fluorescence depolarization martin cole, faraz khan physics 200 professor newman...
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Fluorescence Depolarization
Martin Cole, Faraz Khan
Physics 200
Professor Newman
http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html
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FluorescenceElectrons are excited to higher
energy states, jumping them to a higher energy orbital
Electrons relax to give off heat (non-radiative) and photons (radiative)
Electrons can also spin flip to form a triplet spin-parallel state
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The Jablonski Diagram
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RatesThe 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
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Size and TimeIf 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
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Fluorescent ProbesThree 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
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Steady State DepolarizationConsider 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
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Polarization and AnisotropyA (anisotropy) = (I║ - I┴ ) / (I║ + 2I┴ )
P (polarization) = (I║ - I┴ ) / (I║ + I┴ )
If there were no polarization, I║ =
I┴ and P and A become 0For a perfectly rigid molecule,
Pmax is ½ and Amax is 2/5
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Rigid MoleculeP0= (3cos2ζ –1) / (cos2ζ +3)A0= (3cos2ζ –1) / 5
Where ζ is the angle between absorption and emission dipoles
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Time-Resolved Fluorescence DepolarizationTwo 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
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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
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InstrumentationMethods 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)
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Anisotropy MeasurementsTwo 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
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Axis ModulationWe 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
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CalculationsFrom 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
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Static Polarization Constant
Illumination ◦ Use average
Anisotropy equations2
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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Hopkins et al Probe
http://www.biochemj.org/bj/440/bj4400043add.htm
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τcor and Rotational Diffusion3
http://www.youtube.com/watch?v=A_HyVm6UTM8
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.
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Perrin Equation for Anisotropy
Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence , 130 (11), 2166-2170.
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Anisotropy Decay
Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical
Chemistry , 110, 6001-6009.
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Ellipsoid CorrectionsRelation of
Anisotropy with time can be expanded to three exponentials if macromolecules are viewed as ellipsoids
http://science.yourdictionary.com/ellipsoid
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Anisotropy and Molecular Weight
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).
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Dependence on Lifetime
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.
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Interesting Experiments
8
Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004). Preparation and characterization of Alexa Fluor 594-
labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the
epidermal growth factor receptor. Analytical Biochemistry , 324 (2), 227-236.
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References 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.
2 Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics: Structure, Dynamics, Function. Cambridge: Cambridge University Press.
3 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.
4 Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence , 130 (11), 2166-2170.
5 Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical Chemistry , 110, 6001-6009
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).
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.
8 Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004). Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor. Analytical Biochemistry , 324 (2), 227-236.