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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