Preparation of animal tissues for surface-scanning electron microscopy

This paper surveys common problems associated with the preparation of animal tissues so that natural or artificial surfaces may be studied in the scanning electron microscope (SEM). Problems arise because (1) we need to prepare surfaces free from extraneous solids in solution (e. g. mucus, blood, tissue fluid); (2) surfaces of simple sections generally do not reveal significant information so that other methods of 'opening up' internal organization must be developed; (3) we need to remove the preponderant tissue component-water-without significantly altering the remaining structure, which generally has a very low density, is fragile and is non-conducting. In the case of natural, free surfaces to soft tissues, isolated cells or cultures the most useful approach seems to be (a) to wash with a suitable isotonic medium; (b) to fix with an aldehyde; (c) to harden with Parducz's (1967) OsO4, HgCl2 fixative. Artificial surfaces revealing details of internal organization are best prepared by dissecting and ripping aldehyde-perfused and fixed tissue; the duration of fixation has been found to influence the location of the ripping planes in CNS. Enzymatic digestion may usefully be combined with dissection, but must be conducted before fixation. Wet tissue surface is least distorted following freeze drying from a non-polar solvent (e. g. amyl acetate) which has substituted the tissue water. Critical-point drying requires simple apparatus and is also generally satisfactory, but it does cause some bulk shrinkage. Specimens are best coated with a combination of carbon and gold to render them conducting: carbon because it is easily scattered and thus tends to cover very rough surfaces, and because it is tough and stabilizes the surface; gold because it is the most convenient heavy metal having high secondary-electron emission to evaporate. Dried soft tissues have very low bulk densities, and beam-penetration artifacts (bulk charging and edge artifacts) are therefore troublesome. These may be limited by working at as low an accelerating beam voltage as can be tolerated to achieve the required resolution. The preparation and examination of stereo-pair micrographs is recommended as a routine because images can then be interpreted in terms of topography alone, and brightness variations of artifactual or unknown origin can be mentally excluded.