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 re­releasing the rest of the 
energy as luminescence or fluorescence (the so­called 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 various­sized  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   'non­radiative   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 non­radiative 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 "non­radiative rates", which can include:

 dynamic collisional quenching

 near­field dipole­dipole interaction (or resonance energy transfer), 

 internal conversion 

 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 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 first­order 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 k   is the sum of all rates of excited state decay. 
i

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 self­ballasted 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.

6
 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 envelope
http://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 short­wave ultraviolet, long­wave ultra violet, or X­rays.

Many types of calcite and  amber  will fluoresce under shortwave UV.  Rubies,  emeralds, and the 

Hope Diamond exhibit red fluorescence under short­wave UV light; diamonds also emit light under 
X ray radiation. Fluorescence can also be used to help recognise chirality in minerals.

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

  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   DNA­DNA   or   DNA­RNA   hybridization 
under   high­stringency   conditions   and   fluorophore­based   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 

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 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 
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 (fluorescent­activated 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   sub­cellular   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.

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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 
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 Day­Glo 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.

10
 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.

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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.

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

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

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.

A blue sapphire in black light

www.agta.org/.../images/20050510figure02.jpg

The cathode ray oscilloscope

12
The Cathode Ray Oscilloscope has a fluorescent screen which employs zinc silicate as phosphor,
emitting green light when struck by the electron beam

Phosphorescence

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

fluorescence, a phosphorescent material does not immediately re­emit the radiation it absorbs. The 
slower time scales of the re­emission are associated with "forbidden"  energy state  transitions in 
quantum mechanics. As these transitions occur less often in certain materials, absorbed radiation 
may be re­emitted 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 "glow­in­the­
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 re­emit the light occur less often.

Most photoluminescent events, in which a chemical substrate absorbs and then re­emits a photon of 

13
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 
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 so­called "glow­in­the­dark" 
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.

Phosphorescent   powder   under   visible   light,  

ultraviolet light, and total darkness.
http://en.wikipedia.org/wiki/phosphorescence

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 Glow-in-dark silicone bracelets
http://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.

15
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 10­10 metre

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

proportional to its value.

16
 

17
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/de
monstration_cathode_ray_osci/demonstration_cathode_ray_osci.html

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