Biological Applications: Sample Preparation and Quantitation

The most common source of uncertainty in the use of electron beam analytical techniques with biological materials is the preparation technique: the steps that intervene between the normal in vivo condition of the sample and the state in which it is analyzed. Generally, these steps involve removing material from an aqueous, atmospheric pressure environment to one that is dry and at relatively high vacuum, and may also include sectioning, sawing, or fracturing in order to expose the portions of interest. During this treatment, material must not be lost, redistributed, or gained, and the original structural relationships of the material must be conserved. These are quite restrictive requirements, and are usually satisfied only in part. Indeed, establishing the degree to which they are met is often a major problem in itself. There may be no universally satisfactory preparation technique, although some of the freezing techniques approach this. Thus it is usually the responsibility of each analyst to establish criteria for the problem of interest. In general, the properties of the sample determine which preparative techniques can be employed. Thus, if one wishes to analyze tissue fluids, crystalline inclusions, or cell organelles, it is likely that different methods will be useful. A brief survey of some of the methods that are available follows, along with some suggestions about their advantages as well as the hazards that are likely to be associated with them. A general overview was given by Coleman and Terepka.(1)