Fluorescence

Fluorescent minerals

Fluorescence is the emission of visible light by a substance that has absorbed light of a different wavelength. In most cases, absorption of light of a smaller wavelength induces emission of light with a larger (less energetic) wavelength. A smaller wavelength emission is sometimes observed from multiple photon absorption, but this occurs only under conditions of intense radiation such as are available with laser light. The energy difference between the absorbed and emitted photons is dissipated in the fluorescent material, via internal molecular vibrations and eventually heat. The most striking examples of this phenomenon occur when the absorbed photon is in the ultraviolet region of the spectrum, and is thus invisible, and the emitted light is in the visible region. Practical applications of this effect are found in mineralology, gemology, detection of urine and semen stains, and the detection of scorpions.

The term 'fluorescence' was coined by George Gabriel Stokes in his 1852 paper; the name was given as a description of the essence of the mineral fluorite, composed of calcium fluoride, which gave a visible emission when illuminated with "invisible radiation" (UV radiation).

Equations

Photochemistry

Fluorescence occurs when a molecule, atom or nanostructure relaxes to its ground state after being electrically excited.

Excitation: $S_{0}+h\nu _{ex}\to S_{1}$

Fluorescence (emission): $S_{1}\to S_{0}+h\nu _{em}$, here $h\nu$ is a generic term for photon energy with h = Planck's constant and $\nu$ = 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 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 '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.

$\Phi ={\frac {\rm {\#\ photons\ emitted}}{\rm {\#\ photons\ absorbed}}}$

The maximum fluorescence quantum yield is 1.0 (100%); every 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 rates excited state decay:

${\frac {{k}_{f}}{\sum _{i}{k}_{i}}}$

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

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

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, this will affect both the excited state lifetime and the fluorescence quantum yield.

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

Lifetime

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:

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

where $\left[S1\right]$ is the concentration of excited state molecules at time $t$, $\left[S1\right]_{0}$ is the initial concentration and $\Gamma$ 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:

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

where $\Gamma _{tot}$ is the total decay rate, $\Gamma _{rad}$ the radiative decay rate and $\Gamma _{nrad}$ 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 fluorescent compounds that emit photons 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.

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 always 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. Some deep-sea animals, such as the Greeneye, use fluorescence.

Lighting

Fluorescent paint and plastic lit by UV tubes.

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 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 very energy-efficient compared to incandescent technology, but the spectra produced may cause certain colours to appear unnatural. A compact fluorescent lamp can replace incandescent lighting.

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.

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

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 energy levels in a Boltzmann distribution. There is then a higher probability of lower energy atoms being excited and releasing photons, making analysis more efficient.

Fingerprints can be visualized with fluorescent compounds such as ninhydrin.

Equiptment

Main article: Fluorescence spectroscopy

Usually the setup of a Fluorescence assay involves a Light source, which may emit an array different wavelengths of light. Generally, only one wavelegth 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 various energies due to Stokes shift and various electron transitions. Therefore, the wavelegths are passed through an emission monochromator, and selectively observed by a detector

Biochemistry and medicine

Main article: Fluorescence in the life sciences

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 biological molecules by means of the fluorescent emission at a specific frequency were 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 a extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot, finding a large use in many biological applications.
The quantification of a dye is done with a Spectrofluorometer and finds additional applications in:

• 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. Labeling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image (multiple channels). DNA microarrays are a variant of this.
• 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.

. Ethidium bromide fluoresces orange when intercalating DNA and when exposed to UV light.]]

• FACS (fluorescent-activated cell sorting)
• 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 may be carcinogenic - an arguably safer alternative is the dye SYBR Green.
• 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.

Additionally Fluorescence resonance energy transfer used to study protein interactions, detect specific nucleic acid sequences and used as biosensors, while fluorescent lifetime can give an additional layer of information.

Gemology, mineralogy, geology, 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 and amber 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.

Crude oil (petroleum) fluoresces in a range of colors, from dull brown for heavy oils and tars through to bright yellowish and bluish white for very light oils and condensates. This phenomenon is used in oil exploration drilling to identify very small amounts of oil in drill cuttings and core samples.

Organic liquids

Organic liquids such as mixtures of anthracene in benzene, toluene, 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

• Absorption-re-emission atomic line filters use the phenomenon of fluorescence to filter light extremely effectively.
• Black light
• Blacklight paint
• Evos microscope
• Fluorescence correlation spectroscopy
• Fluorescence in plants: natural and modified
• Fluorescence spectroscopy
• Fluorescent multilayer card
• Fluorescent Multilayer Disc
• Fluorescent lamp
• Fluorometer
• High-visibility clothing
• Laser-induced fluorescence
• List of light sources
• Phosphorescence
• Spectroscopy
• Two-photon absorption
• X-ray fluorescence
• Phosphor thermometry, phosphorescence can be used to detect the temperature of an object eg a gas turbine component ,,,

Further reading

• Lakowicz, J.R. 2006. Principles of Fluorescence Spectroscopy, Third Edition, Plenum Press, New York. ISBN 0-387-31278-1.
• Valeur, B. 2001. Molecular Fluorescence: Principles and Applications, Wiley-VCH. ISBN 352729919X .
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. ISBN 0-8247-8350-6.

External links

• Fluorescence Applications Spanning the UV, Vis, and NIR by Photon Technology International Inc.
• [1] Fluorescence Applications & Instruments Slideshows | HORIBA Jobin Yvon
• Interactive Fluorescence Dye and Filter Database Carl Zeiss Interactive Fluorescence Dye and Filter Database.
• ISS Fluorescence Lifetime Standards Tables
• ISS Fluorescence Probes Data Tables
• The Fluorescence Foundation
• Fluorophores.org The database of fluorescent dyes
• Jablonski diagram
• Fluorescence on Scienceworld
• Basic Concepts in Fluorescence
• Scorpion detection using UV LEDs
• Immunofluorescence Protocol
• An example of use of fluorescence in generating cellular images
• Difference between flourescence and glow in the dark
• More examples how the fluorescence can be used
• Fluorescence in digital Photography
• The Influence of Fluorescence in the World of Art
• Fluorescence control by Photonic Crystals - ICMM
• The Fluorescent Mineral Society
• Fluorescence in Practice
• Laboratory for Fluorescence Dynamics
• "A nano-history of fluorescence" lecture by David Jameson
• Exitation and emmision spectra of various fluorescent dyes
• Manawatu Microscopy - first known collaboration environment for Microscopy and Image Analysis featuring Open DataBase of Fluorescent Dyes and Stains and their applications.