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Ctirad Hofr
Steady State
Fluorescence
Fluorescence methods in life sciences
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Steady state fluorescence
1. Definition
2. Fluorescence sensitivity to environment
3. Effects on fluorescence spectrum
4. Fluorescence quenching
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The steady state and timeresolved
fluorescence
• Steady state fluorescence is measured during by
continuous irradiation by a source of excitation light.
The fluorescence intensity is time-averaged.
• Time resolved fluorescence is measured using
pulse excitation (pulse length is usually shorter than
the fluorescence decay time of the sample) or
phase-modulated excitation radiation. TR
fluorescence enables us to analyze the time
dependence of the fluorescence intensity or
anisotropy.
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The influence of environment on the
absorption and emission spectrum
In solutions, solvation of fluorescent molecules occurs between
fluorophore molecules and solvent due to electrostatic
interactions dipole-dipole or dipole-induced dipole. Because, in
general, the molecules differ in their dipole moments and
polarizability in the ground and excited state, changes in the
optical spectra of different solvation molecules occur during
fluorescence measurements in solutions by different solvation of
molecules. The time required for molecular relaxation (10-10s) is
much longer than the transition rate of the electron - absorption
(10-15s) but usually shorter than the lifetime of the excited state
(10-8s). The emission therefore occurs from the state where
equilibrium configuration has been reached. As part of the
absorbed energy is spent for relaxation of solvent molecules
around the molecules of the fluorophore in the excited and
ground state, energy of emitted fluorescent radiation is lower
than would correspond only to a electron transition
From. Fišar: http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
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Electric dipole
• It consists of a pair of opposite polarity charges in
the distance l
• Dipole moment µ = q . l
vector pointing from the negative charge to
positive charge
Unit: Debye; 1D = 3.3 x 10-30 C.m
q+ q-
µ
l
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Molecular dipoles
• A molecule is a dipole when the distribution of
positive and negative charges overlap. In the
case where the molecule is not mirrorsymmetrical,
charge distribution is irregular and
the molecule is a dipole.
• A molecule having a dipole moment is
polarized.
• Molecules (generally mirror symmetrical - CO2)
that are not dipoles may be transformed to
dipoles when the molecule occurs in an electric
field - induced dipole is formed.
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The dipole moment of
polyatomic molecules
Physical Chemistry Atkins and de Paula, Chapter 17
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Interaction of dipoles
• The polarized molecules prefer the
arrangement with the minimum energy of
dipoles
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Polarizability of molecules
• The ability of molecules to create an induced
dipole due to an external electric field
• The induced dipole is proportional to the
intensity of the electric field E
• Induced dipole µ∗
µ* = α E
α is polarizability of molecules
The greater the polarizability of the molecule,
the greater the influence of electric field on the
molecule.
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Change of the dipole during
interaction of molecules
http://www.theochem.ruhr-uni-bochum.de/~axel.kohlmeyer/cpmd-vmd/part3.html
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Interaction energy of two dipoles
• Interaction between two dipoles µ1 a µ2
3
0
2
21
4
)cos31(
r
V
πε
θµµ −
−=
q2
q1
rl1
θ
For the dipole-dipole interaction, potential energy V depends on
the relative orientation.
Minimal energy is at θ = 0°
attractive interaction (opposite charges are together) θ < 54.7 °
Maximal energy is at θ = 90°
repulsive interaction (same charges are together) θ > 54.7 °
Zero potential energy is at the "magic" angle θ = 54.7°
l2
-q2
-q1
Dependence of potential energy
on position of dipoles
4
12 in degrees
0 45 90 135 180
1-(cosx)^2
-2
-1
0
1
+ - + - + +
-
+ - + -
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Dipole-induced dipole interaction
• Polar molecule with a dipole moment
μ1 can induce a dipole moment in a
polarizable molecule
• An induced dipole interacts with a
permanent dipole of the first molecule
and they are mutually attracting
• An induced dipole (blue arrows)
follows changes in a permanent
dipole orientation (yellow arrow)
6
0
2
2
1
r
V
πε
αµ
−=
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Solvation of the fluorophore during
absorption and emission in solutions
In solutions, solvation of fluorescent molecules
occurs between fluorophore molecules and
solvent due to electrostatic interactions dipoledipole
or dipole-induced dipole. Because, in
general, the molecules differ in their dipole
moments and polarizability in the ground and
excited state, changes in the optical spectra of
different solvation molecules occurs during
fluorescence measurements in solutions by
different solvation of molecules. The time
required for molecular relaxation (10-10s) is
much longer than the transition rate of the
electron - absorption (10-15s) but usually shorter
than the lifetime of the excited state (10-8s). The
emission therefore occurs from the state where
equilibrium configuration has been reached. As
part of the absorbed energy is spent for relaxation
of solvent molecules around the molecules of the
fluorophore in the excited and ground state,
energy of emitted fluorescent radiation is lower
than would correspond only to a electron
transition
1 - equilibrium configuration in the ground state
2 - nonequilibrium configuration it the excited state
(Franc-Condon state)
3 - equilibrium configuration in the excited state
4 - nonequilibrium configuration in the ground state
(Franc-Condon state)
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Influence of solvent polarity
• The dipole moment of the molecule in
the excited state µE is greater than in
the ground state µG
• After excitation, solvent molecules are
oriented (relaxed) around µE, which
reduces the energy of the excited
state
The greater the polarity of the solvent, the greater the
influence of orientation of dipoles and the more energy is
consumed for their orientation and then less energy left for
the emitted light, i.e. the greater the wavelength of the
emitted light
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Interaction of the excited
fluorophore and solvent
The greater the polarity of the solvent, the greater the
influence of orientation of dipoles, the smaller the energy of
the emitted radiation and the larger the shift of emitted light λ .
Polar fluorophores are the most sensitive to solvent polarity.
Nonpolar fluorophores are less sensitive.
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The dependence of the dipole
moment on the shape of the molecule
Change of dipole moment is
higher for longer fluorophores
Aminonaphtalene derivatives with
a phenyl group exhibit greater
sensitivity to the solvent and
greater dipole moments in an
excited state probably due to
greater charge separation along
the long aromatic system
Change of dipole moment upon excitation is higher for longer
molecules.
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Difference of the solvent polarity
effect on absorption and emission
spectrum
Fluorescence spectrum varies more with
increasing polarity of solvents than the
absorption spectrum.
.
ABS
Raising of molar concentration of methanol in
hexane in the range of 0-340 mM (0 -> 6)
Absorption spectrum of 2-acetylantracene in pure hexane (0),
200mM solution of methanol in hexane (1) and pure methanol (2).
2-acetylantracene
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Probe for monitoring of
environment polarity
• Addition of polar groups to fluorophore increases its sensitivity to
solvent polarity
• Addition of more polar groups also increases the Stokes shift
Derivates of DOP (2,5-difenyloxazol) and their emission spectrums
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Why is emission spectrum more sensitive to
environment polarity than the absorption
spectrum?
• Because absorption is faster than emission which is slower
than the relaxation of molecules
• chronological order : Absorption (10-15s) -> environment
relaxation (10-10s) ->emission (10-8s)
• absorption can not detect changes in the local environment of
the molecule because it is quicker than they occur
• Molecule environment is the same before and after the
absorption
• In contrast, the fluorophore molecule is already surrounded by
relaxed (changed) environment during emission
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Dependence of emission
spectrum on solvent polarity
By increasing
polarity :
H – hexane
CH- cyklohexane
T- toluene
EA – ethylacetate
Bu – n-buthanol
polarity
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Practical demonstration
• Prodan (N,N-Dimethyl-6-propionyl-2-naphthylamine)
O
CH3
N
CH3
CH3
C - Cyklohexane
D - dimethylformamide
E - Ethanol CH3CH2OH
V - water H2O
Bu - n- Buthanol CH3CH2CH2CH2OH
G – Glycerol
OH
OH
OH
N
CH3
CH3
O
H
polarity
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Temperature effect on the
emission spectrum
• Reducing the temperature generally
causes an increase in viscosity of the
solvent thereby increas the time required
for orientation of molecules of the solvent
• The lower the temperature, the less the
molecules return to the ground state with
relaxed surrounding solvent molecules the
less energy is consumed and thus the
shift is smaller
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Change of emission spectrum
upon molecule binding
ANS to HSA
Prodan to protein
DAPI to DNA
EtBr to DNA
Sensitivities of fluorescent probes to environment are
used for monitoring of binding and quantifying of the
amount of biological molecules.
Quantum yield is often increased when the fluorophore
binds to the protein or DNA. This is used in monitoring
of binding.
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ANS fluorescence intensity
increase upon binding to human
serum albumin
ANS (1-anilinonaftalén-8-sulfonic acid):
• MW = 321,33
• solvent for the stock solution: dimethylformamide( DMF)
• solvent for spectroscopic measurement: methanol (MeOH)
• longwave absorption maximum in methanol : λexmax = 372 nm
(molar extinction coefficient: 7800 cm-1M-1)
• fluorescent emission maximum in methanol : λemmax = 480 nm
• quantum yield of fluorescence depends on the environment and
is particularly sensitive to the presence of water; emission
depends on the solvent
• detailed description of the characteristics of the ANS can be
found in [Slavík J.: Anilinonaphthalene sulfonate as a probe of
membrane composition and function. Biochim. Biophys. Acta 694,
1-25 (1982)]
The environment polarity change is shown by the emission intensity
change in the visible region of the spectrum
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What happens when PRODAN
binds to BSA ?
Maximum wavelength of 520 nm moves to 460.
Although, the emission intensity increases, we
are not able to observe it because the eye has
a lower sensitivity to light with λ=460 nm.
Gradual bond of PRODAN to BSA can be better
monitored as emission decrease of a free
fluorophore
460 nm 520 nm
PRODAN
PRODAN + BSA
Eye sensitivity
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DAPI binding to DNA
• DAPI (4,6-diamidino-2-phenylindole )
• Ex. 355 nm / Em. 461 nm
• Binding to the minor groove
• The biggest increase in intensity during binding near the AT-rich
regions
• Use for DNA labeling for fluorescence microscopy specimens
(sensitivity of the order of ng of DNA)
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EtBr binding to DNA
Probe characteristics
MW = 394,31
• soluble in water
• it emits fluoresces a little in water. Fluorescence increases about 30 times
upon binding to DNA
• time decay of fluorescence is about 1.7 ns in water and increases to 20 ns
after binding to double-stranded DNA
• binding to DNA is performed by intercalation of the aromatic ring between
base pairs of double-stranded DNA
• absorption maximum in DNA: λexmax = 523 nm
• fluorescent emission maximum in DNA: λemmax = 604 nm
• Sensitivity: in the order of ng of DNA can be detected on the gel
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Other mechanisms of spectral
shift
• Hydrogen bonds in a solvent
• Inner transfer of charge (within a molecule)
• Solvent molecules relaxation rate
• Interaction of probe – probe
• Conformational changes of the fluorescent probe
• Changes in rates of radiative and non-radiative
processes
Dashed arrows represent that the
transitions can be iradiative or non-
radiative
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The dependence of the emission
spectrum on temperature
Temperature reduction increases the time needed to relax solvents.
Temperature reduction has similar effects as reduction of solvent polarity
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Interaction of probe-probe
excimer fluorescence
Fluorophore molecules can create together excited
complex excimer.
Excimer is excited dimer shortly.
It is exiples in case of two different molecules.
Molecules must be in contact to create an excimer .
Excimer fluorescence emission band is shifted to
longer wavelengths compared to the fluorescence of
isolated molecules.
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Use of excimer upon detection of
DNA insertion mutations
• Oligonucleotide with attached
pyrene residues instead of single
base
• When it binds to WT unmutated
DNA: one pyrene residue
intercalates, the latter is outside the
helix
• When it binds to mutated DNA
which contains one extra base:
excimer will create.
• Excimer emission shows that it is
an insertion mutant
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Fluorescence quenching
• Fluorescence quenching can be defined as bimolecular
process which reduces the quantum yield of fluorescence (ie.
the intensity of fluorescence) without changing the
fluorescence emission spectrum. It can be the result of
different processes.
• collisional (dynamic) quenching occurs when the
fluorophore is deactivated in the excited state (i.e. it returns to
the ground state non-radiatively) during collision with
quenching molecule. The molecules in this process are not
chemically changed in contrast to
• static quenching, when non-fluorescent complex is created
after contact of the fluorophore and the quencher.
• selfquenching is the quenching of the fluorophore by itself;
occurs at high concentrations or high degree of labeling.
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Dynamic quenching
Reducing of the fluorescence intensity by dynamic quenching is
described in Stern-Volmer equation :
F0/F = τ0/τ = 1 + kq τ0 Cq
F0 – fluorescence quantum yield in the absence of a quencher,
F – the same in the presence of a quencher at Cq, τ0 – fluorescence
time decay without a quencher, τ – time decay in the presence of a
quencher, kq – bimolecular quenching constant (= bimolecular rate
constant determined by diffusion multiplied by the efficiency of
quenching). kq value gives concentration of a quencher at which
the fluorescence intensity is reduced by half.
Molecular oxygen (O2) is the most common quencher of
fluorescence and phosphorescence. Furthermore, fluorescence is
quenched (due to intersystem conversion) by halogen atoms such
as bromine and iodine. Acrylamide is also a frequently used
quencher.
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Static quenching
• A fluorophore and a quencher creates a complex, which does
not emit fluorescence
• The Stern-Volmer equation is also applied :
F0/F = 1 + Ka τ0 Cq
Ka is association constant of the fluorophore and the quencher
Typical static quenchers:
Nucleic acid bases
Nicotinamide
Heavy metals
Guanine
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Different dependence of dynamic
and static quenching
• Both types of
quenching show the
same dependence on
the concentration of
the quencher.
• A part of fluorophores
forming complexes is
only "invisible„ during
static quenching.
Fluorescence time
decay τ does not alter.
• During dynamic
quenching, time decay
altersτ0=τ
Static quenchingDynamic quenching
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Dependence of both types of
quenching on temperature
• Dynamic quenching increases
with increasing temperature. The
mobility of quencher molecules
increases, thereby they "quench"
more fluorophore molecules in
the same time .
• Static quenching decreases with
increasing temperature because
it is easier to dissociate weakly
bound complexes of a
flourophore and a quencher.
Static quenchingDynamic quenching
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Use of quenching during
fluorophore localization
In the membrane
• If the fluorophore P1 is embedded in
the membrane, it is unavailable for
the quencher Q and it almost does
not lead to quenching.
• Fluorescence intensity is almost
unchanged with increasing
concentration of the quencher.
On the surface
• If the fluorophore P2 is on the
surface, it leads to efficient
quenching.
• Fluorescence intensity declines
significantly with increasing
concentration of the quencher .
Embedded in
the membrane
Exposed on the
surface
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Literature
• Lakowicz J.R.: Principles of Fluorescence
Spectroscopy. Third Edition, Springer +
Business Media, New York, 2006.
• Fišar Z.: FLUORESCENČNÍ SPEKTROSKOPIE
V NEUROVĚDÁCH
http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
Graphics from the book Principles of Fluorescence for the purpose of this lecture
was kindly provided by Professor J.R.Lakowitz.
Acknowledgment
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Next
• What does time resolved fluorescence tell us
more than stable fluorescence?