Fluor. Depol.
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Transcript Fluor. Depol.
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.
1
2
3
4 Albani, J. (2010). Fluorescence
5
6
7
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.