Fluorescence Depolarization

1. Fluorescence Depolarization Martin Cole, Faraz Khan Physics 200 Professor Newman http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html

2. Fluorescence • Electrons 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

3. The Jablonski Diagram

4. Rates • The rate of absorption is extremely fast, on the order of 10-15 seconds • Internal conversion from S2to 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

5. 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

6. 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

7. 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

8. 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

9. Rigid Molecule • P0= (3cos2ζ –1) / (cos2ζ +3) • A0= (3cos2ζ –1) / 5 • Where ζ is the angle between absorption and emission dipoles

10. 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

11. 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

12. 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)

13. 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

14. 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

15. 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

16. 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.

17. Hopkins et al Probe http://www.biochemj.org/bj/440/bj4400043add.htm

18. τcorand 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

19. 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.

20. 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.

21. Ellipsoid Corrections • Relation of Anisotropy with time can be expanded to three exponentials if macromolecules are viewed as ellipsoids http://science.yourdictionary.com/ellipsoid

22. 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).

23. 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.

24. 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.

25. 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. • 2Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics: Structure, Dynamics, Function. Cambridge: Cambridge University Press. • 3Neyroz, P., Menna, C., Polverini, E., & Masotti, L. (1996). Intrinsic Fluorescence Properties and Structural Analysis of p13suc1 from Schizosaccharomycespombe. Journal of Biological Chemistry, 271, 27249-27258. • 4Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence, 130 (11), 2166-2170. • 5Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical Chemistry, 110, 6001-6009 • 6Kay, 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.