PROCEDURE

The procedure requires an Epofix kit, which is available from the Electron Microscopy Sciences (P.O. Box 251, 321 Morris Road, Fort Washington, PA 19034), and Struers (Valhøjs Allé 176, 2610 Rødovre, Denmark). The Epofix kit consists of Epofix resin (contains bisphenol-a-diglycidylether), Epofix hardener (contains triethylenetetramine), measuring syringes, wood stirers, and paper cups. Instructions are included in the kit and should be read carefully (Anonymous 1995). The procedure described here is partially based on these instructions. Suitable gloves, eye/face protection, and clothing protection must be used because potential health hazards may cause health problems (see Wells 1989; Chaney 1989; Smith and Latimer 1989).

The procedure consists of the following steps.

1. Set up the Epofix kit in a proper laboratory. Use a fume hood because of toxic fumes.
2. Dry, in an oven (e.g., 45°C for two days), the material, which will be embedded, to remove moisture before it is placed in the mould.
3. Maintain a mixing ratio of resin and hardener that is 15 and 2 parts by volume, respectively. Otherwise, mix 25 parts by weight of resin with 3 parts by weight of hardener. Mix the resin and hardener in a paper cup and stir carefully for at least 2 minutes. Pot life is about 30 minutes.
4. Pour the mixture carefully over the material in the mould so that only a few air bubbles are caught.
5. Put the mould containing the material and epoxy mixture into a vacuum chamber or vacuum desiccator for no longer than 20 minutes. The time in vacuum is critical to prevent overheating and associated boiling of the epoxy mixture (boiling point 20°C at 40 mm Hg) (see Waters 1975; Wells 1989). To ensure casting of tiny slender cavities, the vacuum may be interrupted and then turned on again. This action, which may be repeated, will force the epoxy further into the cavities and squeeze out air.
6. Let the mixture harden at room temperature (20°C) and atmospheric pressure. The timed required for hardening is dependent on a number of factors, e.g., amount of mounting material used, mixing ratio, and temperature. Hardening time for 30 g of mounting material is about 8 hours at room temperature.
7. Saw the sample into two or more pieces to produce cross sections of the embedded material. Epofix is a transparent epoxy; it is easy to monitor the cutting.
8. A weak acid (e.g., hydrochloric acid [0.5 percent by volume], soda water) may be applied for 10 to 30 minutes to remove the embedded material; depending on its amount and texture. Otherwise a buffered solution of the calcium chelator EDTA, which is a disodium salt of ethylenediamine tetraacetic acid, may be used (Carter and Ambrose 1989). Alternatively, ion-exchange water can be used (H. J. Hansen, personal commun., 1999). Removal of the embedded material has to be slow-acting as to prevent possible breakage of the delicate parts of the cast.
9. Rinse the cast with distilled water to prevent further etching and possible chemical precipitation. After drying and gold coating, the cast is ready for scanning electron microscopy (SEM) (see Carter and Ambrose 1989).