Depolarization due to Rotation

The interpretation of the anisotropy decay can be, in the case of multi-coponent systems, very complicated and require the use of specific software especially when deconvolution is needed. Nevertheless in the case of depolarization due to rotation... 

Rigid structure to avoid depolarization due to rotations of side groups. High and constant ro values when excited in the last electronic absorption band. These properties provide a depolarization that is unaffected by small shifts in the absorption spectrum . 

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... 

Nonadiabatic coupling mixes all terms. However, if Ae is sufficiently high, the location of - and 11-11 nonadiabatic coupling due to radial motion will be different from that of a -11 coupling due to rotational motion. This argument, and also the fact that the probability of nonresonant transition is small, permit depolarization and transition with energy transfer to be treated separately. Let us discuss now the latter. According to the theory of nearly adiabatic perturbations (Section II),... 

In an attempt to measure the rotational correlation time of the protein, we have measured the anisotropy of the Trp residues of cytochrome b2 core at different temperatures and thus for different viscosities. The results are described with the classical Perrin plot (1 / A as a function of T / r ) (Fig. 5.19). The data yield a rotational correlation time equal to 38 ps instead of 5.9 ns calculated theoretically for the cytochrome b2 core, with an extrapolated value A(o) equal to 0.208, lower than that (0.265) usually found for Trp residues at = 300 nm at - 45°C. The fact that the extrapolated anisotropy is lower than the limiting one means that the system is depolarized due to the global and local motions within the protein. In this case, the value of the apparent rotational correlation time (a) calculated from the Perrin plot will be lower than the global rotational time of the protein (Op). However, the fact that Oa is 1000 times lower than Op indicates that a third process different than the global... 

At the beginning of this chapter, we derived the value of n expected due to pfaotoselection (m=0.4), showed how n> was decreased by a factor dqtendent on the angle 0 between the absorption and emission transition mranents (Eq. [10.20]), and demonstrated that the anisotropy was decreased further due to rotational diflusion (Eq. [10.50]). There is a simple relationslup between these different causes of depolarization, whidi is known as Soleillet s rule. The anisotropy is given by the product of the various depolarization factors which occur in a given sample. 

If the rotations are isotropic the depolarization is a function of the relaxation time of the rotation of the direction containing the emission oscillator OE. This relaxation time may be expressed as a function of the principal relaxation times pa and pb. As the rotations can be isotropic only if OE makes a random angle with the axis of revolution, it is necessary to calculate the depolarization due to a collection of molecules in which the angle between that axis and OE assumes all values between 0 and ir/2. This can be done by means of Eq. (26). The following result is thus obtained (Weber, 1952a),... 

Figure 6.17 shows the dependence of the G factor on the wavelength. The curve was obtained using a solution of fluorescein in propylene glycol. This solvent is often used to study fluorescence anisotropy because of its high viscosity which slows down depolarization processes due to rotation allowing to investigate them... 

Fig. 0.2. (a) The comb spectrum of N2 considered as a quantum rotator. The envelope of the rotational structure of the Q-branch slightly split by the rotovibra-tional interaction is shaded, (b) The depolarized rotovibrational spectrum of N2 at corpuscular density n = 92 amagat, T = 296 K and pressure p = 100 atm. The central peak, reported in a reduced (x30) scale is due to a polarized component [5] (V) experimental (—) best fit. 

Light of definite energy and polarization has a selective power to exclusively excite dye molecules whose electronic transition energy and orientation match these parameters. Thus, if a dye is excited by polarized fight, its emission will also be highly polarized. Depolarization occurs only when the time correlation of these selectively excited species is lost due to their rotation or participation in some... 

Values of +0.4 and +0.5 are theoretically expected for r0 and pm respectively, if the absorption and emission transition moments are in the same direction, as is often the case with excitation at the longest-wavelength absorption maximum. However, due to rapid internal rotation of the emission transition moments immediately after excitation, the experimentally determined values of rQ and p0 are always lower than the maximal values. Thus, the highest value ever observed for rQ is+ 0.35. In the common case where the fluorophore undergoes rotational motion during the excited-state lifetime, values of r or p closer to zero are observed depending on the extent of depolarization, and in the case of complete depolarization these parameters assume values of zero. The dependence of the anisotropy on rotational motion is described by eq 9[55]... 

The values of /labs were determined from the experimentally measured absorption anisotropy with Eq. 6. In viscous polyTHF, the rotational movement of dye molecules on a lOOps time scale is assumed to be negligible, and thus, does not reduce the limiting value of anisotropy. For the concentrated fluorene solutions (5 x 10 M, 1 mm cuvette), the anisotropy Tabs was not affected by FOrster depolarization mechanisms [13] due to the short time delay, tD, between probe and pump pulses when td/ti 1 [35]. 

Rotating wire electrode — When a metal wire is rotating in a solution and used as an - electrode for - current measurements, the current will be enhanced due to the enhanced - mass transport of the - depolarizer to the electrode surface. However, since the hydrodynamic conditions are usually rather difficult to control at a rotating wire, especially when the wire is not a highly symmetric cylinder rotating around its own axis, the rotating wire electrode found only some application as an - indicator electrode in - amperometric titrations. 

Spectra of samples in the liquid state (Fig. 2.6-lB) are given by molecules which may have any orientation with respect to the beam of the spectrometer. Like in gases, flexible molecules in a liquid may assume any of the possible conformations. Some bands are broad, since they are the sum of spectra due to different complexes of interacting molecules. In the low frequency region spectra often show wings due to hindered translational and rotational motions of randomly oriented molecules in associates. These are analogous to the lattice vibrations in molecular crystals, which, however, give rise to sharp and well-defined bands. The depolarization ratio p of a Raman spectrum of molecules in the liquid state (Eqs. 2.4-11... 13) characterizes the symmetry of the vibrations, i.e., it allows to differ between totally symmetric and all other vibrations (see Sec. 2.7.3.4). 

Figure 8 Schematic layout of a typical 90° Raman depolarization experiment showing the positions of the polarization analyzer and the scrambler. The analyzer may simply be a polaroid sheet, which can be rotated by 90° to allow the parallel ( ) and perpendicular ( ) components of the scattered light to pass through to the detector. The function of a scrambler is to change linear into circular polarization of the light entering the Raman spectrometer slit in order to avoid measurement errors due to the variable spectrometer transmittance of the light polarized in different directions...

The measured rotational reorientation times, initial value of the fluorescence anisotropy, and fluorescence lifetimes in several solvents are listed in Table 1. The initial value of anisotropy is very close to 0.4, which suggests that the directions of the transition dipole of the absorption and the fluorescence are the same and the depolarization not due to the reorientation of the molecule can be ignored. 

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