Nondestructive Imaging of Pin-Mounted Museum Specimens of Insects Using the Environmental Scanning Electron Microscope (ESEM)

The abilities of researchers to obtain high-quality images and other data from pin-mounted museum insect specimens using conventional scanning electron microscopy (SEM) are hindered by several necessary constraints. The specimens may represent unique exemplars (e.g., holotypes) upon which the taxon name rests. In some cases the specimen may no longer be extant in any environment outside the museum. Thus these insects must always be handled with extreme care, regardless of how they are to be observed.

Normal preparation of an insect for SEM involves sputter coating it with a conductive metal to minimize the effects of charging, and conductive paint must be applied to an obscure or uninteresting area to complete the connection to the specimen mount and thus to ground.1 Generally, unless such specimens have been newly collected, they will have already been killed and allowed to air dry, with a mounting pin inserted through the thorax. The body of the insect shrinks against the pin, which cannot then be removed for observation and later reinserted without damage. Some smaller insects are glued carefully to a triangular paper point, through which the pin is inserted. A museum specimen or other specimen for which further use is intended clearly cannot be sputter coated or painted with a conductive material. Although orientation of the specimen for micrography would be simplified greatly by removing the pin, a means must be found to image as much of the specimen as possible with the pin intact. A further complication is that some types of mounting pins are poorly conductive, perhaps as a result of efforts to prevent oxidation of the pin surface.

Imaging at reduced atmosphere conditions using an "environmental" scanning electron microscope (ESEM) and a high-resolution backscattered (BS) electron detector can reduce or avoid some of the problems listed above. The general necessity that higher accelerating voltages and larger beam diameters be used in BS imaging, however, coupled with the fact that smaller body parts will often still charge under such conditions, results in a loss of some high-resolution imaging capability. The use of an ESEM with a field-emission electron source (FEG) and a gaseous secondary electron detector (GSED) thus far presents the best means of obtaining high-resolution images of uncoated museum specimens of pin-mounted insects. In this laboratory we have applied two further modifications of the ESEM-FEG/GSED system to aid in imaging of such specimens -- or fresh specimens that have not suffered the effects of air drying. These allow us to image at lower magnifications, thus providing a better overall view of the insect, and with better capability to manipulate the pin. The first of the modifications is the LF (large-field) GSED, an innovation by the manufacturer.2 The second modification is a stage-mounted device designed and fabricated by the authors that allows the pin and insect to be rotated during observation. Preliminary results are shown in Figures 1 and 2. Figure 1 shows an open spiracle from an intact pin-mounted museum specimen. Had sputter coating of this specimen been permissible, it is unlikely that the inner portion of the spiracle would have been coated to the extent of image clarity shown here. Implicit in this image is the physical positioning capability conferred by the pin-rotating mechanism. The lower image is a lesser-magnification view of a museum specimen, demonstrating that the LF version of the GSED is capable of larger fields of view than previous models. Both modifications are expected to lead to further uses within the electron microscopy community.

Figure 1. Figure 2.
Figure 1 Figure 2
Figures 1 and 2. The acrocerid fly Coroncodes siculus (Diptera: Acroceridae), imaged using the ESEM-FEG with large-field (LF) GSED. (1, left) A view into the interior of an open spiracle from a pin-mounted specimen that was not sputter-coated or otherwise treated to make it conductive prior to imaging. This image demonstrates the capability of the LF GSED to permit observation of areas that might not even be accessible to sputter coating, in combination with the added maneuverability conferred by the pin-rotating device. (2, right) A lower-magnification micrograph demonstrating the capability of the LF GSED to image larger fields of view than previous versions of the same detector.

References

  1. J. I. Goldstein et al., Practical Electron Microscopy, New York Plenum, 1975.
  2. FEI Co., Portland OR.
  3. Support provided by the NSF (9871103) and the Beckman Foundation for the purchase of the ESEM.

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