Fluorescence Microscopy

When organic or inorganic specimens absorb and subsequently reradiate light, the process is typically a result of fluorescence or phosphorescence. Fluorescence emission is nearly simultaneous with the absorption of the excitation light as the time delay between photon absorption and emission is typically less than a microsecond. When the emission persists long after the excitation light is extinguished, the phenomenon is known as phosphorescence. Stokes coined the term ‘‘fluorescence’’ in the middle of the 19th century when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet (UV) excitation. Stokes noted that the fluorescence emission always occurred at a longer wavelength than that of the excitation light. Early investigations showed that many specimens (minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, inorganic compounds, etc.) fluoresce when irradiated with UV light. In the 1930s, the use of fluorochromes began in biology to stain tissue components, bacteria, or other pathogens. Some of these stains were highly specific and they stimulated the development of the fluorescence microscope. Fluorescence microscopy has become an essential tool in biology as well as in materials science as it has attributes that are not readily available in other optical microscopy techniques. The use of an array of fluorochromes has made it possible to identify cells and submicroscopic cellular components and entities with a high degree of specificity amid nonfluorescing material. The fluorescence microscope can reveal the presence of a single fluorescing molecule. In a sample, through the use of multiple staining, different probes can simultaneously identify several target molecules. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of the respective objects, the detection of fluorescing molecules below such limits is readily achieved. There are specimens that autofluoresce when they are irradiated and this phenomenon is exploited in the field of botany, petrology, and in the semiconductor industry. In the study of animal tissues or pathogens, autofluorescence is often either extremely faint or nonspecific. Of far greater value for such specimens are added fluorochromes (also called fluorophores), which are excited by specific wavelength irradiating light and emit light of useful intensity. Fluorochromes are stains that attach themselves to visible or subvisible structures, are often highly specific in their attachment targeting, and have significant quantum yield (the photon emission/absorption ratio). The growth in the use of fluorescent microscopes is closely linked to the development of hundreds of fluorochromes with known intensity curves of excitation and emission and well-understood biological structure targets.