Angle-resolved Fluorescence Depolarization Experiments Research Articles

Determination of the directions of the transition dipoles in tetrabutylperylene in stretched polymers

The directions of the transition dipole moments of 2,5,8,11,-tetra-butylperylene were determined from angle-resolved fluorescence depolarization experiments on molecules embedded in a stretched anhydrous nitrocellulose matrix. The absorption transition moments lies almost parallel to the elongated axis of the molecule, but the emission transition moment makes an angle of 20° with the axis. The orientational distribution of the molecules in the polymer indicates significant deviations from a circular form.

The orientation of transition moments of dye molecules used in fluorescence studies of muscle systems.

Fluorescence and phosphorescence depolarization techniques can provide information on orientational order and rotational motion of crossbridges in muscle fibres. However the depolarization experiment monitors the orientation and motion of the crossbridges indirectly. The changes in depolarization arise from a change in the orientation of the transition dipoles of the dye attached to the crossbridge. In order to extract the physiologically relevant orientations from the data it is therefore necessary to characterize the orientation of the dye molecule relative to the crossbridge and the orientation of the transition moments in the frame of the dyes. The dyes 1,5-I-AEDANS and eosin-5-maleimide are commonly used for labelling the crossbridge in muscle fibres. The orientations of the absorption and fluorescence emission dipoles of these two dyes in the molecular frame were determined. Angle resolved fluorescence depolarization experiments on the dyes, macroscopically aligned in a stretched polymer matrix of poly vinyl alcohol, were carried out. The data were analyzed in terms of an orientational distribution of the dye molecules in the film and the orientations of the absorption and emission dipoles in the frame of the dye molecule. Experimental data, obtained from a given sample at different excitation wavelengths, were analyzed simultaneously in a global target approach. This leads to a reduction in the number of independent parameters optimized by the non-linear least squares procedure.

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.

Determination of transition moment directions in molecules of low symmetry using polarized fluorescence. I. Theory

A theoretical description of angle-resolved fluorescence depolarization experiments of dye molecules in stretched polymer films is presented. It is shown how the technique can be used for the determination of transition moment directions in organic molecules of low symmetry. The application of these angle-resolved measurements to the determination of the direction of the transition moments in a number of dye molecules is discussed in the following paper. The theoretical treatment implicitly assumes that the motions of the fluorescent molecules are slow on the time scale of their excited state lifetime (in the order of ns) so that their orientation is identical at the times of absorption and emission. Furthermore the molecules are taken to be uniaxially distributed around the stretch direction of the polymer film and it is shown how this can be checked experimentally. The macroscopic orientational distribution is assumed to be invariant for a rotation of 180° of the molecules around a ‘‘mechanical’’ symmetry axis chosen in the molecular frame as well as to a reflection in the molecular plane (C2v D2h symmetry). The treatment presented makes use of the Wigner rotation matrix formalism in the description of the fluorescence intensities in terms of molecular orientational properties (order parameters and angles between transition moments and the mechanical symmetry axis). The advantge of this formalism is the ease with which rotational transformations are carried out between molecular and laboratory frames and further that the symmetry properties of both the molecules and the system are treated in a simple and general way.

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols.

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.

Headgroup hydration in egg-lecithin multibilayers affects the behaviour of DPH probes

Angle-resolved fluorescence depolarization experiments were carried out on DPH molecules embedded in egg-licithin bilayers of different water contents. It was found that the order parameter 〈 P 2〉 of the absorption moment is significantly higher than that of the emission moment and that the difference increases on hydrating the lecithin headgroups. The changes in the order parameter 〈 P 2〉 and 〈 P 4〉 of the DPH molecules induced by hydration are consistent with X-ray diffraction data. Furthermore the reorientational motions of the DPH molecules were found to become faster with increasing hydration.

The static and dynamic behaviour of fluorescent probe molecules in lipid bilayers

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in multibilayers of dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) above their respective phase transitions. The finding that the order parameter 〈 P 2〉 of the absorption moment is significantly higher than that for the emission moment for each probe is shown to arise from a tilt of the emission moment relative to the molecular symmetry axis. It is further shown that while the order parameter 〈 P 2〉 is the same for both probes in DMPC bilayers, it is higher for TMA-DPH than for DPH molecules in POPC bilayers. Considerations of the order parameters 〈 P 4〉, however, show that this difference can be ascribed solely to the higher fraction of DPH molecules lying with their axes parallel to the bilayer surface. Furthermore it is found that TMA-DPH molecules undergo slower reorientational motions than DPH molecules in the same bilayer system. Nevertheless the motion of both probe molecules is faster in DMPC than in POPC bilayers. The results indicate that TMA-DPH is a more useful probe than DPH in the systems investigated.

Angle resolved fluorescence depolarization experiments on smectic mesophases

Second and fourth rank order parameters (<P 2> and <P 4>) have been determined for some smectics from measurements of the angle-resolved fluorescence depolarization profile of a probe embedded in the systems. It is shown that the degree of order can be separated from the preferential molecular orientation when both <P 2> and <P 4> are known. The degree of order could be described by an extended mean field potential.

Determination of orientational order parameters in oriented lipid membrane systems by angle-resolved fluorescence depolarization experiments

Angle-resolved fluorescence depolarization experiments have been performed on some lipid membrane systems in order to test the validity of a recently developed theory describing this type of experiments. Good agreement between theory and experiment was found. The experiments yield the order parameters 〈 P 2〉 and 〈 P 4〉 and a rotational correlation time τ c. On applying an information-theoretic form of the orientational distribution function. 〈 P 2〉 and 〈 P 4〉 were used to estimate the degree of order in the systems studied. Knowledge of 〈 P 4〉 proved useful to get more detailed information on the orientational order. Another finding was that the angle between the absorption and emission moment of the used fluorescent probe, diphenyl-hexatriene, depends on the membrane system in which it is incorporated. Finally, the experimental results point to the fact that for some membrane systems the molecular motion is inadequately described by the simple strong-collision model.

Angle resolved fluorescence depolarization experiments on oriented lipid membrane systems

It is demonstrated that angle resolved steady state fluorescence depolarization experiments on oriented lipid membrane systems can be an useful alternative to time-resolved fluorescence depolarization experiments on vesicles. It is shown that some basic assumptions underlying time-resolved experiments are not always valid. The usefulness of the measurement of an additional order parameter, 〈 P 4 〉 is demonstrated.