Fluorescence rotational depolarization

Under the (fluid) conditions necessary to promote photoassociation, the excimer fluorescence is rotationally depolarized however, measurements of pyrene crystal fluorescence have confirmed81 the predicted direction of polarization along the major axis. 

Most interesting applications of intramolecular energy transfer between nonconjugated chromophores are found in the conformational studies of biomolecules like nucleic acids and proteins. The experiments on rotational depolarization of emission from intrinsic fluorescent groups on externally attached fluorescent probes, have resulted in a vast store of knowledge which has helped to enrich the subject of photobiology. 

Molecules undergo significant rotation during the excited-state lifetime when p is much smaller than x. In this case, the fluorescence is depolarized. In the converse case, when p is larger than x, the emission is highly polarized. This means that one can think of fluorescence polarization as a measurement of the rotation speed (p) in units of the internal molecular clock (x). 

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. 

Steady-state fluorescence anisotropy In low-viscosity solvents the rotational depolarization of low molecular weight compounds occurs on the picosecond timescale [124]. Since in this case the rotation is much faster than the fluorescence, the steady-state emission is unpolarised. If the rotational motion of the fluorophore is on the same timescale as the fluorescence, a steady state polarisation is observed. In the simplest case for a spherical rotor and a single-exponential fluorescence intensity decay (r), the measured anisotropy is given by... 

Kawski A (1993) Fluorescence anisotropy—theory and applications of rotational depolarization. CritRev Anal Chem 23(6) 459-529. doi 10.1080/10408349308051654... 

Fluorescence polarization is frequently expressed in terms of anisotropy (r) due to the comparatively simple equations describing rotational depolarization. The dependence of fluorescence anisotropy on molecular rotation can be described quantitatively with the well-known Perrin equation. 

Photoexcited fluorescence from spread monolayers may be studied [158,159] if the substance has both a strong absorption band and a high emission yield as in the case for chlorophyll [159]. Gaines and co-workers [160] have reported on the emission from monolayers of Ru(bipyridine)3, one of the pyridine ligands having attached C g aliphatic chains. Ruorescence depolarization provides information about the restriction of rotational diffusion of molecules in a monolayer [161], Combining pressure-area... 

Tao T 1969 Time-dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules Biopolymers 8 609-32... 

Depolarization measurements, coupled with fluorescence lifetimes, are correlated with rates of molecular rotation to obtain estimates of molecular conformation, volume, and shape. 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

Time Resolved Fluorescence Depolarization. In Equation 3, it is assumed that the polarization decays to zero as a single exponential function, which is equivalent to assuming that the molecular shape is spherical with isotropic rotational motion. Multiexponential decays arise from anisotropic rotational motion, which might indicate a nonspherical molecule, a molecule rotating in a nonuniform environment, a fluorophore bound to tbe molecule in a manner that binders its motion, or a mixture of fluorophores with different rotational rates. 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

If excited molecules can rotate during the excited-state lifetime, the emitted fluorescence is partially (or totally) depolarized (Figure 5.9). The preferred orientation of emitting molecules resulting from photoselection at time zero is indeed gradually affected as a function of time by the rotational Brownian motions. From the extent of fluorescence depolarization, we can obtain information on the molecular motions, which depend on the size and the shape of molecules, and on the fluidity of their microenvironment. 

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

A. Kasprzak and G. Weber, Fluorescence depolarization and rotational modes of tyrosine in bovine pancreatic trypsin inhibitor, Biochemistry 21, 5924-5927 (1982). 

Analysis of rotational mobility of fluorophores by observation of fluorescence depolarization with nanosecond time resolution(28) or by variation of the lifetime (by the action of quenchers ).(9,29 30)... 

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