Angle-resolved Fluorescence Depolarization Research Articles

The orientation of the phosphorescence dipole moment of erythrosine B within its molecular frame

For an unequivocal interpretation of time-resolved phosphorescence depolarization measurements on dye molecules in biophysical systems, the orientation of the transition dipole moments within the frame of the dye has to be known. The goal of this work is to find the orientation of the phosphorescence dipole moment of erythrosine B within its molecular frame. To this end, we first determine four difference angles, exciting a molecule in two of its absorption bands and measuring the fluorescence and phosphorescence anisotropy for each excitation wavelength. The next step is to position the transition dipole moments within the molecular frame, combining the results of angle-resolved fluorescence depolarization measurements with the measured difference angles. It is shown that the phosphorescence dipole moment is tilted out of the plane of the molecule. Additionally, we find that the phosphorescence dipole moment is located above the fluorescence dipole moment and not above the absorption dipole moment. This induces an extra difference angle between the absorption and the phosphorescence dipole moments which in the past has been interpreted erroneously as a larger tilt out of the plane of the molecule.

Spectral characterization of fluorescent 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative

This study describes a spectroscopic characterization of the fluorescent probe 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative in various solvents. The monomeric character and the purity of the two compounds was etablished in ethanol, ethanol–water, water, dimethylformamide, dimethyl sulfoxide, glycerol, polyvinylalcohol and nitrocellulose films, using a combination of absorption and fluorescence spectroscopy and mass spectrometry. In all the polar solvents studied here, the probes were present in the open form only and thus, from the spectral parameters, the natural lifetime and Förster distance in those solvents could be calculated. Fluorescence lifetimes were determined and the homogeneity of probe distribution in both stretched and unstretched polymer films was checked. For this, a combination of confocal microscopy and polarized fluorescence experiments was used. Finally, applying angle-resolved fluorescence depolarization for immobilized molecules in uniaxially stretched polymer films, the orientation of both rhodamines in stretched polyvinylalcohol films was examined and the orientation of the transition dipole moments within the molecular frame was determined. There were no differences between the 5-isomer and its N-acetylcysteine derivative, but a strong environmental dependence of most optical properties was observed. This dependence was especially intriguing for the difference angle between the absorption and emission dipole moment. The implications of our results and suggestions for future research are discussed.

Physical effects of biologically formed cholesterol oxidation products on lipid membranes investigated with fluorescence depolarization spectroscopy and electron spin resonance.

Planar oriented membranes of 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) containing cholesterol, 19-hydroxycholesterol, 22S-hydroxycholesterol, or 25-hydroxycholesterol in concentrations up to 5 mol % were investigated with angle-resolved fluorescence depolarization and electron spin resonance measurements. Analyses of the data with the Brownian diffusion model show that the oxysterols have structural effects similar to those of cholesterol: an increase in molecular order and no change in the rotational diffusion coefficients of the probe molecules. Time-resolved fluorescence anisotropy measurements on diphenylhexatriene (DPH) in small unilamellar vesicles of POPC and DOPC were performed using oxysterols commonly found in oxidized low density lipoproteins (LDL) in comparison to membranes containing cholesterol or no sterols. Analyses using the Brownian rotational diffusion model show that most LDL-oxysterols affect the vesicle physical structure in a manner similar to cholesterol, viz. an increase in molecular order and a decrease in the dynamics. Cholesterol-alpha-epoxide has a much smaller ordering effect than cholesterol in POPC-vesicles. A similar effect was found for 7 beta-hydroxycholesterol in DOPC-vesicles. The tendency of the oxysterols to influence the molecular order as compared to pure cholesterol may contribute to cell membrane permeability changes affecting crucial cell functions and events leading to vascular cell injury. Increased LDL oxysterol levels may account for some of the structural changes noted for oxidatively modified LDL as well as its toxicity to vascular cells.

Application of angle-resolved fluorescence depolarization in muscle research

Angle-resolved fluorescence depolarization (AFD) experiments have been used for over a decade in studies of fluorescent molecules in macroscopically aligned systems such as lipid bilayers and stretched polymer films. The importance of this technique lies in the fact that it affords the determination of both the second- and the fourth-rank order parameters of the orientational distribution of the probe molecules in the sample. Here we apply the technique to the study of the orientational distribution of crossbridges in muscle fibers. This orientational distribution is particularly relevant in muscle research, as crossbridge rotation is commonly regarded to be the driving mechanism in force development. An unfortunate consequence of the fact that the crossbridges have an average orientation of approximately 45(o) relative to the fiber axis is that the values of the second-rank order parameter [Symbol: see text]P 2[Symbol: see text] of the crossbridge distribution are close to 0. Therefore, knowledge of [Symbol: see text]P 4[Symbol: see text] is essential for a reliable reconstruction of the form of the distribution function. AFD of dyelabeled muscle was measured under rigor and relaxation conditions. The results indicate that no significant changes in depolarization take place upon a transition from the rigor to the relaxed state in the muscle and seem not to support the rotating crossbridge model, which postulates a clear change of orientation of the crossbridges.

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.

Theory of two-photon induced fluorescence anisotropy decay in membranes

We report the first theoretical description for the time-dependent fluorescence anisotropy decay resulted from two-photon excitation (r([2])(t)) for fluorophores in macroscopically isotropic and oriented membranes. In case of two-photon excitation, the initial value of the fluorescence anisotropy r([2])(0) immediately after excitation by a flash of polarized light is a function of the components of the two-photon absorption transition tensor [unk]S and the projections of the emission transition moment to the principal axes of [unk]S. The components of [unk]S depend on the symmetries of all molecular states relevant to the two-photon absorption process. The maximal value of r([2])(0) is proven to be as large as 0.61 in contrast to 0.4 for the conventional one-photon induced fluorescence anisotropy r([1])(0). It is shown that only for some special cases the ratio of the two-photon r([2])(t) over the conventional one-photon r([1])(t) will be a constant at all times for fluorophores in macroscopically isotropic membrane systems. In oriented membrane systems, an additional order parameter <P(6)> can be determined by the use of angle-resolved fluorescence depolarization measurements resulted from two-photon excitation. The advantages of measuring time-resolved fluorescence anisotropy decays or angle-resolved fluorescence depolarization ratios by two-photon excitation for the study of orientational dynamics in isotropic or oriented membranes are discussed from the theoretical point of view.

Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.

Impact of oxidized lipids and antioxidants, such as vitamin E and lazaroids, on the structure and dynamics of unsaturated membranes

The structural and dynamic effects of well defined, synthesized lipid peroxidation products (PLPC-OH and PLPC-OOH), the natural membrane-bound antioxidant vitamin E (α-tocopherol and β-tocopherol) and three synthetic membrane-bound antioxidants (the lazaroids U-74006F, U-75412 and U78518E) on bilayer lipid membranes have been investigated by time-resolved fluorescence depolarization and angle-resolved fluorescence depolarization (AFD) techniques and by electron paramagnetic resonance utilizing probe molecules. The lipid peroxidation products strongly disorder the investigated unsaturated lipid membranes, but they do not affect the lipid dynamics. The disordering effect is compatible with a model where the lipid hydroperoxy of hydroxy moieties reside closer to the polar head-group region of the membrane lipids. Fluorescence depolarization data obtained from the intrinsic fluorescence of α-tocopherol indicate that the trolox moiety of α-tocopherol (the fluorophore and antioxidant part of the molecule) can have different orientations in DOPC membranes depending on the α-tocopherol concentration. These orientational possibilities and the location of the lipid hydroperoxy moieties in lipid bilayers may account for the high reaction efficiency of α-tocopherol with lipid hydroperoxy radicals in membranes. Vitamin E in low and in high concentrations does not affect the molecular orientational order in membranes, but it does affect the dynamics. Because of the lack of structural effects of vitamin E on unsaturated lipid membranes without or with lipid peroxidation products, the hypothesis of vitamin E functioning as a membrane-stabilizing agent is questionable.In contrast with vitamin E, the three lazaroids as synthetic antioxidants have different effects on membrane structure and dynamics. The steroid-based lazaroids U-74006F and U-75412 have a disordering effect in the majority of the lipid systems investigated whereas the effects on dynamics vary depending on the surrounding lipids. The trolox derivative U-78518E, structurally closely related to α-tocopherol, has no or small ordering effects while the effects on dynamics also vary depending on the surrounding lipids. The effects of the different compounds on the molecular orientational order of membranes may be understood by their overall molecular shape. The main question that as yet remains unanswered is: which factors determine the dynamic effects of molecules in membranes?

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. II. Applications to pyranine, perylene, and DPH

The application of angle-resolved fluorescence depolarization techniques to the simultaneous determination of the directions of the transition moments and orientational order of dye molecules incorporated into uniaxially stretched polymer films is discussed and illustrated. The directions of the transition moments of pyranine molecules are determined in a general way, on exciting the molecules in two distinct electronic transitions and monitoring the fluorescence emission under the same conditions. This assumes that the fluorescence emission occurs via the same transition moment for every excited absorption band. Experiments on perylene molecules show that the directions of the transition moments can also be determined from measurements utilizing a single electronic absorption band. In this case the orientational distribution of the molecules is assumed to have the form given by the maximum entropy method. Finally, it is shown the directions of the transition moments of DPH molecules in unstretched polymer films can be determined with this technique. It appears that these molecules are oriented to a very small, yet measurable, extent in unstretched films.

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.

Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: Effects of unsaturation and hydration

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5α-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852–860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe moleciles do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.

On the model-independent determination of 〈P4〉 from time- and angle-resolved fluorescence depolarization on macroscopically oriented lipid multibilayer systems

Analysis of angle- and time-resolved fluorescence depolarization experiments on DPH and TMA-DPH embedded in macroscopically ordered lipid multibilayer systems shows that the order parameters 〈P2〉 and 〈P4〉 and the angle between the transition dipoles of the probe molecule can be obtained without making use of a particular reorientational model. The values found for 〈P4〉 correspond to the higher values obtained from model-dependent analysis of anisotropy experiments on vesicle systems. The angles between the transition dipoles obtained here are in excellent agreement with those obtained from steady-state experiments.

The membrane fluidity concept revisited by polarized fluorescence spectroscopy on different model membranes containing unsaturated lipids and sterols

Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidit) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidty). The plant sterol stigmasterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman [29, 30] attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as ‘membrane fluidity’. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig [19, 21]. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.

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 of macroscopically ordered bilayers of unsaturated lipids

The orientational order and dynamics of diphenylhexatriene molecules incorporated in planar multibilayers of unsaturated lipids were studied by angle-resolved flourescence depolarization at room temperature. Bilayer systems of the phosphatidylcholines soybean PC, dioleoyl PC and egg PC as well as the galactolipid digalactosyl diacylglycerol (DGDG) were used in the experiments. The depolarization ratios of diphenylhexatriene in these systems exhibited a weak angular dependence, in marked contrast to those observed from diphenylhexatriene in multibilayers of saturated phosphatidylcholines (Vos, M.H., Kooyman, R.P.H. and Levine, Y.K. (1983) Biochem. Biophys. Res. Commun. 116, 462–468). The results indicate that the diphenylhexatriene molecules possess a low degree of orientational order. However, the dynamics of the reorientational motions are inconsistent with the assumption that the diphenylhexatriene molecules possess effective cylindrical symmetry about their long axes. The fluorescence depolarization experiment is sensitive to the rotational motions about the long axes as the absorption and emission moments of diphenylhexatriene molecules are not mutually parallel. It is suggested that diphenylhexatriene molecules in bilayers of unsaturated lipids undergo slower reorientational motions around their long molecular axes than in bilayers of saturated phosphatidylcholines. A further conclusion of this study is that the ‘wobbling-in-cone’ model provides a poor description of the behaviour of diphenylhexatriene molecules in lipid bilayers.

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.