is unlikely to be able to detect maximum sustainable levels of damage.     

Novel sample preparation method of polymer emulsion for SEM observation

The aim of this study was to design a simple and reliable method for obtaining the detailed information about the average size, size distribution, and the surface morphology of particles with variation of the sample preparation of a polymer emulsion. In this work, the characteristic features of the particles of rosin size with high viscosity were first described by scanning electron microscopy (SEM). The morphologies of polymer emulsion of solid lipid nanoparticles and of the microspheres were observed. The advantage of the method is that not only the true size and shape of emulsion particles can be shown, but the problem of high-viscosity emulsion that prevents there study with SEM is solved. Using this new method, the micromorphology and size distribution of the emulsion particles with different viscosities have been clearly observed.     

Containment system for the preparation of vitrified-hydrated virus specimens

A guillotine-type quick freezing device and a bio-hazard containment box have been designed, constructed, and used to prepare vitrifiedhydrated specimens of viruses in their native environment. Special design considerations include the preservation of the specimen in its natural state in vitrified ice, prevention of virus aerosols escape, and control of the potentially explosive air-coolant vapor mixtures.