Fluorescence is a luminescence, i.e. optical phenomenon in cold bodies, in which a molecule absorbs a high-energy photon, and re-emits it as a lower-energy (longer-wavelength) photon. The energy difference between the absorbed and emitted photons ends up as molecular vibrations (heat). Usually the absorbed photon is in the ultraviolet, and the emitted light (luminescence) is in the visible range, but this depends on the absorbance curve and Stokes shift of the particular fluorophore. Fluorescence is named after the mineral fluorspar (calcium fluoride), which exhibits this phenomenon.
This means that the system starts in state S1, and after the fluorescent emission of a photon with energy hν, it is in state S2 where:
h = Planck's constant and
ν = frequency of the fluorescing light.
There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of applications:
The common fluorescent tube relies on fluorescence. Inside the glass tube is a partial vacuum and a small amount of mercury. An electric discharge in the tube causes the mercury atoms to emit light. The emitted light is in the ultraviolet range and is invisible, and also harmful to living organisms, so the tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs the UV and re-emits visible light.
Recently, "white LEDs" (light-emitting diodes) have become available which work through a similar process. Typically, the actual light-emitting semiconductor produces light in the blue part of the spectrum, which strikes a phosphor compound deposited on a reflector; the phosphor fluoresces in the orange part of the spectrum, the combination of the two colors producing a net effect of apparently white light.
Biochemistry and medicine
There is a wide range of applications for fluorescence in this field. Large biological molecules can have a fluorescent chemical group attached by a chemical reaction, and the fluorescence of the attached tag enables very sensitive detection of the molecule. Examples:
- automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labelled DNA molecules are separated, the fluorescent label is excited by a UV source, and the identity of the base terminating the molecule is identified by the wavelength of the emitted light.
- DNA detection: the compound ethidium bromide, when free to change its conformation in solution, has very little fluorescence. Ethidium bromide's fluorescence is greatly enhanced when it binds to DNA, so this compound is very useful in visualising the location of DNA fragments in agarose gel electrophoresis
- The DNA microarray
- Immunology: An antibody has a fluorescent chemical group attached, and the sites (e.g. on a microscopic specimen) where the antibody has bound can be seen, and even quantitated, by the fluorescence.
- FACS (fluorescent-activated cell sorting)
- Fluorescence has been used to study the structure and conformations of DNA and proteins. This is especially important in complexes of multiple biomolecules.
- Aequorin, from the jellyfish Aequorea victoria, produces a blue glow in the presence of Ca2+ ions (by a chemical reaction). It has been used to image calcium flow in cells in real time. The success with aequorin spurred further investigation of A. victoria and led to the discovery of Green Fluorescent Protein (GFP), which has become an extemely important research tool. GFP and related proteins are used as reporters for any number of biological events including such things as sub-cellular localization. Levels of gene expression are sometimes measured by linking a gene for GFP production to another gene.
Also, many biological molecules have an intrinsic fluorescence that can sometimes be used without the need to attach a chemical tag. Sometimes this intrinsic fluorescence changes when the molecule is in a specific environment, so the distribution or binding of the molecule can be measured. Bilirubin, for instance, is highly fluorescent when bound to a specific site on serum albumin. Zinc protoporphyrin, formed in developing red blood cells instead of hemoglobin when iron is unavailable or lead is present, has a bright fluorescence and can be used to detect these problems.
Gemology, mineralogy and forensics
Gemstones, minerals, fibers and many other materials which may be encountered in forensics or with a relationship to various collectibles may have a distinctive fluorescence or may fluoresce differently under short-wave ultraviolet, long-wave ultra violet, or X-rays.
Rubies and the Hope Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation.