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Preparing specimens for scanning electron microscopy

The scanning electron microscope is a very useful tool in the taxonomic study of smaller parasitic Hymenoptera. It is of particular value as a means of illustrating three-dimensional structures of sculpture where line-drawings or micrographs may be insufficient. It is also used, more conventionally, as a means of examining structures in more detail because of the greater resolution which can be obtained in comparison with a normal light microscope.
Useful techniques used for scanning electron microscopy have been described in some detail by Gibson (1984). Unfortunately, this is published in a newsletter for those interested in Chalcidoidea and is not generally available. However, a few of the more important subtleties of Gibson's technique are included in the following notes.

Cleaning specimens
Before preparing material for examination with a scanning electron microscope it is essential that it is absolutely clean. Specimens can be cleaned, prior to mounting, using a strong solution of detergent. A 1:1 solution of ammonium hydroxide and water has been recommended for this purpose by Gibson. The specimens should then be thoroughly cleaned in distilled water before transferring to 70% alcohol. Gibson also recommends removing stubborn particles of dirt from dried specimens by using the tip of a minuten pin which has been dragged across the surface of double-sided sticky tape. The small amount of adhesive which attaches itself to the point of the pin can then be used to remove very small particles without damaging the specimen.

Scanning specimens
In the past, the greatest drawback in using scanning electron microscopy has been the necessity for coating specimens, prior to examination, with gold, gold palladium or platinum. This process is diffcult to reverse and thus generally impractical where few specimens are available for examination, particularly if these are important type-specimens. However, in recent years, the advent of the back-scattered/low vacuum technique using an environmental chamber (see Robinson, 1980) has made it possible to examine uncoated specimens without the risk of charging due to poor grounding (charging results in flare from extremities or distortion of the image). The sort of results that are possible using this low vacuum technique are clearly illustrated in a paper by Austin (1984, figs 1-6).

Uncoated material
Electron micrographs resulting from use of this technique are ideal for illustrating the general habitus of smaller species, macro-structure of the integument or sculpture.

Uncoated material must be examined in an environmental chamber (but see also Murphy, 1978). Specimens mounted on card rectangles or points as described above (pp.249-253) can be examined directly, provided there is sufficient contact of the specimen with the card. Removing the wings, head and legs and mounting them separately on the card will reduce the risk of charging as well as making examination easier.

Coated material
This is most useful for examining the fine detail of smaller structures, eg sensillae, where high magnification (500x +) is essential.

Prior to examination, specimens are attached to an aluminium or brass stub using double-sided sticky tape especially made for the purpose. Alternatively the specimens can be mounted onto a 6mm glass coverslip (itself mounted onto a stub) using an epoxy resin such as Araldite®. This gives a very smooth even background to the specimen. These are then coated with gold, gold palladium or platinum. Gibson recommends the use of a "sputter" coater in preference to a "vaporiser" because all surfaces will be evenly coated. A "vapouriser" or "shadow caster" leaves some surfaces uncoated increasing the risk of charging. The risk of charging will also be reduced, and examination will be made easier, if the head and all the appendages are removed and mounted separately.

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Last updated 04-Sep-2003 Dr B R Pitkin