The fluorescent microscope operates by filtering through only light with a specific wavelength for the fluorescent sample, usually from a mercury or xenon lamp. The light collides with the sample, and electrons become excited and move to a higher orbital level. As electrons return to their normal level, light energy is emitted. This is less bright with a longer wavelength than the original excitation light, and is made visible by use of a second filter in the microscope that separates the emitted light from the original excitation light.
Samples are first prepared prior to being viewed on a fluorescent microscope. For cells and tissues, this is done by fixation. Fixation allows for the structural organization of the sample to be maintained prior to staining with a fluorescent probe. The fixation technique use depends on factors such as the type of fluorescent probe used, and the size or thickness of the sample. There are several fixation techniques including precipitation, cryofixation and cross-linking.
Cross-linking is a common fixing technique, where a reagent that binds to intracellular components of the sample is added. Aldehydes such as paraformaldehyde and glutaraldehyde are commonly used as fixing reagents, and they form covalent links with amine groups from the sample, via the Schiff acid-based reaction. Aldehydes are considered a carcinogenic and should be handled within a fume hood. Cross-linking reagents are generally dissolved in an appropriate buffer such as phosphate buffered saline (PBS), which helps to maintain the cell close to its normal physiological state without disrupting its cellular structure. Amine-containing buffers are not recommended because they bind to the cross-linking reagent.
Fluorochromes are generally designed around aromatic, organic compounds that bind to the biological sample. In cell samples, fluorochromes also monitor cell viability, including apoptosis, membrane fluidity, endocytosis, exocytosis and enzymatic activity. Common fluorochromes used in fluorescent microscopy are rhodamine and Hoechst stains. Rhodamine is used to label a range of biological molecules, including nucleic acids, proteins, lipids, carbohydrates, hormones and toxins. Rhodamine has a high photostability, extinction, fluorescent quantum yield, and a low degree of triplet formation. The two types of Hoechst stains, 33258 and 33342, are mainly used to label DNA. They can be used to stain living cells and can also visualize mitochondria and nuclei. Hoechst stains disrupt DNA replication during cell division as a result of their DNA binding and are thus considered carcinogenic and mutagenic.