A method for preparing powdered specimens for transmission electron microscopy

A new method of preparing TEM samples from powders is described. The method consists of compacting epoxy-powder composite mixtures in special molds by centrifuging prior to polymerization. The compact is then ion milled by standard methods. Samples prepared in this way are currently being used to study dislocation structures developed during processing of ceramic powders.     

The limitations to resolution in the observation of radiation-sensitive specimens by electron microscopy

When radiation-sensitive materials are observed in the electron microscope, the existence of a critical radiation dose introduces two types of limitations, which we have calculated, in addition to the resolution limit of the microscope. The first limitation is due to the statistical noise of the image which becomes important at low doses. The second one arises from the sensitivity of the photographic emulsion. It is shown that the second factor is responsible for the effective resolution, irrespective of the accelerating voltage of the temperature of the specimen. A distinction is made between very radiation-sensitive materials (critical dose smaller than ≈2500 C/m²), for which the resolution is dictated by the sensitivity of the emulsion and moderately radiation-sensitive materials (critical dose ≈2500 C/m²), where, depending on experimental conditions, either the sensitivity of the emulsion or the characteristics of the microscope itself dictate the resolution. A method is given which determines in practice the best experimental conditions for an optimisation of the resolution. Numerical values are calculated for the case of polyethylene single crystals.     

A simple method for the handling and orientation of small specimens for electron microscopy

Cyanoacrylic glue (Eastman 910) was used to affix small pieces of nasal scrapings to lens paper immediately before fixation in the glutaraldehyde. The lens paper not only served to hold specimens together so that they were not lost during tissue processing, but also functioned as a ‘landmark’ for the specimens, so that specimens could be oriented in a specific manner during embedding and subsequent sectioning.     

A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.     

A new rapid method of air-drying for scanning electron microscopy using tetramethylsilane

A new rapid air-drying technique which gives results comparable to critical point-drying is described for scanning electron microscopy, using a trematode parasite, Homalogaster paloniae, as a test specimen.     

Relocation of minute specimens for scanning electron microscopy

A relocatable stub-holder assembly for SEM that can record the position of small specimens by x-y-coordinates is described. It enables the accurate pin-pointing of objects for further study later.     

Preparation of lithium specimens for transmission electron microscopy

A method of preparing undeformed thin lithium specimens for TEM is described. A solution of dehydrated methanol and toluene is used both for initial dishing of the foil by chemical polishing and also for final thinning. Under electron beam irradiation in the TEM, new pure Li crystals can grow out of the existing Li specimen. These in situ crystals can be used for the study of the microstructure and electronic structure of Li using TEM and electron energy loss spectrometry.     

A new slide rule for electron microscopy

The principle and the mechanism of a new slide rule is explained and its possible application to solving all of the tasks in electron microscopy with respect to the calculation of sizes, magnifications etc.     

Electroless plating—A new technique for the preparation of hard tissue specimens for scanning electron microscopy

Specimens with complicated, intricate three-dimensional structures, which are otherwise difficult to coat adequately by conventional means (sputter or evaporative coating) for scanning electron microscopy, can quickly and simply be rendered conductive by electroless plating. The technique can be recommended only when studying specimens at low magnification because fine detail may be lost.     

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Stain density is an important parameter for optimising the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue and liver tissue.     

The preparation of cross-section specimens for transmission electron microscopy

The structure and chemistry of thin solid films are best studied by transmission electron microscopy (TEM) when they are viewed in cross-section—that is, when the surface normal of the film is made perpendicular to the electron beam. In this orientation, the substrate, the thin film layers, and the interfaces between them can be imaged either simultaneously or individually. Further, information from each of these regions remains distinct from that obtained from the others, eliminating the problems of superimposition that are a consequence of viewing a layered structure in the conventional manner (i.e., parallel to the surface normal). A technique for fabricating TEM specimens that can be viewed in cross-section is described here. Although the majority of our work is with silicon-based materials, the technique can be readily adapted to the study of other systems.     

A simple vacuum pipette for processing groups of specimens for electron microscopy.

A standard sink filter pump attached to a special glass adaptor, holding pipettes, enables groups of specimens to be fixed and dehydrated for electron microscopy with speed and safety.     

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.     

Ion milling of materials science specimens for electron microscopy: A review

Ion bombardment to perforation is a common technique in the materials sciences by which thin specimens can be prepared for transmission electron microscopy. The process is not without complication and involves radiation damage to the specimen and tends not to preserve the initial specimen topology. Some of the more important facets of the ion-milling process, pertinent to such specimen preparations, are described.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

Processing small delicate biological specimens for scanning electron microscopy

To overcome the problems of collecting and handling delicate cells while dehydrating them for scanning electron microscopy, a method is suggested whereby cells are collected on solvent resistant Millipore filters. The schedule used for successfully processing a wide range of algae, protozoa and some other organisms is outlined as is the construction of a simple carrier to hold the filter in the critical point drying apparatus.     

A technique for preparing human dermal and scar specimens for scanning electron microscopy

The aims in a current study of dermis and scar tissue using the scanning electron microscope are:

• 1 to maintain the orientation of the specimen
• 2 to preserve the in vivo architecture, and
• 3 to expose a satisfactory surface.

A technique which best fulfils these requirements involves glutaraldehyde fixation of a splinted specimen, freeze-fracturing using a modified pair of pliers, freeze-drying, incubation in a buffered saline solution of hyaluronidase and freeze-drying again after washing. Consistently satisfactory specimens have been obtained, which provide information about fibre architecture and cell distribution.