A gripping stub* for the examination of multilaminous biological specimens in the scanning electron microscope

A simple specimen holder has been developed which expedites study of multilaminous biological specimens in the scanning electron microscope. It has the interesting additional property of allowing examination of the top and bottom of a single sample.     

The selection of conditions for examination of specimens in a scanning electron microscope

In the past not much attention has been paid to the use of the most desirable conditions for examining a particular kind of specimen in a scanning electron microscope (SEM). The various factors affecting resolution in the SEM, namely those of beam diameter, beam penetration, contrast, signal-to-noise ratio and depth of focus, are examined. It is not possible in practice to eliminate all detrimental effects and compromises have to be made in instrumental settings, such as gun potential, lens currents, etc. in order to obtain the best image for a given specimen. This paper deals with the problems involved in examining textile fibres, metal surfaces and thin films with particular emphasis being made on the choice of gun potential and the amount of image detail observed. Examples are shown comparing micrographs taken using gun potentials of 5 kV and 20 kV, showing that due to electron-penetration effects much surface detail is lost at the high gun potential. It is, therefore, useful to examine specimens initially at different accelerating voltages to determine if any desirable information is being lost due to the penetration of the electron beam. Some operating conditions are also given for use as a guideline for working at low accelerating voltages.     

A versatile multi-specimen holder for processing and critical point drying of materials for examination in the scanning electron microscope (SEM)

A specimen holder has been designed specifically for use in the Polaron E.3000 critical point drier (CPD) and is capable of drying up to twenty different specimens within a size range of 4.5 mm to 30 μm by utilization of a variable grid system. The principle, however, could be employed in designs for most other critical point dryers. A large number of designs have been produced for handling small specimens during preparation and CPD procedures prior to observation in the scanning electron microscope (SEM). In a great number of cases these have been specific to one particular organism or preparation method. Marchant (1973) suggested culturing organisms on Millipore filters which could then be used in conjunction with a plain filter as a sieve mechanism to contain the specimens, the complete unit being housed in a BEEM embedding capsule. Scott et al. (1973) produced another design, again utilizing the BEEM capsule together with 100 grade copper mesh as the specimen retaining section. Both these designs were flexible in that different grade filters and meshes could be employed. The disadvantage of using BEEM capsules was noted by Taylor (1975) in that the chemical plasticizers present in the capsule material could leak out in the presence of substitution solvents. To overcome this he produced an all metal design for a container employing a similar type of grid system as in this multispecimen holder. His design was for a single chamber specimen holder with a maximum specimen thickness of 1 mm and was such that the complete chamber could be placed directly into the SEM. The need for a multispecimen holder arose when large numbers of specimens, each specific in its preparation required critical point drying without tedious repetition of critical point drying runs. It was necessary to consider the inflexible features of the design. These were, the external dimensions of the container, which had to fit within the existing aluminium specimen-holder of the Polaron E.3000 CPD, and the dimensions of the transmission electron microscope (TEM) grids used as the specimen retainers. Transmission electron microscope grids are available in two sizes, 3.01 mm and 2.3 mm, with a large range in patterns and materials to choose from to suit most types of specimen preparation. From these fixed dimensions a design was drawn up which allowed specimens of up to 4.5 mm across to be prepared and yet still gave the large number of individual chambers required. The construction of the holder can be seen, from the drawing (Fig. 1) and the photographs (Fig. 2) to be comprised of large dimension chambers with a TEM grid housed at each end to contain the specimens and yet still allow the free flow of dehydrating and substitution agents. The complex arrangement of screws was necessary to facilitate the assembly and use of a container which has a separate lid section that can be dismantled to allow different grids to be inserted depending on the dimensions of the material under preparation. The specimens are supported on the lower grid system which can also be varied and for the ease of removal of larger specimens the chamber section divides leaving the specimens readily accessible in the lower half. Where the specimens are very small, the chamber section can be completely removed by carefully removing all screws and then screwing a stud down the centre thread and extracting the chamber section leaving all the specimens supported on their grids on the base plate. These can easily be transferred directly onto the SEM stub and secured either by double-sided sellotape or careful application of an adhesive such as Durafix. It has even been found that discs punched out of Visking tubing can be used in place of the TEM grids to provide a finer sieve mechanism. It was noted, however, that the tubing was hardened by substitution solvents but this still did not seem to impair the results as satisfactory preparations of Penicillium expansum sporangiophores and pollen of Dactylis glomeratus have been achieved (Medi-Cine, 1976). The container has been made out of high quality brass because of its good machining properties necessary when such fine work has to be carried out. The metric design utilizes standard milling cutters with the inclusion of the ?th cutter to produce satisfactory grid housings allowing free movement to ensure that they always settle on the base plate. The versatility of the design can be further increased with the production of two accessory structures (see Fig. 1). The first, simply produced by cutting brass tubing, is for the preparation of small numbers of specimens per chamber, and ensures that the specimens are deposited onto the grid. The holes drilled through the tube wall allow easy removal with fine forceps. The second structure simply partitions the large chambers increasing the capacity to eighty specimens where such a large specific separation is required. The production of artifacts resulting from specimen handling is reduced considerably with this specimen holder. Once the specimens have been loaded, all the processing stages can be carried out on all the material at once and the specimens are ready for mounting prior to observation.     

Splitting wood specimens for observation in the scanning electron microscope

This report deals with the principles of splitting wood specimens for observation in the scanning electron microscope and gives some examples in which the splitting is a crucial step in specimen preparation.     

Cutting wood specimens for observation in the scanning electron microscope

This report deals with the cutting of wood specimens for observation in the scanning electron microscope. Several cutting devices and types of knives are described and critically evaluated.     

A simple technique for examining frozen hydrated specimens in the scanning electron microscope.

The use of a wide angle backscattered electron detector in a scanning electron microscope, which has the capability of the specimen chamber pressure being controlled independently of the column pressure, provides a simple technique for examining frozen hydrated specimens. Large specimens have been examined within 1 min of being placed on the stub and have been examined for many hours without charging artefacts or distortion due to dehydration.     

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 technique for the examination of polar ice using the scanning electron microscope

The microstructure and location of impurities in polar ice are of great relevance to ice core studies. We describe a reliable method to examine ice in the scanning electron microscope (SEM). Specimens were cut in a cold room and could have their surfaces altered by sublimation either before (pre‐etching) or after (etching) introduction to the cryo‐chamber of the SEM. Pre‐etching was used to smooth surfaces, whilst etching stripped away layers from the specimen surface, aiding the location of particles in situ, and allowing embedded structures to be revealed. X‐ray analysis was used to determine the composition of localized impurities, which in some cases had been concentrated on the surface by etching. Examining uncoated surfaces was found to be advantageous and did not detract from qualitative X‐ray analysis. Imaging uncoated was performed at low accelerating voltages and probe currents to avoid problems of surface charging.     

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 simple technique for examining fresh, frozen, biological specimens in the scanning electron microscope

A technique is described for the examination of frozen biological specimens on a modified stub, and a clamping device for examining freeze-fractured specimens.     

Preparation of tough,fibrous material for examination in the scanning electron microscope

A simple method has been devised which minimizes distortion of fibrous material during the cutting needed for examination of cross-sections in the SEM. Camphene, suggested for use in ‘freeze-drying’ at room temperature (Watters & Buck, 1971) is used as an embedding material with a surrounding layer of ice for reinforcement and sectioning can then be carried out. The technique has been successfully applied to hides and skins in several stages of processing to leather, i.e. in both wet and dry initial states.     

Improved handling of structural fragile cell-biological specimens during electron microscopic preparation by the exchange method

An exact method of preparation of soft biological specimens for electron microscopic analysis of surface fine structures is described. It allows routine preparations of fragile specimens for SEM and TEM imaging modes. With this procedure physical preparation parameters such as mechanical loads on the specimen surface or changes of temperature are controlled. The wet specimens are premounted in cheap disposable BEEM-containers or glass boats and are constantly kept under liquid in a closed system. The exchange of preparation media is performed continuously and, if necessary, over gradients. For comparative investigations with different EM-modes, at each step of the procedure parts of the specimens may be removed for individual processing. Conventionally prepared critical-point dried specimens are compared to those processed by the exchange technique and preservation of surface fine structures is demonstrated. Shadow-casted clathrin cages and stereo-replicas of virus infected cell cultures are shown in TEM preparations. For SEM, coverslip cell cultures and isolated glomerulus basement membranes are prepared and an additional flat embedding for TEM ultrathin sections is demonstrated.     

A balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

Hybrid scanning transmission electron/scanning tunnelling microscope system for the preparation and investigation of biomolecules

A hybrid scanning transmission electron/scanning tunnelling microscope vacuum system is introduced, which allows freeze drying and metal coating of biological samples and their simultaneous observation by scanning transmission electron microscopy and scanning tunnelling microscopy (STM). Different metal coatings and STM tips were analysed to obtain the highest possible resolution for such a system. Bovine liver catalase was used as a test sample and the STM results are compared to a molecular scale model.     

Nanotip electron gun for the scanning electron microscope

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.     

A robust micromanipulator for the scanning electron microscope.

A simple device for holding and moving mechanical tools in the region of a sample being viewed in the scanning electron microscope is described. The unit has a 20:1 mechanical reduction and when fitted with a tungsten carbide dental chisel, it is sufficiently rigid to cut biological hard tissues. Alternately, when fitted with an electro-etched tungsten needle, it can be used, in conjunction with specimen stage controls, to remove individual cells from the surface of soft tissues. Examples of these applications are illustrated.