Depolarization of fluorescence

Rahman T S, Knox R S and Kenkre V M 1979 Theory of depolarization of fluorescence in molecular pairs Chem. Phys. 44 197-211... 

Thus, when a population of fluorophores is illuminated by a linearly polarized incident light, those whose transition moments are oriented in a direction close to that of the electric vector of the incident beam are preferentially excited. This is called photoselection. Because the distribution of excited fluorophores is anisotropic, the emitted fluorescence is also anisotropic. Any change in direction of the transition moment during the lifetime of the excited state will cause this anisotropy to decrease, i.e. will induce a partial (or total) depolarization of fluorescence. 

Fig. 5.9. Rotational motions inducing depolarization of fluorescence. The absorption and emission transition moments are assumed to be parallel.

Another experimental method that has been used to determine orientational correlation functions in macromolecular systems is based on measurements of the time-dependence of the depolarization of fluorescence 26 From these measurements rotational diffusion coefficients and the shape of the rotating macromolecule have been determined.27... 

All of these methods yield information about the time evolution of the specific correlation functions. What is usually measured, except in the case of the depolarization of fluorescence, is the power or frequency spectrum of the respective correlation functions over a wide range of frequencies. 

Raman scattering depolarization of fluorescence Spin-rotation relaxation time... 

Figure B3.6.12 Depolarization of fluorescence indicates rotation of the chromophore. Monochromatic radiation from the source (S) has all but the vertically polarized electric vector removed by the polarizer (P). This is absorbed only by those molecules (see Fig. B3.6.5) in which the transition dipole of the chromophore is aligned vertically. In the case where these molecules do not rotate appreciably before they fluoresce ( no rotation"), the same molecules will fluoresce (indicated by shading) and their emitted radiation will be polarized parallel to the incident radiation. The intensity of radiation falling on the detector (D) will be zero when the analyzer (A) is oriented perpendicular to the polarizer. In the case where the molecules rotate significantly before fluorescence takes place, some of the excited chromophores will emit radiation with a horizontal polarization ( rotation ) and some with a vertical polarization. Finite intensities will be measured with both parallel and perpendicular orientations of the analyzer. The fluorescence from the remainder of the excited molecules will not be detected. The heavy arrows on the left of the diagram illustrate the case where there is rotation.

In the present context, fluorescence is an important tool for the rapid comparison of a protein with a standard sample of the recombinant or the authentic protein, as part of the process of characterizing and authenticating a recombinant protein. Coupled with measurements of the depolarization of fluorescence, the method can provide information on the dynamics of a protein and on the change in dynamics in response to, for example, binding of ligands. 

Thomas, J. C., Allison, S. A., Appellof, C. J., and Schurr, J. M. (1980). Torsion dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements of a clean viral phi 29 DNA. Biophys. Chem. 12, 177-188. 

Weber, G (1989) Perrin revised parametric theory of the motional depolarization of fluorescence, J. Phys. 

Energy migration may reveal itself as a fast component in the decay of fluorescence of M, being often in the pfcosecond region, but possibly in some polymer systems on the nanosecond time scale. The phenomenon can certainly contribute greatly to the observed depolarization of fluorescence ... 

Fredrickson3 has formulated expressions for the concentration depolarization of fluorescence in the presence of molecular rotation. A theoretical examination of diffusion influenced fluorescence quenching by nearest possible quenching neighbours in liquids has been made35. A modified version of Smoluchowski - Collins - Kimball formulation of the Stern - Volmer equations has been matched with experimental data for quenching of anthraquinone derivatives by N,N-dimethyl- -toluidine. Another paper discusses this work on the basis of the kinetics of partly diffusion controlled reactions3 . 

The depolarization of fluorescence has been observed at temperatures around 100 K not only for 1, but also for the two derivatives, lb and Ic. in contrast, the measurements performed under the same conditions for le revealed no sign of depolarization. The textbook values of the anisotropy were obtained, i.e. 0.4 and about -0.2 for excitation into Sj and S2, respectively (Fig. 8.11). The octaethyl derivative le is the porphycene with the largest separation (2.80 A) between the hydrogen-bonded nitrogen atoms and should therefore exhibit the slowest tautomerization kinetics. It was thus concluded that the reduced anisotropy values observed in three different porphycenes are caused by excited state tautomerization [30, 80]. As shown in Fig. 8.12, the interconversion between the two trans tautomers changes the direction of the transition moment. Therefore, only a part of the excited state population emits fluorescence polarized parallel to that of the transition moment in Sg-Sj absorption (this fraction should approach 0.5 if the tautomerization is fast compared to the excited state lifetime). For the remaining frac-... 

Researches on theoretical topics have not been reported very extensively. A few papers are mentioned here and some others at appropriate points later in the article. Weber has re-examined the famous Perrin equation for quantifying the rotational depolarization of fluorescence. The arguments presented in the paper are applied to the temperature dependence of the local motions of tyrosine and tryptophan residues observed in proteins. 

Depolarization of fluorescence experiments can be performed with two independent configurations of polarizers and analyzers. For example, if n4 and n/ are parallel we find... 

Raman scattering depolarization of fluorescence u, unit vector along molecular transition dipole ... 

Both absorption and emissirm processes may be intramolecular, localized in a single molecule. On the other hand, they can also involve whole crystals that may act as absorbers and emitters. Such energy transfers can manifest themselves in different ways that include sensitized fluorescence or phosphorescence, concentration depolarization of fluorescence, photo-conduction, and formation of triplet acceptor molecules. 

Perrin, F., Brownian movement of an ellipsoid. 11. Free rotation and depolarization of fluorescence. Translation and diffusion of ellipsoidal molecules, /. Phys. Radium, 7,1,1936. 

The consistency of these various methods may be evaluated from the results listed in Table I of data obtained with polystyrene in solvents of low viscosity by the sound absorption (SA) and NMR relaxation methods, for poly(p-chloro-styrene) by dielectric dispersion (DD), for polystyrene labelled with nitroxyl by the ESR method and for a styrene copolymer with a small concentration of 9-p-vinylphenyl-lO-phenylanthracene residues by depolarization of fluorescence (DF). It may be seen that sound absorption and dielectric dispersion yield similar transition frequencies in the range of 12-35 MHz at 10-25 C,... 

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