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Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. Fluorite under white (left) and shortwave UV-Light (right)

Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which a molecule absorbs a high-energy photon, and re-emits it as a lower-energy 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, 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 exhibits this phenomenon.

Equations

Chemical Process

Fluorescence occurs when a molecule or quantum dot relaxes to its ground state after being electronically excited.

Excitation:

Fluorescence (emission):

hν is a generic term for photon energy where: h = Planck's constant and ν = frequency of light. (The specific frequencies of exciting and emitted light are dependent on the particular system.)

State S₀ is called the ground state of the fluorophore (fluorescent molecule) and S₁ is its first (electronically) excited state.

A molecule in its excited state, S₁, can relax by various competing pathways. It can undergo 'non-radiative relaxation' in which the excitation energy is dissipated as heat (vibrations) to the solvent. Excited organic molecules can also relax via conversion to a triplet state which may subsequently relax via phosphorescence or by a secondary non-radiative relaxation step.

Relaxation of an S₁ state can also occur through interaction with a second molecule through fluorescence quenching. Molecular oxygen (O₂) is an extremely efficient quencher of fluorescence because of its unusual triplet ground state.

Molecules that are excited through light absorption or via a different process (e.g. as the product of a reaction) can transfer energy to a second 'sensitizer' molecule, which is converted to its excited state and can then fluoresce. This process is used in lightsticks.

Fluorescence Quantum Yield

The fluorescence quantum yield gives the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed.

The maximum fluorescence quantum yield is 1.0 (100%); every photon absorbed results in a photon emitted. Compunds with quantum yields of 0.10 are still considered quite fluorescent. Most substances have zero fluorescence (Φ = 0).

Fluorescence quantum yield are measured by comparison to a standard with known quantum yield; the quinine salt, quinine sulfate, in a sulfuric acid solution is a common fluorescence standard.

Fluorescence Lifetime

The fluorescece lifetime refers to the time the molecule stays in its excited state before emitting a photon. Fluorescence typically follows first-order kinetics:

is the remaining concentration of excited state molecules at time = t, is the initial concentration after excitation, and k is the first-order rate constant. The lifetime is defined as:

The lifetime is related to the facility of the relaxation pathway. If the fluorescence relaxation pathway is very efficient, the relaxation is very fast and the lifetime is short (picoseconds). If the relaxation pathway is 'forbidden' (disfavored by quantum mechanical rules), then the lifetime is long (microseconds). The longer the lifetime, the greater the chance that relaxation will occur by a different (non-fluorescent) pathway; therefore long fluorescence lifetimes often correspond to small fluorescence quantum yields. The fluorescence lifetime is an important parameter for practical applications of fluorescence such as Fluorescence resonance energy transfer.

Rules

There are several rules that deal with fluorescence. The Kasha–Vavilov rule dictates that the quantum yield of luminescence is independent of the wavelength of exciting radiation.

This is not quite true and is violated severely in many simple molecules. A somewhat more reliable statement, although still with exceptions, would be that the fluorescence spectrum shows very little dependence on the wavelength of exciting radiation.

The Jablonski diagram describes most of the relaxation mechanism for excited state molecules.

Applications

There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of applications:

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 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 ultraviolet and re-emits visible light.

Recently, "white light-emitting diodes" ("white LEDs") 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.

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 are highly efficient with high CRI and good color temp index rating.

The modern mercury vapor streetlight is said to have been evolved from the fluorescent lamp.

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 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
• 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. 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.

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.

Many types of calcite will fluoresce under shortwave UV.

Rubies, emeralds, and the Hope Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation.

Organic liquids

Organic liquids such as mixtures of anthracene in benzene or toluol, or stilbene in the same solvents, fluoresce with ultraviolet or gamma ray irradiation. The decay times of this fluorescence is of the order of nanoseconds since the duration of the light depends on the lifetime of the excited states of the fluorescent material, in this case anthracene or stilbene.

See also

• Phosphorescence
• Laser-induced fluorescence

External links

• How Fluorescent Lamps Work
• Jablonski diagram
• Fluorescence on Scienceworld
• Basic Concepts in Fluorescence
• Scorpion detection using UV LEDs
• Immunofluorescence Protocol
• Handbook on fluorescent probes used in biology from the company Molecular Probes

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