Fluorescence depolarization emission anisotropy

The fluorescence depolarization technique excites a fluorescent dye by linearly polarized light and measures the polarization anisotropy of the fluorescence emission. The fluorescence anisotropy, r, is defined as... 

The plane-polarized light pulses characteristic of mode-locked lasers also provide an ideal excitation source for time-dependent fluorescence depolarization studies although conventional excitation sources can be used. If the rotational relaxation time of the excited molecule is comparable to its fluorescence decay time, then the vertical (I ) and horizontal (Ix) components of the fluorescence decay observed through suitable polarizers following excitation by polarized li t pulses, may be analysed to provide information concerning the size and motion of die molecule and Sect. 5. However, if only the true fluorescence decay characteristics are of interest it is necessary to compensate for these emission anisotropy effects Perhaps the simplest technique is to analyse only that component of fluorescence emitted at 54.7° to the direction of pdarization of the excitation source, the so-called magic-angle ... 

Thus, as mentioned earlier, time-resolved depolarization measurements afford a means of recording the time profile of the rotational autocorrelation function. The steady state technique, with continuous sample excitation, produces merely the time average of the emission anisotropy, F. For a rotating chromc hore with a sin e fluorescence decay time Tf, F is related to r(t) by the following expression... 

A popular probe molecule which has b n employed for such studies is 1,6 diphenyl 1,3,5 hexatriene (DPH). This molecule has both absorption and emission along the long molecular axis and is thought to dissolve in the hydrocarbon interior. Time-resolved fluorescence depolarization studies with DPH probe molecules have been performed on the following bflayer syrtems dKdihydrosteraculoyl)pho halidyl choline dipalmitoyl phosphatidyl choline L-a-dimyristollecithin residues egg lecithin residues and mouse leukaemic L 1210cells In all reports the time-dependence of the emission anisotropy was found to decay non xponentially indkat-ing either... 

The influence of cholesterol on the fluidity of lipid bilayers is well known and has been investigated using time-resolved fluorescence depolarization . The decays of the emission anisotropy recorded for the probe molecule DPH dispersed in liposome of di-(dihydrosterculoyl) phosphalidyl choline with various added amounts... 

An independent verification of this procedure was possible for lb [82]. The four n-propyl substituents ensure that this molecule can be oriented to a high degree in stretched polymer sheets. Linear dichroism (LD) measurements on the aligned samples resulted in the determination of transition moment directions. A pattern similar to that of parent 1 was obtained. Both LD and emission anisotropy procedures yielded similar values, which, additionally, confirmed that the assignment of fluorescence depolarization to excited state tautomerization was correct. 

The fluorophore was modeled by two beads that are attached as a short pendant side-chain (tag). Both the absorption and emission dipole moments of the fluorophore are defined by the direction of the tag (parallel), as indicated by the vector in Fig. 19, and the fluorescence anisotropy was calculated from its orientation autocorrelation function. For simplicity, we assumed that the reorientaional motion of the fluorophore is the only source of fluorescence depolarization. We neglected energy transfer and other processes that might occur in real systems. The fluorescence anisotropy decays were interpreted using the mean relaxation time, defined as ... 

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... 

Fluorescence of porphycene embedded in rigid media was found to be depolarized, both at room temperature in the poly(vinyl butyral) matrix [79] and at low temperatures in glasses [30, 80[ (Fig. 8.11). In a rigid environment, where the reorientation of an excited chromophore is not possible, the direction of the Sq-Sj transition moment should be the same in absorption and emission, leading, for excitation into Sj, to the anisotropy value of 0.4. Instead, the observed values... 

Fluorescence anisotropy measurements can also be used to obtain the rates of the excited state tautomerization. Two variants can be applied. The first is based on the analysis of time-resolved anisotropy curves. These are constructed from measurements of the fluorescence decay recorded with different positions of the polarizers in the excitation and emission channels. The anisotropy decay reflects the movement of the transition moment and thus, the hydrogen exchange. For molecules with a long-lived Sj state, the anisotropy decay can also be caused by rotational diffusion. In order to avoid depolarization effects due to molecular rotation, the experiments should be carried out in rigid media, such as polymers or glasses. When the Sj lifetime is short compared to that of rotational diffusion, tautomerization rates can be determined even in solution. This is the case for lb, for which time-resolved anisotropy measurements have been performed at 293 K, using a... 

The possible fluorescence anisotropies r thus range from - 0.2 (for perpendicular absorption and emission transition moments, c = 0) to a maximum of +0.4 (for parallel transition moments, c = 1). Complete depolarization (r = 0) occurs only when the absorption and emission moments are separated by the magic angle 54.7°, for which (3c - 1) = 0. This example illustrates the sensitivity of the anisotropy to chromophore architecture in the protein this emerges as a consequence of the well-defined chromoprotein structure. 

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... 

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