Fluorescence

Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. The energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat. Usually the absorbed photon is in the ultraviolet range, and the emitted light 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 fluorite, composed of calcium fluoride, which often exhibits this phenomenon.

Fluorescence was described in scientific literature in 1845 by John Herschel, son of the famous astronomer William Herschel, who observed a peculiar behavior by a colorless solution of quinine sulfate, which when exposed to sunlight developed a blue surface color, but only in the twentieth century came to the knowledge of the atomic structure sufficient to describe the nature of the phenomenon, and its first uses in spectroscopy represented by the work of Aleksander Jabłoński.

Factors affecting fluorescence

There are several factors that determine the most likely mechanism: the phase of the sample, the way energy is delivered to the molecule, the nature of the excited electronic state and PES, the pressure (if it is a gas-phase molecule), and the presence of other chemical species that may promote or inhibit quenching or intermolecular energy transfer.

For example, fluorescence readily occurs for atoms in the gas phase at low pressure. Since there are no rotational or vibrational energy levels in an atomic system, non-radiative mechanisms are highly unlikely, especially at low pressure. In addition, chemical relaxation (isomerization reactions, dissociations, and more) is not possible.

Fluorescence is also affected by the structure of the molecule. For example rigid molecules that have systems of conjugated double bonds, lend themselves very well to fluorescence: in particular molecules where there are aromatic structures, in which for the phenomenon of resonance double bonds are scattered throughout the structure, if excited give rise to transitions π→π* and then promote fluorescence.

The temperature is another element that influences the fluorescence, in fact from the temperature depends on the vibrational state of the molecule: therefore it can promote the internal conversion.

Finally it is important to mention the molar absorption coefficient, from which depends the average life time of the excited state. The higher the coefficient, the shorter the average lifetime, the greater the probability of fluorescence.

Applications

There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of applications. Some deep-sea animals, such as the Greeneye, use fluorescence.

Lighting

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 (UV) range, is invisible, and is harmful to most living organisms. The tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs the ultraviolet and re-emits visible light. Fluorescent lighting is very energy efficient compared to incandescent technology, but the spectra produced may cause certain colours to appear unnatural.

In the mid 1990s, white light-emitting diodes (LEDs) became 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 the chip; the phosphor fluoresces from the green to red part of the spectrum. The combination of the blue light that goes through the phosphor and the light emitted by the phosphor produce a net emission of white light.

The modern mercury vapor streetlight is said to have been evolved from the fluorescent lamp. Glow sticks oxidise phenyl oxalate ester in order to produce light.

Compact fluorescent lighting (CFL) is the same as any typical fluorescent lamp with advantages. It is self-ballasted and used to replace incandescents in most applications. They produce a quarter of the heat per lumen as incandescent bulbs and last about five times as long. These bulbs contain mercury and must be handled and disposed with care.

Analytical chemistry

Fluorescence in several wavelengths can be detected by an array detector, to detect compounds from HPLC flow. Also, TLC plates can be visualized if the compounds or a coloring reagent is fluorescent. Fluorescence is most effective when there is a larger ratio of atoms at lower levels in a Boltzman distribution because then there is more of a chance those atoms will be excited then release a photon and can be analyzed.

Fingerprints can be visualized with fluorescent compounds such as ninhydrin.

Biochemistry and medicine

Biological molecules can be tagged with a fluorescent chemical group (fluorophore) by a simple chemical reaction, and the fluorescence of the tag enables sensitive and quantitative detection of the molecule. Examples:

  • Fluorescence microscopy of tissues, cells or subcellular structures is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labeling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image.
  • Automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labeled 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. Ethidium bromide can be toxic – a safer alternative is the dye SYBR Green.
  • 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 quantified, by the fluorescence.
  • FACS (fluorescent-activated cell sorting)
  • Fluorescence has been used to study the structure and conformations of DNA and proteins with techniques such as Fluorescence resonance energy transfer, which measures distance at the angstrom level. 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 extremely 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.

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