Fluorescence depolarization orientational averaging

Similar to fluorescence depolarization and NMR, two limiting cases exist in which the molecular motion becomes too slow or too fast to further effect the ESR lineshape (Fig. 8) (35). At the fast motion limit, one can observe a narrow triplet centered around the average g value igxx + gyy + giz with a distance between lines of aiso = Axx- -Ayy- -A2,z)l3, where gu and Ajj are principal values of the g-tensor and the hyperflne splitting tensor A, respectively. At the slow motion limit, which is also referred to as the rigid limit, the spectrum (shown in Fig. 8) is a simple superposition of spectra for all possible spatial orientations of the nitroxide with no evidence of any motional effects. Between these limits, the analysis of the ESR lineshape and spectral simulations, which are based on the Stochastic Liouville Equation, provide ample information on lipid/protein dynamics and ordering in the membrane (36). 

Let us consider the hyperfine depolarization effect caused by the presence of nuclear spin that is not oriented or aligned by light but acts as a randomly oriented flywheel to reduce the average polarization of fluorescence. The emission process can be effected by the presence of nuclear spin J, even if the hyperfine structure is unresolved by the detection apparatus. For this purpose one has to multiply each polarization moment fq in Eq. (5.34) by a coefficient g(K which is equal to [177, 402]... 

In the case of Forster resonance energy transfer (FRET) [13], i.e. energy transfer by the dipole-dipole mechanism, and for randomly oriented donor-acceptor pairs, the depolarization after one transfer step (ensemble average) is almost complete [27]. For this reason, fluorescence anisotropy is a good indicator of energy transfer between identical fluorophores, hence of relative distances. Existence of efficient FRET may therefore reflect an association process. 

Comment on collisional depolarization studies. The resonance fluorescence experiments described above have served to stimulate and test the development of recent sophisticated theories of collision broadening. However, the hope that new information on interatomic forces would be obtained by these experiments has not been realized. This is largely because the observed broadening is the mean effect of collisions averaged over all possible relative orientations and over the thermal velocity distribution of the ensemble. It may be that in the future more detailed information on the interatomic potentials could be obtained by combining tunable dye laser excitation and atomic beam scattering techniques. 

---