Fluorescence

Introduction

Fluorescence is a luminescence, which 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.

When a molecule or atom absorbs light, it enters an excited electronic state. The Stokes shift occurs

because the molecule loses a small amount of the absorbed energy before re-releasing the rest of the

energy as luminescence or fluorescence (the so-called Stokes fluorescence), depending on the time

between the absorption and the reemission. This energy is often lost as thermal energy. Stokes

fluorescence is the reemission of longer wavelength (lower frequency) photons (energy) by a

molecule that has absorbed photons of shorter wavelengths (higher frequency).

This is shown in the graph below.

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http://en.wikipedia.org/wiki/Image:Stokes_shift.png

Fluorescence is named after the mineral fluorite, composed of calcium fluoride, which often

exhibits this phenomenon.

Fluorescence induced by exposure to ultraviolet light in vials containing various-sized cadmium

selenide (CdSe) quantum dots

http://en.wikipedia.org/wiki/fluorescence

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Photochemistry

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

electronically excited.

Excitation:

Fluorescence (emission):

Hν: generic term for photon energy where: h = Planck's constant and ν = frequency of light.

S0: ground state of the fluorophore

S1: 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 as the product of a reaction can transfer

energy to a second 'sensitized' molecule, which is converted to its excited state and can, then

fluorescence. This process is used in lightsticks.

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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. 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:

kf: rate of spontaneous emission of radiation and ∑ i ki 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

Inter-system 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 quantum yield.

The quinine salt, quinine sulphate, in a sulphuric acid solution is a common fluorescence standard.

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Fluorescence lifetime

The fluorescence lifetime refers to the time the molecule stays in its excited state before emitting a

photon. Fluorescence typically follows first-order kinetics:

[S1]: remaining concentration of excited state molecules at time

[S1]0: initial concentration after excitation.

This is an instance of exponential decay. The lifetime is related to the rates of excited state decay

as:

∑ i ki is the sum of all rates of excited state decay.

Thus, 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). Thus, the lifetime is related to the facility of the

relaxation pathway. 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.

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Applications of fluorescence

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. Fluorescent lighting is very energy efficient

compared to incandescent technology, but over-illumination and unnatural spectra can lead to

adverse health effects.

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 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 incandescent in most applications. They are highly efficient.

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

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The unfiltered ultraviolet glow of a germicidal lamp is produced by a low pressure mercury vapour discharge (identical to that in a fluorescent lamp) in an uncoated fused quartz envelopehttp://en.wikipedia.org/wiki/Fluorescent_lamp

Assorted types of fluorescent lamps. Top, two Compact fluorescent lamps, bottom, two regular tubes.http://en.wikipedia.org/wiki/Fluorescent_lamp

Fluorescent minerals

Gemstones, minerals, fibres 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. Fluorescence can also be used to help recognise chirality in minerals.

Fluorescent Minerals http://en.wikipedia.org/wiki/fluorescence

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Organic oils

Crude oil (Petroleum) fluoresces in a range of colours, 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

sample.

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.

1. 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.

2. 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. However, ethidium bromide can be toxic.

3. A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene

array) is a collection of microscopic DNA spots, commonly representing single genes,

arrayed on a solid surface by covalent attachment to chemically suitable matrices. DNA

arrays are different from other types of microarray only in that they either measure DNA or

use DNA as part of its detection system. Qualitative or quantitative measurements with

DNA microarray utilise the selective nature of DNA-DNA or DNA-RNA hybridization

under high-stringency conditions and fluorophore-based detection. DNA arrays are

commonly used for expression profiling, i.e., monitoring expression levels of thousands of

genes simultaneously, or for comparative genomic hybridization. DNA microarray is used in

monitoring expression levels for thousands of genes simultaneously in many areas of

biology and medicine, such as studying treatments, disease, and developmental stages. For

example, microarray can be used to identify disease genes by comparing gene expression in

diseased and normal cells. It is also used to assess large genomic rearrangements. DNA

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micoarray is applied when looking for single nucleotide polymorphism in the genome of

populations. It is also used to determine protein binding site occupancy throughout the

genome.

4. 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.

5. FACS (fluorescent-activated cell sorting)

6. 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.

7. 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 localisation. 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 haemoglobin when iron is

unavailable or lead is present, has a bright fluorescence and can be used to detect these problems.

The number of fluorescence applications is growing in the biomedical biological and related

sciences. Nowadays, methods of analysis also include the use of fluorescence microscopes. These

microscopes use high intensity light sources, usually mercury or xenon lamps, LEDs, or lasers, to

excite fluorescence in the samples under observation. Optical filters then separate excitation light

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from emitted fluorescence, to be detected by eye, or with a camera or other light detectors

(photomultiplier tubes, spectrographs, etc).

Fluorescence in plants

Some plants are naturally fluorescent, such as the Day-Glo flower. These contain pigments like

betaxanthis which absorb shorter wavelengths of light exciting electrons to a higher energy state

and emit longer wavelengths of light as the electrons return to the ground state.

Plants have also been genetically modified to fluoresce. Fluorescing transgenes are used in plants as

reporters of gene expression in vivo. Fluorescent antibodies are used to visualize protein

localization in vitro. There are many types of fluorescent proteins such as green fluorescent protein

that absorb and emit at different wavelengths. This enables the production of many differently

labelled fluorescent molecules in a single plant.

Plant fluorescence is nowadays being used by the NASA. They are working together to learn more

about the planet Mars. These scientists and engineers have chosen the Arabidopsis mustard plant,

for many reasons, to go to Mars. Reporter genes have been added to this plant to glow for different

environmental “stressors”. These stressors include temperature, drought, disease, metal content in

the soil, peroxides, etc. Each stressor will glow at a different wavelength that will be monitored. By

doing such an experiment more will be learned about the environment on Mars in order to modify

plant life to be able to survive there.

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Tobacco plant exhibiting fluorescence in uv light.

http://www.molbiotech.rwth-aachen.de/Groups/cereal

biotechnology group/red-fluorescent-protein-expressed in

tobacco plants.jpg

Fluorescence around us

Club Soda or Tonic Water

The bitter flavoring of tonic water is due to the presence of quinine, which glows blue-white when

placed under a black light.

Body Fluids

Many body fluids contain fluorescent molecules. Forensic scientists use ultraviolet lights at crime

scenes to find blood, urine, or semen which are all fluorescent.

Vitamins

Vitamin A and the B vitamins thiamine, niacin, and riboflavin are strongly fluorescent.

Chlorophyll

Chlorophyll makes plants green, but it fluoresces a blood red colour when exposed to ultraviolet

light.

Antifreeze

Manufacturers purposely include fluorescent additives in antifreeze fluid so that black lights can be

used to find antifreeze splashes to help investigators reconstruct automobile accident scenes.

Laundry Detergents

Some of the whiteners in detergent work by making clothing a bit fluorescent. Even though clothing

is rinsed after washing, residues on white clothing cause it to glow bluish-white under a black light.

Blueing agents and softening agents often contain fluorescent dyes, too. The presence of these

molecules sometimes causes white clothing to appear blue in photographs.

Tooth Whiteners

Whiteners and some enamel contain compounds that glow blue to keep teeth from appearing

yellow.

Postage Stamps

Stamps are printed with inks that contain fluorescent dyes.

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Jellyfish

Jellyfish contains some proteins which are intensely fluorescent on exposure to ultraviolet light

Some Minerals and Gems

Fluorescent rocks include fluorite, calcite, gypsum, ruby, talc, opal, agate, quartz, and amber.

Minerals and gemstones are most commonly made fluorescent or phosphorescent due to the

presence of impurities. The Hope Diamond, which is blue, phosphoresces red for several seconds

after exposure to shortwave ultraviolet light.

The cathode ray oscilloscope

The Cathode Ray Oscilloscope has a fluorescent screen which employs zinc silicate as phosphor,

emitting green light when struck by the electron beam

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Jelly fish

http://en.wikipedia.org/wiki/Aequorea victoria,

A blue sapphire in black lightwww.agta.org/.../images/20050510figure02.jpg

Phosphorescence

Phosphorescence is a specific type of photoluminescence related to fluorescence. Unlike

fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. The

slower time scales of the re-emission are associated with "forbidden" energy state transitions in

quantum mechanics. As these transitions occur less often in certain materials, absorbed radiation

may be re-emitted at a lower intensity for up to several hours.

In simpler terms, phosphorescence is a process in which energy absorbed by a substance is released

relatively slowly in the form of light. This is in some cases the mechanism used for "glow-in-the-

dark" materials which are "charged" by exposure to light. Unlike the relatively swift reactions in a

common fluorescent tube, phosphorescent materials used for these materials absorb the energy and

"store" it for a longer time as the subatomic reactions required to re-emit the light occur less often.

Most photoluminescent events, in which a chemical substrate absorbs and then re-emits a photon of

light, are fast, on the order of 10 nanoseconds. However, for light to be absorbed and emitted at

these fast time scales, the energy of the photons involved (i.e. the wavelength of the light) must be

carefully tuned according to the rules of quantum mechanics to match the available energy states

and allowed transitions of the substrate.

In the special case of phosphorescence, the absorbed photon energy undergoes an unusual

intersystem crossing into an energy state of higher spin multiplicity, usually a triplet state. As a

result, the energy can become trapped in the triplet state with only quantum mechanically

"forbidden" transitions available to return to the lower energy state. These transitions, although

"forbidden", will still occur but are kinetically unfavoured and thus progress at significantly slower

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time scales. Most phosphorescent compounds are still relatively fast emitters, with triplet lifetimes

on the order of milliseconds. However, some compounds have triplet lifetimes up to minutes or

even hours, allowing these substances to effectively store light energy in the form of very slowly

degrading excited electron states. If the phosphorescent quantum yield is high, these substances will

release significant amounts of light over long time scales, creating so-called "glow-in-the-dark"

materials.

Most examples of "glow-in-the-dark" materials do not glow because they are phosphorescent. For

example, "glow sticks" glow due to a chemiluminescent process which is commonly mistaken for

phosphorescence. In chemi-luminescence, an excited state is created via a chemical reaction. The

excited state will then transfer to a "dye" molecule, also known as a (sensitizer, or fluorophor), and

subsequently fluoresce back to the ground state.

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Phosphorescent powder under visible light, ultraviolet light, and total darkness.

http://en.wikipedia.org/wiki/phosphorescence

Glow-in-dark silicone braceletshttp://global-b2b-network.com/direct/dbimage/50239461/Glow_In_Dark_Silicone_Bracelets.jpg

Equation

Where S is a singlet and T a triplet whose subscripts denote states (0 is the ground state, and 1 the

excited state). Transitions can also occur to higher energy levels, but the first excited state is

denoted for simplicity.

In order for toys, athletic balls, etc to glow, they must be placed under light for a desired amount of

time.

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Glossary

Photon: A quantum of electromagnetic energy; a particle of light

Fluorophore: a component of a molecule which causes the molecule to be fluorescent

Quenching: any process which decreases the fluorescence intensity of a given substance

Luminescence: the emission of light by a substance other than as a result of incandescence

Quantum dot: a particle of matter so small that the addition or removal of an electron changes its

properties in some useful way.

Chiral molecule: one which s not superimposable on its mirror image

Electrophoresis: movement of charged particles in a fluid or gel under the influence of an electric

field

Angstrom: a unit of length equal to 10-10 metre

Exponential decay: A quantity is said to be subject to exponential decay if it decreases at a rate

proportional to its value.

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References

1. Fluorescence- Wikipedia the free encyclopaedia. [Online]. April 2007.

Available at http://en.wikipedia.org/wiki/fluorescence.

2. Fluorescence- from Eric Weisstein’s world of physics. [Online]. March 2007.

Available at http://scienceworld.wolfram.com/physics/fluorescence.html

3. Molecular Expressions Microscopy Primar: Specialised Microscopy

Techniques- fluorescence-basic concepts in fluorescence. [Online]. March

2007. Available at

http://micromagnet.fsu.edu/pimer/techniques/fluorescence/fluorescenceintro.h

tml.

4. DNA microarray- Wikipedia the free encyclopaedia. [Online]. April 2007.

Available at http://en.wikipedia.org/wiki/DNA_microarray

5. Phosphorescence- Wikipedia the free encyclopaedia. [Online]. April 2007.

Available at http://en.wikipedia.org/wiki/phosphorescence

6. Fluorescence in plants: natural and modified- Wikipedia the free

encyclopaedia. [Online]. April 2007. Available at

http://en.wikipedia.org/wiki/ Fluorescence in plants: natural and modified

7. Fluorescent lamp- Wikipedia the free encyclopaedia. [Online]. April 2007.

Available at http://en.wikipedia.org/wiki/Fluorescent_lamp

8. Cathode ray oscilloscope demonstration. [Online]. April 2007. Available at

http://www.klingereducational.com/products/modern_physics_experiments/

demonstration_cathode_ray_osci/demonstration_cathode_ray_osci.html

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