The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
British scientist Sir George G. Stokes first described fluorescence in 1852 and was responsible for coining the term when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. Stokes noted that fluorescence emission always occurred at a longer wavelength than that of the excitation light.
Early investigations in the 19th century showed that many specimens (including minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, and inorganic compounds) fluoresce when irradiated with ultraviolet light. However, it was not until the 1930s that the use of fluorochromes was initiated in biological investigations to stain tissue components, bacteria, and other pathogens. Several of these stains were highly specific and stimulated the development of the fluorescence microscope.
The technique of fluorescence microscopy with the use of fluorescence microscope became an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Through the use of multiple fluorescence labeling with a fluorescence microscope, different probes can simultaneously identify several target molecules simultaneously.
Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved with the fluorescence microscope. A variety of different specimens exhibit auto fluorescence without the application of fluorochromes when they are irradiated with the use of a fluorescence microscope, a phenomenon that has been thoroughly exploited in the fields of botany, petrology, and the semiconductor industry. In contrast, the study of animal tissues and pathogens with the use of a fluorescence microscope is often complicated with either extremely faint or bright, nonspecific auto fluorescence. Of far greater value for the latter studies are added fluorochromes, also termed fluorophores, which are excited by specific wavelengths of irradiating light and emit light of defined and useful intensity.
Fluorochromes, when used with a fluorescence microscope, are stains that attach themselves to visible or sub-visible structures, are often highly specific in their attachment targeting, and have a significant quantum yield the ratio of photon absorption to emission. The widespread growth in the utilization of a fluorescence microscope is closely linked to the development of new synthetic and naturally occurring fluorophores with known intensity profiles of excitation and emission along with well-understood biological targets.
