Anisotropy experiments

The obtained average value Yp = (25.74 1.0)% (by mass) seems to be higher than the optical data, allowing the presence of 1-2 unknown light particles, and to be in agreement with the results from CMB anisotropy experiments [1],... 

Time-resolved emission anisotropy experiments provide information not only on the fluidity via the correlation time rc, but also on the order of the medium via the ratio rco/ro. The theoretical aspects are presented in Section 5.5.2, with special attention to the wobble-in-cone model (Kinosita et al., 1977 Lipari and Szabo, 1980). Phospholipid vesicles and natural membranes have been extensively studied by time-resolved fluorescence anisotropy. An illustration is given in Box 8.3. 

Many spectroscopic methods have been employed for the investigation of such systems For example, wide-band, time-resolved, pulsed photoacoustic spectroscopy was employed to study the electron transfer reaction between a triplet magnesium porphyrin and various quinones in polar and nonpolar solvents. Likewise, ultrafast time-resolved anisotropy experiments with [5-(l,4-benzoquinonyl)-10,15,20-triphenylpor-phyrinato]magnesium 16 showed that the photoinduced electron transfer process involving the locally-excited MgP Q state is solvent-independent, while the thermal charge recombination reaction is solvent-dependent . Recently, several examples of quinone-phtha-locyanine systems have also been reported . 

Most recently, Robinson and co-workers (72) used time-resolved decay of anisotropy experiments to probe the AOT reverse micelle system in ethane and propane. These authors conclude there was no local solvent density augmentation about the reverse micelle. In addition, the rotational dynamics of their probe (perylene tetracarboxylate) was independent of fluid density. This observation was consistent with results from Johnston and co-workers and Smith and co-workers (61-65,72). 

Molecules that possess magnetic anisotropy experience a slight tendency toward alignment with an imposed magnetic field, but in normal isotropic solvents random thermal motions dominate, and no effects of orientation are usually... 

The structural dimension at a water/DCE interface is d — 2.48, while short-range structural information about the interface obtained by the fluorescence dynamic anisotropy experiments suggests that the interface is three-dimensional-like. Taking the results obtained by molecular dynamics simulations into account, these results can be understood only by the fact that the water/DCE interface is thin ( 1 nm), but is rough with respect to the spatial resolution of the excitation energy transfer quenching method ( 7 nm), as shown in Figure 12.7. 

There are several analytical procedures available for derivation of relaxation information from time-resolved anisotropy experiments, the merits of which have been discussed at length elsewhere [25,112,114]. The salient points are covered here direct analysis of r(t) using a function such as Equation 2.31 is the most straightforward method but can become particularly problematic if the motion under study is comparable to the width of the excitation pulse [25,112,114]. Furthermore, as r(t) can suffer contamination from the polarizing effects of stray excitation from the source, particularly in weakly fluorescent samples, other methods are required to overcome such artifacts. Impulse reconvolution [115] allows mathematical removal of the instrumental pulse from the experimental data and involves an analysis of s(t) by a statistically adequate model function (e.g., Eq. 2.8). The best fit to s(t) is... 

Early time-resolved anisotropy experiments on PMAA used a combination of anthryl-based labels and probes [18,46,60,76] in an effort to fully characterize the conformational switch of the polyelectrolyte in aqueous solution. In their study of probes dispersed in and labels incorporated into PMAA, Treloar and coworkers [60,76] derived information from anisotropy experiments pertinent not only to the cluster size of the rotating units, but also to the structure of the hypercoil itself. 

Time-resolved anisotropy experiments reveal the dynamics of tumbling, and fast components may be assigned to local motions of the probe within the double helix (see below). The intensities of parallel and perpendicular polarized light are monitored as a function of time, and the time-resolved anisotropy is calculated as in Equation 2. Lakowicz [188] has described in great detail the data analysis for these kinds of experiments. Many data analyses rely on fitting the time-resolved data to sums of exponentials and considering limited physical states of the probe molecule (e.g., free vs. bound, solvent-exposed vs. buried) as appropriate. 

In the fluorescence intensity quenching (thermal and with iodide), it is the fluorescein environment consisting of amino acids (thermal quenching) and of amino acids and solvent dipoles that is relaxing around the excited fluorescein. In the fluorescence anisotropy experiments, on the other hand, the displacement of the emission dipole moment of the fluorescein is monitored. In the first approach, it is the environment that is either fluid or rigid. In the second approach, the restricted reorientational motion of the fluorophore is followed. 

The mean fluorescence lifetime is used to calculate the rotational correlation time from the Perrin plot (quenching resolved emission anisotropy experiment). 

Red-edge excitation spectra and anisotropy experiments indicate clearly that the Trp residues and the calcofluor displays free local motions, while TNS is tightly bound to the protein. Also, experiments with calcofluor showed that the sialic acid residue displays free motion while the carbohydrate residues that are close to the glycosylation site are tightly rigid. 

Quenching resolved emission anisotropy experiments could be performed at emission wavelengths in the blue (< 330 nm) and red (> 330 nm) portions of the spectrum to yield a more consistent data surface. However, this could be possible if at each edge of the fluorescence spectrum the emission occurs mainly from the buried or the surface Trp residues. Unfortunately, this is not the case since for example at 315 nm, the fractional contribution to the total fluorescence of the surface Trp residue is 42%. 

Some extremely unfortunate situations can also occur in systems with several parallel detection paths. Often light reflected back from one detection path can enter the other one. A typical example is the T geometry" often used for anisotropy experiments, see Fig. 7.26. 

Coronene has also been conjugated to lipids." Both pyrene and coronene display low initial anisotropies and are only moderately useful for anisotropy experiments. However, as described in the following sections, diae are two types of organmnetallic fluorophores which display long lifetimes and other unique features which allow new types of experiments. 

Figure 2. Experimental geometry of a 90 fluorescence anisotropy experiment. Excitation is along the X-axis, fluorescence is detected along the Y-axis.

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