SPI Critical Point Drying Apparatus

1. Wash tissue (see previous section for details).
2. Fix tissue.
3. Dehydrate with acetone or ethanol.
4. If using ethanol, substitute with amyl acetate or Freon^® 113.
5. Place tissue in baskets taking care not to allow drying; this operation should be carried out under the solvent.
6. Fill transfer boat with transfer liquid (acetone, amyl acetate, Freon^® 113) and put baskets in boat.
7. Ensure apparatus is correctly installed (Section 2.1.3) and run cold water to cool chamber to 20° C.
8. Load specimen boat, close door and open inlet valve. Filling with liquid gas should be rapid; open vent valve to avoid back-pressure.
9. Leave inlet valve fully open (with vent valve slightly open to maintain liquid level) and open drain valve to remove substitution liquid. This flushing action should be kept up for 3-5 minutes. If the noise is found to be objectionable, a 'silencer' in the form of a length of plastic pipe may be fitted to the drain valve; this also serves to direct the flow of exhausting solvent.
10. After flushing the bulk of the substitution liquid, fill the chamber and close all valves. Leave for 1 hour to allow impregnation. (The times mentioned refer to a piece or pieces of tissue less than 1mm in thickness. For larger pieces or very dense tissue allow longer time).
11. Repeat operation 9.
12. Close the inlet valve and allow the level of liquid to fall to about the level of the top of the boat.
13. Complete the drying run (see page 13 for details) by heating slowly to 36-38° C WITH ALL VALVES CLOSED.
14. Carefully vent the carbon dioxide gas. Avoid condensation effects. NOTE: Condensation can be caused by rapid venting or by the presence of acetone or amyl acetate.
15. Remove the tissue baskets from the chamber, mount the specimens on microscope stubs and proceed with vacuum coating.

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