Conceptual Overview

For at least the past two decades, studies in plant anatomy have come to rely on a variety of analytical techniques as much as the descriptive text that has been classically used. These analytical approaches have involved:

While techniques are constantly changing, current research approaches involve efforts to relate structure to function in as much a quantitative manner as possible. One technique that we will explore in this regard is morphometry. This technique, first employed by the French geologist Delesse in 1847, was used to determine the amount of different mineral substances in rocks. Essentially, it involved determining the amount of each kind of mineral in a slice, or face view, through a rock and then extrapolating two-dimensional data into three-dimensional information. Of course, averaging data from many slices through rock structure gave even more reliable information. The data could be presented as either relative amounts of mineral substances, based on the percent composition in the rock, or as absolute amounts based on the amount of mineral substance contained within a given three-dimensional volume, such as a cubic centimeter. Techniques in biology make use of the fact that sections are used through properly prepared specimens, and that the structures exposed in these sections can be assessed in a manner similar to that of mineral substances in rocks. The technique of morphometry can be applied at either the light or electron microscopy level. Morphometry yields quantitative information about the extent of space occupied by different components. Understanding of three-dimensional form, however, comes from the use of various techniques to make models, either real or virtual through reconstruction.

Physical preservation of cell and tissue structure (as opposed to chemical preservation), is sometimes very useful. However, by far, the majority of plant anatomical studies are still performed on chemically fixed and embedded specimens as discussed in the unit on Preparation & Staining. Physical fixation almost always refers to freezing the specimen by one manner or another. When accomplished properly it has the advantage of retaining structures that would normally be extracted through the process of chemical fixation, dehydration and embedment. By cryogenically stabilizing biological material, it is much more suitable for the localization of enzyme and antigenic properties and determinations of natural elemental composition. Effective freezing for transmission electron microscopy generally requires extremely rapid freezing using cryogens (freezing fluids) such as ethane or propane chilled to near their freezing points by a bath of liquid nitrogen.

Liquid nitrogen itself is not a particularly rapid cryogenic agent since its boiling point is -196° C, and its freezing point is -210° C. Thus, with only a 14° C temperature range in the liquid state, there is very little ability to absorb heat without vaporizing. As a consequence, liquid nitrogen is a slow freezing agent which produces many ice crystals of significant size in the cells. These ice crystals, upon formation, may tear through membranes, organelles and even cell walls leaving very poor preservation of structure.

Ethane and propane have a much greater temperature range between their boiling and freezing points, and thus can more quickly absorb heat with the result that extremely small (or even no) ice crystals are formed. This provides for a much better quality of specimen preservation. Frozen specimens may then be used for direct sectioning in light and electron microscopy, or for freeze-substitution in which the frozen water of hydration is dissolved away in a bath of methanol or acetone which substitutes for the native water. (Thus, the name "freeze-substitution.")

Localization may be a technique that creates a precipitate at the site of an antigenic agent, enzyme or other active substance. It may also involve capturing the radioactivity of isotopes that have been selectively incorporated into the microscopic structure of plant cells. The latter approach is termed autoradiography.

In this unit we will examine some of these techniques which are useful in extending the descriptive approach of conventional plant anatomy. Try to imagine various experimental situations in which they may be of use in plant research. After having studied the previous units of plant anatomy, you should be in a better position to imagine such applications. While the emphasis of this unit is on electron microscopic techniques, light microscopy analyses can often be conducted in a similar manner.