Solvent Relaxation in Phospholipid Bilayers: Principles and Recent Applications

Although there exist a number of methods, such as NMR, X-ray, e.g., which explore the hydration of phospholipid bilayers, the solvent relaxation (SR) method has the advantage of simple instrumentation, easy data treatment and possibility of measuring fully hydrated samples. The main information gained from SR by the analysis of recorded “time-resolved emission spectra” (TRES) is micro-viscosity and micro-polarity of the dye microenvironment. Based on these parameters, one can draw conclusions about water structure in the bilayer. In this review, we focus on physical background of this method, on all the procedures that are needed in order to obtain relevant parameters, and on the requirements on the fluorescence dyes. Furthermore, a few recent applications (the effect of curvature, binding of antibacterial peptides and phase transition) illustrating the versatility of this method are mentioned. Moreover, limitations and potential problems are discussed.     

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC⁴ bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values () obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above T _c as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid melt profiles are broad and shifted to higher midpoint EA pressure values (P _1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported fluid–gel lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP _1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P _1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P _1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.     

Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.     

Low-Temperature Molecular Motions in Phospholipid Bilayers in Presence of Glycerol as Studied by Spin-Echo EPR of Spin Labels

Glycerol is used as a cryoprotective agent to protect biological systems under freezing conditions. Electron spin echo (ESE) spectroscopy, a pulsed version of EPR, is capable of studying low-temperature molecular motions of nitroxide spin labels. ESE technique was applied to study molecular motions in phospholipid bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with added spin-labeled lipids 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (n-PCSL, n was optionally 5 or 16). Bilayers were hydrated (solvated) either in pure water or in a 1:1 v/v water–glycerol mixture. In the used ESE approach, there were studied stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations). The anisotropic contribution to the echo decay rate, W _anis, was measured, which is proportional, according to theory, to the product of the mean-squared angular amplitude \(\langle \alpha^{ 2} \rangle\) and the correlation time τ _c. W _anis was found to be small below and to sharply increase above 200 K, for the both types of solvents and the both label positions. As compared with hydration by pure water, in presence of glycerol W _anis was larger for the 5th label position while for the 16th one it did not change. Also, for the 5th label position W _anis values were found to be nearly the same as those for a polar spin probe 3,4-dicarboxy-PROXYL which was separately added to the bilayer as a reference and which is assumed to be partitioned only into the solvating shell. These results indicate that motions at the surface of bilayer are governed by the motion of solvating shell while motions in the bilayer interior occur independently. The relation of the obtained data with the dynamical transition phenomenon that is known for biological substances near 200 K from neutron scattering and Mössbauer absorption is discussed.     

Molecular Motion in Frozen Phospholipid Bilayers in the Presence of Sucrose and Sorbitol Studied by the Spin-Echo EPR of Spin Labels

Electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), was applied to spin-labeled stearic acids in phospholipid bilayers hydrated in the presence of sucrose and sorbitol, which are known for their cryoprotective action on biological membranes. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Stearic acids were labeled by nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th specific carbon positions. ESE detects fast stochastic small-angle restricted molecular rotations (stochastic molecular librations) with correlation times on the nanosecond timescale. These motions are believed to have the same nature as the anharmonic motions of hydrogen atoms in biological substances detected by neutron scattering and Mössbauer spectroscopy, which become active above 200 K. To ensure that the echo decays indeed originate from fast stochastic molecular librations, a three-pulse stimulated spin echo was employed. It was found that the presence of sucrose or sorbitol suppresses the observed molecular motions. The observed effect was nearly the same for both label positions, indicating that the motions are similarly suppressed near the bilayer surface and in the bilayer interior. This finding suggests non-specific interactions of sugars with bilayer surface, which are likely to influence only the bulk physical properties of hydrated membranes. The results obtained show the usefulness of spin-echo EPR of spin labels when applied to investigate the molecular mechanisms of action of cryoprotective agents on biological systems.     

Bilayers of neutral lipids bear a small but significant charge

Many experiments done on neutral lipid bilayers in pure water show weak repulsions. These weak forces prevent vesicles from adhering and are generally overcome by adding some salt in the aqueous medium. They also appear as stray repulsions in surface forces measurements made on lipid bilayers. Using a surface forces apparatus in pure water and in salt solution, we have measured the forces between two stearoyl-oleoyl-phosphatidyl-choline (SOPC) bilayers and between two dimiristoyl-phosphatidyl-ethanolamine (DMPE) bilayers. The results show that the repulsions are due to a small amount of negative charges coming from impurities in SOPC. This was quantitatively confirmed by electrophoretic measurements. There are 3 times less charges in the case of DMPE layers. The effect of these charges which is negligible at high salt concentration may significantly affect the adhesion energy and behaviour of neutral lipid bilayers between 0 and salt. Received 18 December 1998     

Weakly Bound States of Polar Molecules in Bilayers

We investigate a system of two polarized molecules in a layered trap. The molecules reside in adjacent layers and interact purely via the dipole?Cdipole interaction. We determine the properties of the ground state of the system as a function of the dipole moment and polarization angle. A bound state is always present in the system and in the weak binding limit the bound state extends to a very large distance and shows universal behavior.     

On the Spectrum of Interlayer Roughnesses in a Phospholipid Multilayer

JETP Letters - The spectral properties of the correlation function of the heights of interlayer boundaries in a lamellar 1,2‑distearoyl-sn-glycero-3-phosphocholine film deposited on the...     

普通X射线荧光光谱仪分析铜精矿样品在储存过程中化学态的变化

采用普通X射线荧光光谱仪观察了经不同方式保存的铜精矿样品的S K_α谱、S K_β伴线谱的谱峰位置和谱线形状,以及(S K_α/S K_β),(Cu L_α/Cu K_α)和(Fe L_α/Fe K_α)的谱线强度比率,根据这些谱线及谱线强度比率存在的一些变化来推断,铜精矿中的硫、铜、铁的化学态在储存过程中发生了变化。研究表明,主要矿相为黄铜矿(含有少量黄铁矿)的铜精矿样品储存2年后,发生了较明显的氧化,有部分S2-,Cu+和黄铁矿中的Fe2+被氧化为高价态,硫原子与氧原子相结合。样品的保存方式对样品的氧化程度有很大影响。     

Radiation from a Charge Supported in a Gravitational Field

We examine whether a charge supported statically in a gravitational field radiates, and find the answer to this question to be positive. Based on our earlier results we find that the important condition for the creation of radiation is the existence of a relative acceleration between the charge and its electric field, where such an acceleration causes the curving of the electric field and the creation of a stress force due to this curvature. This stress force is the reaction force, which creates the radiation. Later we find that this condition do exist for a charge supported statically in a gravitational field, where the electric field of the charge falls in the gravitational field, it curves, and the stress force raised in this curved field, creates electromagnetic radiation.     

Fluorescence of anthracene in a perfluorosulfone membrane

The fluorescence spectrum of anthracene introduced into the pore space of a perfluorosulfone membrane reflects the localization of a guest material in carrier nanochannels with the formation of closely spaced molecules able to form either excimers or stable dimers depending on the irradiation conditions.     

铁磁/反铁磁双层膜系统中交换各向异性及其厚度依赖性

研究铁磁/反铁磁双层膜系统中交换偏置场和矫顽场的冷却磁场依赖性.结果表明,随着冷却磁场的增加,交换偏置场由负值向正值转变.在转变点附近,矫顽场有-个特别的增强,并达到最大值.结果同相关实验-致.研究铁磁层和反铁磁层厚度对交换偏置场和矫顽场的影响.发现,正负交换偏置场和矫顽场随着铁磁层厚度的增大而减小,但随反铁磁层厚度的变化关系复杂.在正交换偏置场的情形,随反铁磁层厚度的增大,交换偏置场增强,矫顽场减弱;在负交换偏置场的情形,随反铁磁层厚度的增大,交换偏置场减弱,矫顽场增强.     

Fluorescence Emission Centres and the Corresponding Infrared Fluorescence Saturation in a Bismuth-Doped Silica Fibre

We investigate the fluorescence characteristics of bismuth doped silica fibres with and without A1 co-dopant which are fabricated by means of modified chemical vapour deposition （MCVD） technique, and find that the fluorescences in the red region （centred around 750nm） and in the infrared region （centred around llOOnm） may originate from different emission sites in the fibre. Strong upconversion phenomena are observed in both Al-codoped and non A1 codoped bismuth fibres when the fibres are excited by an acoustic-optic Q-switched Nd：YVO4 laser. Both the aspects indicate that the upper energy level absorption reported in the work of the bismuth doped silica fibre lasers may result from the fluorescence emission sites that are not responsible for the infrared emission. It is thus expected that optimizing the compositions and the fabrication conditions of the fibre and then transferring more fluorescence emission centres are helpful for the infrared emission.     

张应力对铁磁/反铁磁体系交换偏置的影响

 采用能量极小原理及Stoner-Wohlfarth模型,研究了张应力对铁磁/反铁磁双层薄膜交换偏置的影响。在不施加外磁场时,根据体系能量与铁磁层磁化强度方向之间的关系,得到了内禀易轴与内禀难轴的位置。交换各向异性与单轴各向异性之间的竞争使体系存在单稳态与双稳态两种不同的状态,直接决定了交换偏置的角度依赖关系。分析磁化过程发现,外磁场在沿内禀易轴及内禀难轴方向施加时,磁滞回线的一支转换场发生突变,另一支转换场保持不变,最终导致交换偏置场和矫顽场出现阶跃行为。在阶跃点处,体系具有较大的交换偏置场和矫顽场。数值计算表明：张应力的大小与方向对交换偏置均有很大的影响,均可以使体系在单稳态与双稳态之间相互转变并导致角度依赖关系发生显著变化。研究表明,应力可作为一种可行的方法来控制和调节铁磁/反铁磁体系的交换偏置。     

Photo-induced Fluorescence of Fluometuron in a Continuous-flow Multicommutation Assembly

This paper deals with the photo-induced fluorimetric determination of the herbicide Fluometuron with the aid of a continuous-flow assembly of the emergent and new methodology known as Multicommutation which was provided with an on-line photoreactor. Maximum fluorescence intensity was observed at basic pH solutions, 1×10⁻⁴ mol l⁻¹ NaOH, after 1.4 min of irradiation and being the maximum at λ_exc 247.0 nm and λ_em 325.0 nm. The influence of different experimental parameters either chemical (pH, surfactants presence, solvent polarity and temperature) or hydrodynamic (time of photo-degradation, size and number of different segments and flow-rate) was tested. The linear dynamic range was from 0.01 to 4.0 mg l⁻¹ of Fluometuron; the inter-day reproducibility (as R.S.D.) of the slope was 0.001% and 1.7% from the peaks intra-day reproducibility. A large series of potential interferents was studied and finally the method was applied to human urine, soil, formulation and water samples.     

Fluorescence in a Quantum System with Violated Symmetry

The model of a single multilevel one-electron atom with violated symmetry such that its transition dipole-moment operator has constant diagonal matrix elements, among which not all are pairwise equal to each other, has been studied. It has been shown that the expression for the far electromagnetic field of such an atom does not contain any appreciable contributions from the diagonal matrix elements of the transition dipole moment in an explicit form; thus, these matrix elements have an effect on fluorescence via the time dependence of non-diagonal matrix elements due to quantum non-linear processes of higher orders. It has also been demonstrated that a two-level quantum system, whose transition dipole operator has constant unequal diagonal matrix elements, can continuously fluoresce under excitation with monochromatic laser radiation at a much lower frequency than the frequency of the exciting radiation. The possibility of the experimental detection and practical application of this effect are discussed.