Before the theoretical background and the actual different techniques used in microscopic study of bryophytesare addressed the “why” should be explained. The procedures necessary to get from a plant to a completed slide can be tedious, and the dozens upon dozens of used reagent mixtures lend an almost mystifying esoteric aura to the practice. The question is, why? Why go through fixation, embedding, sectioning, removal of embedding medium, staining and preservation of the completed slide, when native material would give results much closer resembling the natural state of the organism? Microscopy is fundamentally an optic method. This rather trivial statement is followed by another trivial one: This means it can only be used to investigate things which can be seen. Sample preparation in microscopy is centred around making things visible. The first hurdle is that light (and electrons in the case of transmission electron microscopy TEM) can only penetrate thin slices of most matter, making most botanical structures opaque. Furthermore, they are often buried beneath other tissues. In order to investigate them with microscopic methods one has to expose the structures of interest and section them into sufficiently thin slices. This usually kills the tissue, leading to a rapid degradation of cellular structures. To prevent this loss of structural information fixation is employed. Furthermore, in their natural state, many tissues would disintegrate while being sectioned, or yield slices of poor quality. The necessary embedding is also incompatible with life, leading back to fixation as a prerequisite. Another, and probably the most pressing reason for fixation is that death is part of every living organisms live cycle. Freshly collected the organism might be alive and all structures intact, but it will die sooner or later. Besides these more “geometric” obstacles another mayor one is how we see things. Humans can only visually differentiate structures which produce enough contrast. However, most structures in cells are basically transparent. Contrast enhancement can be achieved via special microscopic methods like phase contrast or difference-interference, or by staining specimens. Staining employs simple dyes, stains and metal salts with a high affinity for certain tissues or structures in cells to highly sophisticated fluorescent probes employing the high selectivity of antibodies (immuno-localisation) or complementary nucleic acids (in situ hybridisation).
From this description, one can infer a general order of steps when preparing a sample for microscopic investigation:
However, before one even starts with collecting sample material, one should already know what one wants to observe in the finished slide. Not only does a method equally applicable to every objective not exist, but often methods prohibit each other. Depending on the goal significant deviations from the outline above are possible. Investigations conducted on life material forgo fixation and embedding, cryo-microtomes likewise have no need for them. When one observes moss leaves for purposes of identification one just plucks the leaves of the gametophyte and puts them on a slide with a drop of water. Electron microscopy does not really “stain” but uses heavy metal salts for contrast enhancement or produces reliefs, basically copies, of the structures in question. The purpose of the outline is just to roughly familiarise a novice with the overall steps.
The preliminary structure is as follows: