Dyes and other colorants in microtechnique and biomedical research

Coloration, known in biological study as staining, enhances the visibility of living and dead objects under the microscope. This study reviews some of the colorants, chromogenic compounds and reactions exploited in staining, with special attention given to techniques that impart different colours to specific components of cells and tissues. A major mechanism, the first to be recognised, is the uptake of dye cations or anions by oppositely charged macromolecules. Modern investigations have shown that weaker electrical attractions are also important, especially in techniques that use more than one dye and methods for staining uncharged materials. The sizes and shapes of dye molecules or aggregates can also influence where they will lodge in a stained tissue. Reactive textile dyes have few uses in biological study, but there are histochemical methods that can covalently bind chromogens to DNA and to some macromolecular carbohydrates. Metal complexing dyes are universally used in microtechnique. Their mechanisms of action are not all fully understood, especially in the case of the routine haemalum and eosin (H & E) method that is routinely used for pathology. Staining a tiny quantity of a specific substance requires amplification to generate a visible signal. This is achieved with antibodies and catalysts. An immunoglobulin (antibody) molecule bound by a specific tissue component (antigen) can itself serve as antigen to more than one anti-immunoglubulin molecules that are labelled with an enzyme or with biotin. Enzyme labels catalyse the production of insoluble coloured materials. Biotin has a strong and specific affinity for either avidin or streptavidin, proteins with four biotin-binding sites that can be detected with biotinylated enzymes. Antibodies carried on colloidal gold particles are detected directly by electron microscopy or, for light microscopy, by a process known as physical development. The gold particles are catalytic nuclei for deposition of silver in chemical reactions that have also been used in black and white photography. There are some dyes that can be taken up by living cells, and of these some attach to particular organelles or enter specific types of cells. Fluorescent probes can lodge in specific sites such as mitochondria, lysosomes or the cell membrane; other probes can serve as indicators of chemical or electrical changes in living cells. Some lectins (carbohydrate-binding proteins from plants, which can be labelled with histochemically detectable enzymes) are taken up by neurones, and are useful for tracing the trajectories of the axons of these cells. Certain fluorescent dyes are used for the same purpose, as are labelled derivatives of dextrans.