Fluorescence Depolarization

Martin Cole, Faraz Khan

Physics 200

Professor Newman

http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html

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

The Jablonski Diagram

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

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

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

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

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

Rigid MoleculeP0= (3cos2ζ –1) / (cos2ζ +3)A0= (3cos2ζ –1) / 5

Where ζ is the angle between absorption and emission dipoles

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

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

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)

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

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

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

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.

1

Hopkins et al Probe

http://www.biochemj.org/bj/440/bj4400043add.htm

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.

Perrin Equation for Anisotropy

Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence , 130 (11), 2166-2170.

4

Anisotropy Decay

Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical

Chemistry , 110, 6001-6009.

5

Ellipsoid CorrectionsRelation 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

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).

6

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.

7

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.

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.