Fluorescence Depolarization
http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html
Martin Cole, Faraz Khan
Physics 200
Professor Newman
Fluorescence
Electrons are excited to higher energy
states, jumping them to a higher energy
orbital
 Electrons relax to give off heat (nonradiative) and photons (radiative)
 Electrons can also spin flip to form a
triplet spin-parallel state

The Jablonski Diagram
Rates
The rate of absorption is extremely fast,
on the order of 10-15 seconds
 Internal conversion from S2 to S1 takes
more time, on the order of 10-12 seconds,
but is still very fast
 The emission process can take as long as
10-8 seconds, still fast, but slower than the
other two processes by quite a lot

Size and Time
If a fluorescent group is oriented in a rigid
manner, it emits light with polarity
 As the group spins, the polarity is reduced and
becomes more random
 Large macromolecules spin slowly relative to
emission rates, and produce largely polar
photons
 Small molecules rotate in the time it takes to
emit, and produce a more randomized
spectrum of photons

Fluorescent Probes

Three categories:
◦ Intrinsic: naturally occurring, includes NADH,
FAD, tryptophan and tyrosine
◦ Intrinsic Analogs: residue replacement with a
fluorescent and synthetic molecule
◦ Extrinsic: Probes added that bind to the target
molecule to fluoresce, very common
Steady State Depolarization
Consider a plane of polarized light, moving in
direction x with electric vector in z direction
 We call I║ the intensity of light polarized in the z
direction and I┴ the intensity of light polarized
in the x direction
 We can determine anisotropy (lack of uniform
directionality) and polarization my measuring
the intensities

Polarization and Anisotropy

A (anisotropy) = (I║ - I┴ ) / (I║ + 2I┴ )

P (polarization) = (I║ - I┴ ) / (I║ + I┴ )
If there were no polarization, I║ = I┴ and P
and A become 0
 For a perfectly rigid molecule, Pmax is ½
and Amax is 2/5

Rigid Molecule
 P0= (3cos2ζ –1) / (cos2ζ
 A0= (3cos2ζ –1) / 5

+3)
Where ζ is the angle between absorption
and emission dipoles
Time-Resolved Fluorescence
Depolarization

Two main types:
◦ Decay of emission: measures fluorescence
after excitation pulse to determine
fluorescent lifetime of fluorophore
◦ Anisotropic decay: measures reorientation of
emission dipole to give information of
translational and rotational movement of
molecule
Perrin Equation
A0= AF/ (1+τF/τc)
◦ τF is lifetime of fluorophore
◦ τc is the rotational correlation time

If we find that τc is much bigger than τF,
we find that A0= AF
Instrumentation

Methods of obtaining time-resolved
fluorescent data
◦ Harmonic response - measures emission from
a sinusoidally modulated excitation
◦ Impulse-response – directly observes
emission decay following a short excitation
impulse
 Uses titanium-sapphire lasers to produce extremely
brief pulses (subpicosecond)
Anisotropy Measurements

Two main instrument formats:
◦ T - faster method that measures both parallel
and orthogonal to incoming polarized beam
◦ L - single emission channel is used, emission is
detected at a right angle to the excitation
beam from scattering

Introduces the correlation factor G to the
perpendicular component of the A and P
equations described before
Axis Modulation
We can flip the polarization of our
excitation beam between horizontal and
vertical
 For vertical excitation, we sum emitted
intensities IVH and IVV to get that
AV = IVH + IVV
 For horizontal excitation, we find that
AH = 2IVH

Calculations

From Av and AH, we can calculate the
anisotropy
A=(Av-AH) / (Av+ ½(AH))

This method of anisotropic determination
does not require the G factor correction
Static Polarization

Constant Illumination
◦ Use average
Anisotropy equations2
1
Hopkins, S., Sabido-David, C., Corrie, J., Irving, M., & Goldman, Y. (1998). Fluorescence Polarization
Transiets from Rhodamine Isomers on Myosin Regulatory Light Chain in Skeletal Muscle Fibers.
Biophysical Journal , 74, 3093-3110.
Hopkins et al Probe
http://www.biochemj.org/bj/440/bj4400043add.htm
τcor and Rotational Diffusion3
http://www.glycoforum.gr.jp/science/word/glycotechnology/GT-C06E.html
Neyroz, P., Menna, C., Polverini, E., & Masotti, L. (1996).
Intrinsic Fluorescence Properties and Structural Analysis of
p13suc1 from Schizosaccharomyces pombe. Journal of
Biological Chemistry , 271, 27249-27258.
http://www.youtube.com/watch?v=A_HyVm6UTM8
Perrin Equation for Anisotropy
4
Albani, J. (2010). Fluorescence properties of porcine odorant
binding protein Trp 16 residue. Journal of Luminescence , 130
(11), 2166-2170.
Anisotropy Decay
5
Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast
Förster Transfer in Highly Doped Matrices. Journal of Physical
Chemistry , 110, 6001-6009.
Ellipsoid Corrections

Relation of
Anisotropy with time
can be expanded to
three exponentials if
macromolecules are
viewed as ellipsoids
http://science.yourdictionary.com/ellipsoid
Anisotropy and Molecular Weight
6
Kay, L., Torchia, D., & Bax, A. (1989). Backbone dynamics of proteins as studied by 15N
inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease.
Biochemistry , 28 (8972).
Dependence on Lifetime
7
Pope, A., Haupts, U., & Moore, K. (1999). Homogeneous fluorescence readouts for
miniaturized high-throughput screening: theory and practice. Drug Discovery Today
, 4 (8), 350-362.
Interesting Experiments
8
Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004).
Preparation and characterization of Alexa Fluor 594labeled epidermal growth factor for fluorescence
resonance energy transfer studies: application to the
epidermal growth factor receptor. Analytical
Biochemistry , 324 (2), 227-236.
References
Hopkins, S., Sabido-David, C., Corrie, J., Irving, M., & Goldman, Y. (1998). Fluorescence Polarization Transiets
from Rhodamine Isomers on Myosin Regulatory Light Chain in Skeletal Muscle Fibers. Biophysical Journal , 74,
3093-3110.
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1
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2
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3
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4 Albani, J. (2010). Fluorescence

5
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6
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7
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8 Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004). Preparation and characterization of Alexa Fluor 594labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal
growth factor receptor. Analytical Biochemistry , 324 (2), 227-236.
Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics: Structure, Dynamics, Function.
Cambridge: Cambridge University Press.
Neyroz, P., Menna, C., Polverini, E., & Masotti, L. (1996). Intrinsic Fluorescence Properties and Structural Analysis
of p13suc1 from Schizosaccharomyces pombe. Journal of Biological Chemistry , 271, 27249-27258.
properties of porcine odorant binding protein Trp 16 residue. Journal of
Luminescence , 130 (11), 2166-2170.
Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped
Matrices. Journal of Physical Chemistry , 110, 6001-6009
Kay, L., Torchia, D., & Bax, A. (1989). Backbone dynamics of proteins as studied by 15N inverse detected
heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry , 28 (8972).
Pope, A., Haupts, U., & Moore, K. (1999). Homogeneous fluorescence readouts for miniaturized highthroughput screening: theory and practice. Drug Discovery Today , 4 (8), 350-362.