6 1
Fluorescence methods in life sciences
Ctirad Hofr
Fluorescence
anisotropy
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The phenomenon of anisotropy
• When excitation is caused by light vibrating in
one plane, it is linearly polarized,
fluorescence emission becomes polarized.
• The level of emission polarization is
described by anisotropy (heterogeneity).
Substances that show certain degree of
heterogeneity emit polarized fluorescence.
• Why is emitted light polarized?
Anisotropy around us: monitor
• Anisotropy occurs in LCD monitor
• What does cause it?
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http://www.youtube.com/watch?v=imQnWrLW2i0
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Transition dipole moment
• Transition dipole moment is a quantum mechanical issue. It is not a real
dipole moment. It is given by immediate state of electron shell of the molecule.
The size of the dipole moment of transfer indicates the ability of the given
molecule state to absorb or emit the light. The direction of the dipole moment of
transfer indicates the direction in which the light is preferably absorbed or
emitted by the molecule.
• The molecules preferentially absorb the light whose electric component
oscillates in the same plane as absorption transition dipole of electron into a
higher energetic level.
• Molecules preferably emit light in the same plane as the emission transition
dipole of electron transition into a lower energetic level.
Absorption
Emission
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Photoselection
• If solution of fluorophores is
excited by linearly polarized
light then molecules with
nonzero projection of their
absorption transition moment
into the direction of
polarization of excitation
radiation will be excited
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Fluorescence anisotropy
polarizer
analyzer detector
⊥
⊥
+
−
=
2II
II
r
ΙΙ
ΙΙ
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Intensity of fluorescence
The theory of depolarization of fluorescence shows that the
fluorescence intensity observed using the analyzer, which is rotated by
an angle and from the direction of parallel polarization is
Iα(t) = cos2α III (t) + sin2α Iv(t)
polarizer
analyzer
α
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At what angle of polarizer rotation α
can we measure the total
fluorescence intensity of rotation?
Total intensity of fluorescence
⊥+= 2III ΙΙtotal
⊥⋅+⋅= III ΙΙ ααα
22
sincos
1cossin 22
=+ αα
3
1
cos =α
α = 54,7 °
2
1
sin
cos
2
2
=
α
α
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The total intensity is measured at
the magic angle
Total intensity I(t) = III (t) + 2 Iv(t) depends on
the rotational motion of the fluorophore and
is measured under "magic" angle of 54.7 °
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The time dependence of polarized
fluorescence
and for time dependence of anisotropy we apply
r(t) = (3 cos2 γ(t) – 1)/5
γ is – a dipole reorientation angle at time from 0 to t
– an angle between the dipole moment of absorption and emission
γ Absorption
Emission
Dipol moments
τ
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The maximal value of anisotropy
From the equation, it follows that in the absence of
depolarization (e.g. in dilute frozen solutions in the
absence of depolarizing mechanisms and
assuming that the transition moments of absorption
and emission are parallel) the limit value of
anisotropy of fluorescence excited by polarized
radiation is given only by photoselection
5
)1cos3( 2
0
−
=
γ
r
r0
max = 2/5 = 0.4
Anisotropy of frozen sample
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http://thelab.photophysics.com/circular-dichroism/fluorescence-polarization-darkness-visible
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Anisotropy measurements at steady-state
fluorescence
• Anisotropy – measures polarized emission
• The polarizer is on the track of the excitation
light and makes it plane polarized
• Polarizer - analyzer is in the path of emitted light
– fluorescence
• We measure the fluorescence intensity at
rotation of both the polarizer and the analyzer
vertically (VV), then at rotation of the polarizer
vertically and the analyzer horizontally (VH)
• The value of anisotropy <r> is calculated from
measured intensities
VHVV
VHVV
II
II
r
⋅+
−
=
2http://www.youtube.com/watch?v=imQnWrLW2i0
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L and T
measurement arrangements
6 15V
H V
x
z
y
IVV
IVH
Measurement of fluorescence
anisotropy
r=
IVV
IVH
-
+2
x
x
Provided by Horiba Jobin Yvon
http://www.youtube.com/watch?v=NTpWDp_3W_4 3rd minute
http://www.nordskip.com/vfog.html
Who did use the anisotropy of light for
the first time?
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The principle of monitoring of
macromolecule binding
Excitation
low
anisotropy
high
anisotropy
t ~ ns
Emission
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AF detection of protein-DNA
interaction
KD = 2 . 10-6 M
0.17
0.22
0.27
0.32
0 5 10 15
protein (µM)
FluorescenceAnisotropy
KD
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0
0.1
0.2
0.3
0.4
0.5
0 20 40 60 80 100 120
[Protein], µM
Anisotropy,r
Monomers:
Small
Rapid rotation
Unhindered
Low anisotropy
Depolarized emission
Dimers:
Larger
Slow rotation
Hindered by Viscosity
High anisotropy
Polarized emission
Poskytnuto Horiba Jobin Yvon
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Monitoring of DNA unwinding
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Repressor binding to DNA
• TRP repressor binds to DNA after
activation by tryptophan and
controls the synthesis of
tryptophan.
• Anisotropy increases upon
binding of TR to fluorescently
labeled DNA
• At increasing concentrations of
tryptophan, TRP repressor
binding to DNA increases and
prevents further synthesis of
tryptophan
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Time-resolved fluorescence
anisotropy
• Measurement of time-resolved fluorescence anisotropy
upon the pulse excitation gives much more information
about rotational motion of the fluorophore than steadystate
fluorescence.
• The steady-state fluorescence provides averaged values
of the measured parameters and for their interpretation it
is necessary to perform a number of measurements at
different temperatures. In contrast, the method of timeresolved
fluorescence anisotropy provides necessary
information from measurement at a constant
temperature
• When using the method of time-resolved fluorescence
anisotropy, time-dependent intensity components III (t)
and Iv(t) are measured. Total intensity I(t) = III (t) + 2 Iv(t)
does not depend on the rotational motion of the
fluorophore and is measured under a "magic" angle 54.7
°
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What kind of information can timeresolved
fluorescence anisotropy
provide?
• If the time decay of fluorescence τ is comparable with molecular
reorientation rate, then the fluorescence polarization is modulated by
molecular motion and analysis of time dependence of emission anisotropy
will provide information about the anisotropy of the system where the
fluorophore is located.
• Measurement of fluorescence polarization provides information about the
molecular orientation and mobility and processes that are modulated by
them, e.g.:
1. protein-DNA interaction
2. ligand-receptor interaction
3. biopolymers flexibility
4. fluidity of membranes
5. proteolysis
6. muscle contractions
7. activity of protein kinases
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Perrin equation for the relationship of
anisotropy and rotation of spherical molecules




+
=
><
φ
τ1
1
0r
r
r0 anisotropy at time 0
τ fluorescence decay time
φ rotational correlation time – describes the rotation of
molecules
the time in which the molecule rotates by 1 radian ~ 57.3°
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When can time-resolved anisotropy
give us information about the
movement of molecules?
τ = 10 ns
φ1 = 0.1 ns
φ2= 10 ns
φ3 = 1000 ns




+
=
><
φ
τ1
1
0r
r
?
0
=
><
r
r
When the motion of molecules is happening at a time comparable with
the time decay of fluorescence φ ~ τ
0.0009
0.5
0.99
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Use of Perrin equation for
determination of molecule properties
r0 anisotropy at time 0
τ fluorescence decay time
φ rotation correlation time
Dr rotation diffusion constant
V volume of a molecule
η viscosity of environment
k Boltzman constant (=1.38 .10-23JK-1)
T absolute temperature in Kelvin
η
τ
τ
φ
τ V
kT
D
r
r
r +=+=




+
=
><
161
1
1
0
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The time dependence is more
complex for molecules with complex
shapes
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Time-resolved anisotropy
• Dependence of anisotropy is calculated from the time dependence of the vertically
and horizontally polarized emission
• In the case that the absorption and emission transition moments are parallel, initial
anisotropy is r0=0.4
• Vertically polarized emission decreases faster because the number of molecules
emitting in the direction decreases by rotation and decay.
• Horizontally polarized emission decreases slower, because the population of
molecules is increased by molecules that change their position from vertical to
horizontal arrangement by rotation.
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Measurement of time-resolved
fluorescence anisotropy
• The sample is excited by
short pulses with durations
much shorter than the
decay time τ
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30
Change of rotational correlation
time during multimer formation
• The time dependent
fluorescence
anisotropy can be
used to monitor
changes in the
dynamics of
molecules
• Monitoring of
formation of
mutlimers by
phosphofructokinase
0 10 20 30 40
Čas (ns)
θ = 5 ns
θ = 20 ns
M
T
logr(t)
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DNA flexibility
• How does the flexibility of
DNA depend on
environmental conditions?
• What is the smallest pore
diameter which can the
DNA pass?
http://www.ks.uiuc.edu/Research/nanopore/
PICTURES/ds2.0-V3.2.mpg
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32
Monitoring of the movement of
DNA after intercalation of EtBr
• After increasing the ionic strength (dashed) the structure of DNA
is more rigid.
• The first part of the curve of the fluorescence decay describes
torsional movement of DNA
• The latter part of the curve describes the bent of DNA structures
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Comparison of steady-state and
time-resolved intensity and
fluorescence anisotropy
IntensityI
Wavelength λ
Anisotropyr
Time (min)
logI(t)
Steady-state Time-resolved
More information!
Time (ns)
logr(t)
Time (ns)
pulse
excitation
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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 of Principles of Fluorescence for the purpose of
this lecture was kindly provided by Professor JR Lakowitz.
Acknowledgment