# Fluorescence

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Fluorescent minerals emit visible light when exposed to ultraviolet light
Fluorescent marine fish

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. The most striking examples of fluorescence occur when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, and the emitted light is in the visible region.

Fluorescence has many practical applications, including mineralogy, gemology, chemical sensors (fluorescence spectroscopy), fluorescent labelling, dyes, biological detectors, cosmic-ray detection, and, most commonly, fluorescent lamps. Fluorescence also occurs frequently in nature in some minerals and in various biological states in many branches of the animal kingdom.

## History

Lignum nephriticum cup made from the wood of the narra tree (Pterocarpus indicus), and a flask containing its fluorescent solution
Matlaline, the fluorescent substance in the wood of the tree Eysenhardtia polystachya

An early observation of fluorescence was described in 1560 by Bernardino de Sahagún and in 1565 by Nicolás Monardes in the infusion known as lignum nephriticum (Latin for "kidney wood"). It was derived from the wood of two tree species, Pterocarpus indicus and Eysenhardtia polystachya.[1][2][3][4] The chemical compound responsible for this fluorescence is matlaline, which is the oxidation product of one of the flavonoids found in this wood.[1]

In 1819, Edward D. Clarke[5] and in 1822 René Just Haüy[6] described fluorescence in fluorites, Sir David Brewster described the phenomenon for chlorophyll in 1833[7] and Sir John Herschel did the same for quinine in 1845.[8]

In his 1852 paper on the "Refrangibility" (wavelength change) of light, George Gabriel Stokes described the ability of fluorspar and uranium glass to change invisible light beyond the violet end of the visible spectrum into blue light. He named this phenomenon fluorescence : "I am almost inclined to coin a word, and call the appearance fluorescence, from fluor-spar [i.e., fluorite], as the analogous term opalescence is derived from the name of a mineral."[9] The name was derived from the mineral fluorite (calcium difluoride), some examples of which contain traces of divalent europium, which serves as the fluorescent activator to emit blue light. In a key experiment he used a prism to isolate ultraviolet radiation from sunlight and observed blue light emitted by an ethanol solution of quinine exposed by it.[10]

## Physical principles

### Photochemistry

Fluorescence occurs when an orbital electron of a molecule, atom or nanostructure relaxes to its ground state by emitting a photon of light after being excited to a higher quantum state by some type of energy:[11]

Fluorescence (emission): ${\displaystyle S_{1}\to S_{0}+h\nu _{em}+heat}$

here ${\displaystyle h\nu }$ is a generic term for photon energy with h = Planck's constant and ${\displaystyle \nu }$ = 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, 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 just because of its unusual triplet ground state.

In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. However, when the absorbed electromagnetic radiation is intense, it is possible for one electron to absorb two photons; this two-photon absorption can lead to emission of radiation having a shorter wavelength than the absorbed radiation. The emitted radiation may also be of the same wavelength as the absorbed radiation, termed "resonance fluorescence".[12]

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 'sensitized' molecule, which is converted to its excited state and can then fluoresce. This process is used in lightsticks to produce different colors.

### 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.[13][14]

${\displaystyle \Phi ={\frac {\text{Number of photons emitted}}{\text{Number of photons absorbed}}}}$

The maximum fluorescence quantum yield is 1.0 (100%); each photon absorbed results in a photon emitted. Compounds with quantum yields of 0.10 are still considered quite fluorescent. Another way to define the quantum yield of fluorescence, is by the rate of excited state decay:

${\displaystyle \Phi ={\frac {{k}_{f}}{\sum _{i}{k}_{i}}}}$

where ${\displaystyle {k}_{f}}$ is the rate constant of spontaneous emission of radiation and

${\displaystyle \sum _{i}{k}_{i}}$

is the sum of all rates of excited state decay. Other rates of excited state decay are caused by mechanisms other than photon emission and are, therefore, often called "non-radiative rates", which can include: dynamic collisional quenching, near-field dipole-dipole interaction (or resonance energy transfer), internal conversion, and intersystem crossing. Thus, if the rate of any pathway changes, both the excited state lifetime and the fluorescence quantum yield will be affected.

Fluorescence quantum yields are measured by comparison to a standard. The quinine salt quinine sulfate in a sulfuric acid solution is a common fluorescence standard.

Jablonski diagram. After an electron absorbs a high energy photon the system is excited electronically and vibrationally. The system relaxes vibrationally, and eventually fluoresces at a longer wavelength.

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

${\displaystyle \left[S1\right]=\left[S1\right]_{0}e^{-\Gamma t}}$

where ${\displaystyle \left[S1\right]}$ is the concentration of excited state molecules at time ${\displaystyle t}$, ${\displaystyle \left[S1\right]_{0}}$ is the initial concentration and ${\displaystyle \Gamma }$ is the decay rate or the inverse of the fluorescence lifetime. This is an instance of exponential decay. Various radiative and non-radiative processes can de-populate the excited state. In such case the total decay rate is the sum over all rates:

${\displaystyle \Gamma _{tot}=\Gamma _{rad}+\Gamma _{nrad}}$

where ${\displaystyle \Gamma _{tot}}$ is the total decay rate, ${\displaystyle \Gamma _{rad}}$ the radiative decay rate and ${\displaystyle \Gamma _{nrad}}$ the non-radiative decay rate. It is similar to a first-order chemical reaction in which the first-order rate constant is the sum of all of the rates (a parallel kinetic model). If the rate of spontaneous emission, or any of the other rates are fast, the lifetime is short. For commonly used fluorescent compounds, typical excited state decay times for photon emissions with energies from the UV to near infrared are within the range of 0.5 to 20 nanoseconds. The fluorescence lifetime is an important parameter for practical applications of fluorescence such as fluorescence resonance energy transfer and Fluorescence-lifetime imaging microscopy.

### Jablonski diagram

The Jablonski diagram describes most of the relaxation mechanisms for excited state molecules. The diagram alongside shows how fluorescence occurs due to the relaxation of certain excited electrons of a molecule.[11]

### Fluorescence anisotropy

Fluorophores are more likely to be excited by photons if the transition moment of the fluorophore is parallel to the electric vector of the photon.[15] The polarization of the emitted light will also depend on the transition moment. The transition moment is dependent on the physical orientation of the fluorophore molecule. For fluorophores in solution this means that the intensity and polarization of the emitted light is dependent on rotational diffusion. Therefore, anisotropy measurements can be used to investigate how freely a fluorescent molecule moves in a particular environment.

Fluorescence anisotropy can be defined quantitatively as

${\displaystyle r={I_{\parallel }-I_{\perp } \over I_{\parallel }+2I_{\perp }}}$

where ${\displaystyle I_{\parallel }}$ is the emitted intensity parallel to polarization of the excitation light and ${\displaystyle I_{\perp }}$ is the emitted intensity perpendicular to the polarization of the excitation light.[16]

### Fluorence

Strongly fluorescent pigments often have an unusual appearance which is often described colloquially as a "neon color." This phenomenon was termed "Farbenglut" by Hermann von Helmholtz and "fluorence" by Ralph M. Evans. It is generally thought to be related to the high brightness of the color relative to what it would be as a component of white. Fluorescence shifts energy in the incident illumination from shorter wavelengths to longer (such as blue to yellow) and thus can make the fluorescent color appear brighter (more saturated) than it could possibly be by reflection alone.[17]

## Rules

There are several general rules that deal with fluorescence. Each of the following rules has exceptions but they are useful guidelines for understanding fluorescence (these rules do not necessarily apply to two-photon absorption).

### Kasha–Vavilov rule

#### Atmosphere

Fluorescence is observed in the atmosphere when the air is under energetic electron bombardment. In cases such as the natural aurora, high-altitude nuclear explosions, and rocket-borne electron gun experiments, the molecules and ions formed have a fluorescent response to light.[47]

## Applications of fluorescence

### Lighting

Fluorescent paint and plastic lit by UV tubes. Paintings by Beo Beyond

The common fluorescent lamp 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 ultraviolet light. 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 more energy-efficient than incandescent lighting elements. However, the uneven spectrum of traditional fluorescent lamps may cause certain colors to appear different than when illuminated by incandescent light or daylight. The mercury vapor emission spectrum is dominated by a short-wave UV line at 254 nm (which provides most of the energy to the phosphors), accompanied by visible light emission at 436 nm (blue), 546 nm (green) and 579 nm (yellow-orange). These three lines can be observed superimposed on the white continuum using a hand spectroscope, for light emitted by the usual white fluorescent tubes. These same visible lines, accompanied by the emission lines of trivalent europium and trivalent terbium, and further accompanied by the emission continuum of divalent europium in the blue region, comprise the more discontinuous light emission of the modern trichromatic phosphor systems used in many compact fluorescent lamp and traditional lamps where better color rendition is a goal.[48]

Fluorescent lights were first available to the public at the 1939 New York World's Fair. Improvements since then have largely been better phosphors, longer life, and more consistent internal discharge, and easier-to-use shapes (such as compact fluorescent lamps). Some high-intensity discharge (HID) lamps couple their even-greater electrical efficiency with phosphor enhancement for better color rendition.{{ safesubst:#invoke:Unsubst||date=__DATE__ |$B= {{#invoke:Category handler|main}}{{#invoke:Category handler|main}}[citation needed] }} White light-emitting diodes (LEDs) became available in the mid-1990s as LED lamps, in which blue light emitted from the semiconductor strikes phosphors deposited on the tiny chip. The combination of the blue light that continues through the phosphor and the green to red fluorescence from the phosphors produces a net emission of white light.{{ safesubst:#invoke:Unsubst||date=__DATE__ |$B= {{#invoke:Category handler|main}}{{#invoke:Category handler|main}}[citation needed] }}

Glow sticks sometimes utilize fluorescent materials to absorb light from the chemiluminescent reaction and emit light of a different color.[48]

### Analytical chemistry

Many analytical procedures involve the use of a fluorometer, usually with a single exciting wavelength and single detection wavelength. Because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured.[49]

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 energy levels in a Boltzmann distribution. There is, then, a higher probability of excitement and release of photons by lower-energy atoms, making analysis more efficient.

### Spectroscopy

{{#invoke:main|main}} Usually the setup of a fluorescence assay involves a light source, which may emit many different wavelengths of light. In general, a single wavelength is required for proper analysis, so, in order to selectively filter the light, it is passed through an excitation monochromator, and then that chosen wavelength is passed through the sample cell. After absorption and re-emission of the energy, many wavelengths may emerge due to Stokes shift and various electron transitions. To separate and analyze them, the fluorescent radiation is passed through an emission monochromator, and observed selectively by a detector.[50]

### Biochemistry and medicine

{{#invoke:main|main}}

Endothelial cells under the microscope with three separate channels marking specific cellular components

Fluorescence in the life sciences is used generally as a non-destructive way of tracking or analysis of biological molecules by means of the fluorescent emission at a specific frequency where there is no background from the excitation light, as relatively few cellular components are naturally fluorescent (called intrinsic or autofluorescence). In fact, a protein or other component can be "labelled" with an extrinsic fluorophore, a fluorescent dye that can be a small molecule, protein, or quantum dot, finding a large use in many biological applications.[51]

The quantification of a dye is done with a spectrofluorometer and finds additional applications in:

#### Microscopy

• When scanning the fluorescent intensity across a plane one has fluorescence microscopy of tissues, cells, or subcellular structures, which is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labelling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image (multiple channels). DNA microarrays are a variant of this.
• Immunology: An antibody is first prepared by having 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.
• FLIM (fluorescence lifetime imaging microscopy) can be used to detect certain bio-molecular interactions that manifest themselves by influencing fluorescence lifetimes.
• Cell and molecular biology: detection of colocalization using fluorescence-labelled antibodies for selective detection of the antigens of interest using specialized software, such as CoLocalizer Pro.

#### Other techniques

• FRET (fluorescence resonance energy transfer or Förster resonance energy transfer) is used to study protein interactions, detect specific nucleic acid sequences and used as biosensors, while fluorescence lifetime (FLIM) can give an additional layer of information.
• Biotechnology: biosensors using fluorescence are being studied as possible Fluorescent glucose biosensors.
• 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.
• FACS (fluorescence-activated cell sorting). One of several important cell sorting techniques used in the separation of different cell lines (especially those isolated from animal tissues).
• DNA detection: the compound ethidium bromide, in aqueous solution, has very little fluorescence, as it is quenched by water. Ethidium bromide's fluorescence is greatly enhanced after it binds to DNA, so this compound is very useful in visualising the location of DNA fragments in agarose gel electrophoresis. Intercalated ethidium is in a hydrophobic environment when it is between the base pairs of the DNA, protected from quenching by water which is excluded from the local environment of the intercalated ethidium. Ethidium bromide may be carcinogenic – an arguably safer alternative is the dye SYBR Green.
• FIGS (Fluorescence image-guided surgery) is a medical imaging technique that uses fluorescence to detect properly labeled structures during surgery.
• SAFI (species altered fluorescence imaging) an imaging technique in electrokinetics and microfluidics.[52] It uses non-electromigrating dyes whose fluorescence is easily quenched by migrating chemical species of interest. The dye(s) are usually seeded everywhere in the flow and differential quenching of their fluorescence by analytes is directly observed.

### Forensics

Fingerprints can be visualized with fluorescent compounds such as ninhydrin. Blood and other substances are sometimes detected by fluorescent reagents, like fluorescein. Fibers, and other materials that may be encountered in forensics or with a relationship to various collectibles, are sometimes fluorescent.

### Mechanical engineering

Fluorescent penetrant inspection is used to find cracks and other defects on the surface of a part. Dye tracing, using fluorescent dyes, is used to find leaks in liquid and gas plumbing systems.

### Signage

Fluorescent colors are frequently used in signage, particularly road signs. Fluorescent colors are generally recognizable at longer ranges than their non-fluorescent counterparts, with fluorescent orange being particularly noticeable.[53] This property has led to its frequent use in safety signs and labels.

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