Yehudi K. Levine

Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant.

Solution of the Percus−Yevick approximation of the multicomponent adhesive spheres system applied to the small angle x-ray scattering from microemulsions

This paper describes the application of the numerical solution of the Percus–Yevick approximation for a multicomponent system of sticky hard spheres to small angle x‐ray scattering experiments. The effect of polydispersity on the pair correlation function is quantitatively described, thus enabling the analysis of experimental data. In this way quantitative estimates of the size distribution width, interaction radii and the measure of attraction between particles can be obtained. The attractive interaction in a system of sticky spheres is known to give rise to a transition analogous to a gas–liquid phase transition. The effect of polydispersity on this transition is discussed. Finally it is shown how the model can be used to describe small angle x‐ray scattering from an AOT microemulsion system.

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

Abstract 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 〈P2〉 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 〈P2〉 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 〈P4〉, 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.

Study of the orientational ordering of carotenoids in lipid bilayers by resonance-Raman spectroscopy.

The orientational ordering of beta-carotene and crocetin embedded in lamellar model membranes has been investigated by angle-resolved resonance Raman scattering at a temperature well above the phase transition of the lipid chains. It is shown that the ordering of the carotenoids is dependent on the chemical composition of the lipid bilayers. The orientational distribution functions found clearly show that beta-carotene is oriented parallel to the bilayer plane (dioleoyl lecithin) or perpendicular to it (soybean lecithin). For dimyristoyl lecithin at 40 degrees C, egg-lecithin, and digalactosyl diacylglycerol two maxima were found in the orientational distribution: one parallel and one perpendicular to the bilayer surface. Crocetin embedded in soybean lecithin bilayers yields a similar bimodal distribution function. Because of rapid photodegradation no results could be obtained for spirilloxanthin.

Percolation in oil‐continuous microemulsions. A dielectric study of aerosol OT/water/isooctane

The percolation transition in AOT/water/isooctane microemulsions has been studied by means of dielectric spectroscopy. At the percolation threshold the conductivity of the system increases sharply and the static dielectric permittivity attains a large maximum. It is shown that the frequency dependence of the permittivity can be well described by simple scaling relations of percolation theory. The critical exponent u of the frequency dependence has been found to be equal to u=0.62±0.02 and independent of temperature and microemulsion composition. The temperature dependence of the critical volume fraction for percolation has been related to the temperature dependence of the permittivity of the system far away from the percolation threshold.

A theory of fluorescence depolarization in macroscopically ordered membrane systems

Abstract A theory of fluorescence depolarization experiments in macroscopically ordered membrane systems is presented. The theory is applicable to fluorescing molecules of arbitrary symmetry. It is shown that second- and fourth-rank order parameters can be extracted from the steady-state measurements in which the angle of incidence, as well as the direction of observation, are varied.

Measurement of second and fourth rank order parameters by fluorescence polarization experiments in a lipid membrane system

Abstract It is theoretically and experimentally shown that steady-state measurements of the angular dependence of the fluorescence depolarization in macroscopically ordered membrane systems form an alternative for fixed-geometry time-resolved experiments in such systems. It is found that the experimentally determined fourth rank order parameter is consistent with that calculated from Maier—Saupe theory.

Reorientational dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation

Electron spin resonance experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in multilamellar liposomes and small unilamellar vesicles (SUVs) of palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). The experimental spectra were analyzed by a numerical solution of the stochastic Liouville equation. Effects of temperature, presence of unsaturated bonds and high bilayer curvature on the dynamic behaviour of the lipid molecules were studied. Our results, combined with results from planar multibilayers with a varying hydration rate (Korstanje et al. (1989) Biochim. Biophys. Acta 980, 225-233), give a consistent picture of the orientational order and rotational dynamics of CSL molecules embedded in lipid matrices with various geometrical configurations. Increase of hydration or temperature reduces molecular ordering and increases molecular dynamics. In highly curved vesicle configurations, SUVs, molecular order is found to be lower than in multilamellar liposomes. In contrast, rotational motion is not affected by increase of curvature. In all lipid configurations studied, increase of the number of unsaturated bonds in the fatty acid chains reduces molecular ordering. We find, however, no effect of unsaturation on the rotational mobility of the CSL probe molecules. These results clearly show that changes in molecular orientational order and reorientational dynamics have to be considered separately, and that they are not necessarily correlated as implied by the common concept of membrane fluidity. Comparing our results with data from a motional narrowing analysis shows that the latter approach seriously overestimates the rate of molecular reorientation.

Fluorescence lifetime imaging of free calcium in single cells

It is shown here that the intensity probe CalciumGreen is suitable for quantitative Ca2+ concentration determinations using fluorescence lifetime imaging. The probe is characterized by two distinct fluorescence lifetimes over a large Ca2+ concentration range. This indicates that the probe exists in two forms, one free and one bound to Ca2+. This behaviour is exploited for the determination of the absolute free Ca2+ concentration from fluorescence lifetime measurements. Fluorescence lifetime confocal imaging affords the determination of an effective lifetime of the probe in buffers and single rat myocytes. The images are used for the quantification of the local concentrations of free Ca2+. We find CalciumGreen to be a useful lifetime probe at physiological Ca2+ concentrations. A similar photophysical behaviour has also been found for the intensity probe Fluo-3. However, it is less suitable for the determination of Ca2+ concentrations in cells.

A dissipative particle dynamics description of liquid-crystalline phases. I. Methodology and applications

Simulations of nematic and smectic mesophases based on a dissipative particle dynamics approach are discussed. Mesogenic units are built in the form of standard semirigid bead-spring chains. It is shown that nematic phases can be formed for chains containing at least eight beads, provided that the conservative soft-repulsive potential between nonconnected beads is sufficiently strong. Smectic phases are observed only by modifying the repulsive interaction between the main-chain and terminal beads. The simulations indicate that the smectic-nematic and smectic-isotropic phase transitions take place through the buckling of the smectic layering in the system.