Membrane Permittivity Research Articles

Study of normal and malignant white blood cells by time domain dielectric spectroscopy

The aim of this paper is to present a comprehensive theoretical and experimental study by means of time domain dielectric spectrometer (TDDS) of static and dynamic dielectric properties of normal and malignant blood cells. The successful use of the TDDS method as a tool of human cell study required a special protocol and algorithms for all stages of cell preparation, measurements and data treatment. The routine developed in this study was used in the experimental analysis of nine lines of malignant, transformed and normal lymphocytes. It was shown that dielectric permittivity, capacitance and conductivity values of the cell membrane are higher for normal lymphocytes in comparison with malignant ones.

A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane

For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity σ mt. On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the field component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz–MHz range) with a marked increase of the radial membrane permittivity ε mr at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells.

Time domain dielectric spectroscopy study of human cells: II. Normal and malignant white blood cells

The dielectric properties of human lymphocyte suspensions were studied by time domain dielectric spectroscopy (TDDS). Nine populations of malignant and normal lymphocytes were investigated. Analysis of the dielectric parameters of cell structural parts were performed in the framework of Maxwell–Wagner mixture formula and the double-shell model of cell. The specific capacitance of the cell membranes was estimated by the Hanai–Asami–Koisumi formula. It was shown that the dielectric permittivity, capacitance and conductivity values of cell membranes are higher for normal lymphocytes than for the malignant ones. The difference of the same parameters for normal B- and T-cells is also discussed.

Cytoskeletal rearrangement in K562 erythroleukaemic cells forced to grow on a positively charged polymer surface.

We have recently demonstrated that if human K562 erythroleukaemic cells, which normally grow in suspension, are grown on a positively-charged surface composed of polylysine, a transient reorganization of CD54 (ICAM-1), CD58 (LFA-3) and alphanubeta3 (vitronecin receptor), three important CAMs located on the cell membrane, takes place. In addition, changes of longer duration in membrane conductivity (ionic transport across the cell membrane) and membrane permittivity (static distribution of charges across the cell membrane), indicating more permanent structural as well as functional alterations in the cell membrane, were also observed [2]. Because of the close interrelationship which exists between the cell membrane, CAMs and the cytoskeleton, changes in this intracellular network as well as in the surface morphology of K562 cells grown on the positively-charged polymer, polylysine, were examined. In particular, actin and tubulin were investigated qualitatively and quantitatively by immunofluorescence microscopy and flow cytometry, respectively, while the cell surface was studied by scanning electron microscopy (SEM). The data indicate that when K562 cells are grown onto polylysine no quantitative changes occurred to the cytoskeletal elements even if these were rearranged and that the cell membrane surface is also greatly altered. These results are discussed in light of the pivotal role played by CAMs and the cell cytoskeleton in transducing environmental stimuli, in this case those provided by a positive charge, from the cell membrane to the inside of the cell.

Positively charged polymer polylysine-induced cell adhesion molecule redistribution in K562 cells.

We have recently demonstrated that if human K562 erythroleukemic cells, which normally grow in suspension, are grown in polylysine-coated culture flasks for 48 h, they adhere to these flasks and grow in an anchorage-dependent like manner. Important changes in both membrane conductivity (ionic transport across the cell membrane) and membrane permittivity (static distribution of charges across the cell membrane) were also observed, indicating perturbations in membrane lipids, proteins and polysaccharides. In order to better understand the changes occurring in K562 cells exposed to polylysine and because of the important role played by cell adhesion molecules (CAMs) in cell/cell and cell/substratum interactions, and in cellular adaptation to the surrounding environment, the possible redistribution of these molecules after exposure to polylysine were investigated. In particular, the CD54 (ICAM-1), CD58 (LFA-3) and alphaVbeta3 (vitronectin receptor) molecules were investigated at different times of growth both quantitatively and qualitatively utilizing flow cytometry and immunofluorescence microscopy, respectively. The data indicate that there were no significant quantitative variations in the CAMs examined at all the times tested. In addition, no qualitative changes were observed at 48 h (as well as 24 h) of exposure. However, shorter treatment times (30 min, 1 and 2 h) did induce important CAM reorganization. The results seem to demonstrate that this cycle of CAM redistribution may, in part, be responsible for cellular adaptation to the new growth environment of K562 cells and for the variations in membrane electrical properties observed.

Conductometric properties of human erythrocyte membranes: dependence on haematocrit and alkali metal ions of the suspending medium.

The electrical properties of the cytoplasmatic membrane of human erythrocyte cells have been evaluated by means of dielectric spectroscopy measurements in the radiowave frequency range, using the so-called "suspension method". Measurements have been carried out at different volume fractions of the corpuscular phase (the cell haematocrit) in order to investigate the influence of the cell-cell interactions on the electrical parameters (the membrane permittivity epsilon and the membrane conductivity sigma) of the cell membrane and a set of new values are proposed. Moreover, the influence of different alkali metal ions (Na+, K+, Cs+, Li+) on the ion permeation properties of the membrane are investigated and the structural alterations in the membrane organized briefly discussed.

Polylysine induces changes in membrane electrical properties of K562 cells.

The ionic environment of the cell membrane is of extreme importance in maintaining cell integrity and the numerous functions necessary for cell growth, differentiation, etc., as well as in cell-biomaterial interactions. In this study, the effects of polylysine (a basic poly-amino acid with a net positive charge which is often used to coat biomaterials surfaces) on the erythroleukemic K562 cell membrane were investigated. In particular, the effects of this polycation were evaluated using dielectric relaxation studies in the radiofrequency range with which it is possible to measure both active ionic transport across the cell membrane (membrane conductivity) and the static charge distribution present on the cell surface due to the structural components of the cell membrane (membrane permittivity). The conductivity of the cytosol can also be determined. The results demonstrate that while the conductivity of the cytosol is not significantly altered, both the conductivity and permittivity of the K562 cell membrane are varied by exposure of these cells to polylysine. These observations indicate that both active ionic transport and the type, quantity, or distribution of membrane components such as lipids, proteins, and polysaccharides may also be altered. Although the precise mechanisms by which these variations in K562 cells occur are unknown, it can be hypothesized that changes in the growth characteristics of these cells may be in part responsible. In particular, as demonstrated by light microscopic examination of K562 cells directly in the culture flasks, the cells in polylysine-coated flasks do not grow in suspension as do the controls, but rather show anchorage-dependent-like behavior. It is this important change from suspension to monolayer growth induced by polylysine that may be responsible for the changes in membrane electrical parameters.

Two mechanisms of permeation of small neutral molecules and hydrated ions across phospholipid bilayers

The Gibbs free energy of dipole or iron permeation of lipid bilayers is calculated as the sum of all electrostatic, solvophobic and specific interactions. Partitioning models are consistent with dipole permeation and some features of ionic permeation, particularly if the solvophobic energy is taken into account. Ionic and dipole permeabilities are extremely sensitive to the ionic/dipolar radius. Despite this sensitivity, calculations of the permeability can be carried out for typical monovalent cations, and provide reasonable estimates, but only for hydrated species. An alternative mechanism proposed for ionic permeation involves the occurrence of transient pore-like defects in lipid bilayers which permit ions to bypass the Born energy barrier. The two alternative hypothesis, partitioning vs. transient pores, can be tested by measuring the ionic and dipolar permeation through bilayers of varying thickness. Experimental observations for both potassium and proton permeability are consistent with the transient pore mechanism for shorter chain lipids, but tend towards the theoretical line for partitioning models for longer chain lipids. Results for small neutral solutes are best explained by the solubility-diffusion mechanism. The proposed method of calculation of the Gibbs free energy of ion or dipole membrane transfer and the liquid membrane permittivity can be effectively used not only in describing the biophysical properties of membranes, but also in extraction processes, pharmaceutical applications and liquid membrane separations.

Dielectric dispersion of a membrane consisting of a large number of solid bilayers containing mobile ions

The complex permittivity and the impedance of a lamellar membrane consisting of a repetition unit of two conducting dielectrics is calculated using the linearized Nernst–Planck–Maxwell electrodynamics previously developed for isolated interfaces, lamellar films with identical cation and anion mobility inside each type of lamella and with identical Nernst distribution coefficients and for homogeneous polymer films with ionic impurities. Any intrinsic dielectric dispersion of the two (ion-free) dielectrics is neglected and periodic boundary conditions are assumed in a infinite array of identical bilayers. The anion and the cation may be of any charge type and their diffusion coefficients may have any values in the two dielectrics. The Nernst distribution coefficient may be different for the cation and the anion within limits determined by the validity of the linearisation procedure. The calculated complex permittivities of the composite material may differ substantially from the permittivities calculated by a simple Maxwell–Wagner–Sillars (MWS) procedure because of the dynamical electrical double layers formed at 1–2 Debye lengths from the interfaces. These polarisation layers give rise to the so-called excess impedance. The MWS results are, however, always exact in the high-frequency limit. Furthermore, the MWS approach is correct for the dielectric loss at low frequencies. A frequency weighted ‘Cole–Cole plot’ of the real and imaginary components of the excess impedance has a characteristic ‘apple shape’ starting in the origin at very low frequencies and moving clockwise around the origin with increasing frequency. In the high-frequency limit, the origin is always approached in the fourth quadrant along a line with slope-1. The low-frequency asymptote of the frequency weighted Cole–Cole plot for the excess impedance is sensitive to the special conditions, however. The Cole–Cole plots of the total membrane impedance (MWS+excess) exhibit sometimes two partially overlapping semi-circular arcs, one for each type of lamella. When the thickness of the lamellae becomes of the same order of magnitude as the Debye length, the distinction between the two relaxations is wiped out and only a single, flattened arc is seen. A model lamellar ‘cellulose acetate’ (CA) membrane, consisting of alternating layers of ‘wet CA phase’ and ‘aqueous microphase’ with NaCl dissolved, is investigated. The parameters of the membrane are chosen quite realistically (except the one-dimensional geometry) as those pertaining to dense CA membranes, in accordance with previous emf and impedance spectroscopical studies of dense and asymmetric cellulose acetate membranes. The usual electrochemical ‘equivalent circuit’ approach, connecting in parallel the membrane resistance and the membrane capacitance, is put to a test. From the time constant related to the maximum negative reactance the so-called ‘geometric capacitance’ is calculated. From this, the ‘geometric’ membrane permittivity of the membrane is found. At low salt concentrations, the geometric permittivity coincides with the low-frequency limit of the membrane permittivity, but with increasing salt concentrations the geometric permittivity increases less rapidly with concentration than does the low-frequency membrane permittivity. The geometric membrane permittivity seems to be a complicated mean value between the low-frequency membrane permittivity and the high-frequency membrane permittivity (independent of concentration), since the characteristic frequency for the impedance relaxation is many orders of magnitude higher than the characteristic frequency of the dielectric relaxation. The former is related to the diffusional relaxation time of the CA layer, the second to the relaxation time in the aqueous microphase.

Changes in membrane electrical parameters of yeast following chemical treatment for protoplast isolation

The yeast strains Saccharomyces species and Candida tropicalis were used. In each case, the electrorotation spectra were recorded for normal untreated yeast cells, β-mercaptoethanol-treated cells and isolated protoplasts. In order to obtain realistic information on the passive electrical membrane parameters associated with each step of protoplast isolation, a theoretical analysis of the simulated electrorotation spectra for shelled ellipsoids was performed. As a consequence, the parameters chosen for fitting of the experimental spectra were the electrical conductivities of the cell compartments, the membrane permittivity and the scaling factor. β-Mercaptoethanol was found to have significant action at the membrane level: (1) at high concentration (2%), σ m was increased dramatically; (2) in mild incubation conditions (0.1%), σ m was decreased slightly. Treatment with the enzyme complex led to a decrease in internal conductivity, probably due to a loss of the ionic equilibrium balance.

Nernst–Planckian Electrodynamics, the Excess Interfacial Impedance, and the Complex Permittivity of a Lamellar Membrane with Overlapping Dynamic Electric Double Layers II. The Case of Negligible End Effects, Identical Ionic Diffusion Coefficients in Each Phase, and Identical Nernst Distribution Coefficients

The analysis in Part I [J. Colloid Interface Sci.168,437 (1994)] is extended to obtain analytical expressions for the excess impedance (deviations from Maxwell–Wagner–Sillars theory) (MWS) and for the complex permittivity of lamellar membranes with two phases and with negligible effects from the finite extension of the membrane. In the present paper, we deal exclusively with the case of all ionic diffusion coefficients being equal in each phase and having the same Nernst distribution coefficient between the two phases. In cases, where the MWS relaxations corresponding to the two phases are clearly distinguished, the effect of the excess impedance is to wipe away the difference. The Debye length (1/κ) acts as a “coarse graining distance,” and with lamellar width (L) below this distance, the heterogeneous structure is not discernible in the impedance spectrograms anymore. In the Cole–Cole plot of the reactance against the resistance only a single semi-arc survives. In an example close to the properties of a porous “cellulose acetate membrane,” there is only a small effect of excess impedance even forLκ ≈ 1. In contrast, the MWS theory is plainly wrong for such a membrane when dielectric measurements are considered (relaxing at much higher frequencies). The Debye plot of the complex vs the real membrane capacitance deviates completely from the MWS theory whenLκ ≈ 1 (except at very high frequencies), and the characteristic relaxation frequency is much higher than that predicted from MWS. Indeed,Lκ must be more than 50 in order for the calculated complex permittivities to approach the MWS result for all frequencies.

Time-domain reflectometry studies on Halobacterium halobium and Halobacterium marismortui

The dielectric properties of Halobacterium halobium and Halobacterium marismortui measured over the frequency range 1 MHz to 1 GHz are compared with a single-shell model for interfacial polarization. In the case of Halobacterium halobium, the model shows excellent agreement with the experimental data for reasonable values of membrane and cytoplasmic conductivity and permittivity. For Halobacterium marismortui however, an acceptable fit to the data can only be achieved by invoking a plasma membrane conductivity some three orders of magnitude larger than that of Halobacterium halobium and adding a second high-frequency dielectric dispersion. These observations confirm the findings of a previous study and are consistent with a scheme for Halobacterium marismortui involving thermodynamic compartmentalization of Na and K ions and the existence of a conductive plasma membrane.

A 50 Hz sinusoidal magnetic field induces changes in the membrane electrical properties of K562 leukaemic cells

The cell membrane appears to be one of the major targets of extremely low frequency (ELF) magnetic fields. To determine whether the membrane electrical properties of K562l leukaemic cells can be affected by ELF fields, these cells were exposed to a sinusoidal 50 Hz 2.5 mT magnetic field for 48 h. It should be recalled that the field intensity used is three or four orders of magnitude greater than the values found in homes and in many workplaces. Analyses of dielectric relaxation measurements in the r.f. range demonstrate that both the membrane electrical conductivity and permittivity of K562 cells decrease considerably after exposure of these cells to ELF fields, whereas the conductivity of the cytosol does not vary. Since both membrane conductivity (a measure of the dynamic ionic movement across cell membranes) and membrane permittivity (an indicator of the ionic charges present on cell membranes) vary, these data seem to suggest that exposure of K562 leukaemic cells to ELF fields affects both static and dynamic properties of these cells.

４電極法による膜に吸着した高分子物質の誘電率と導電率の測定

In order to solve the problems in fouling and antithrombus, it is important to measure the permittivity and the conductivity of polymers adsorbed on hydrophobic and hydrophilic membranes. However, it has not been able to make acculate measurement of the capacitance of membranes adsorbing polymers at low frequency, because of the electrode polarization. We, thus, have utilized the 4 terminals method where the voltage and the electric current were mesured by using different elecrodes, to minimize the effect of electrode polarization. Using the above method, we measured the dielectric relaxation of Polycarbonate (PC) and Poly (tetrafluoroetylene) (PTFE) membranes adsorbing Poly (vinylpyrrolidone). In case of the PC membranes, the dielectric relaxation was not observed. On the other hand in case of the PTFE membranes, the relaxation was observed only when the amount of Polyvinylpyrrolidone was small. The permittivity of membrane was independent of the amount of adsorbed polymer, on the other hand, the conductivity of the membrane increased with increasing the adsorbed polymer, indicating that the hydrophobic membranes turned to hydrophylic.

To what extent are the passive electrical parameters of lymphocyte membranes deduced from impedance spectroscopy altered by surface roughness and microvillosity?

The electrical conductivity of human lymphocyte suspensions in NaCl electrolyte solutions of varying osmolarities has been measured in the frequency range from 1 MHz to 1 GHz, where the ion polarization at the membrane-extracellular medium interface influences markedly the electrical behaviour of the cell suspension. To take into account the complexity of the cytoplasmic membrane, where protrusions and microvillous structures extend towards the extracellular medium, an approach based on scaling concepts typical of the fractal analysis of complex systems is proposed. The passive electrical parameters, i.e. the membrane conductivity σ s and the membrane permittivity ϵ s, obtained on the basis of this procedure, differ significantly from those derived from conventional methods. The possible relevance of this circumstance from a biological point of view is indicated.

Application of Chronoamperometry to Evaluation of Membrane Porosity

Abstract Conductance and electric permittivity of Epidian-5 epoxy resin membranes modified with benzene have been determined by the chronoamperometric method. In addition, membrane porosity was evaluated. The membrane porosity was found to undergo different changes in two benzene concentration ranges: in the 0-20% (wt) range the porosity decreases, in the 20-40% (wt) range it increases. The differences in porosity were found to be associated with structural differences of the membrane-induced benzene. Chronoamperometry was found to be an appropriate technique for the evaluation of membrane porosity. The proposed method allows for a convenient analysis of porosity variations in conditions matching the real conditions of membrane work.

A static magnetic field does not affect the dielectric properties of chick embryo myoblast membranes.

We have recently demonstrated using dielectic relaxation studies in the radiowave frequency range that sinusoidal 50 Hz magnetic fields (with intensities ranging from 1 to 10 mT) induce a nonlinear change in both membrane conductivity and permittivity of primary chick embryo myoblasts in vitro. It was the aim of the present study to determine if a DC-induced static magnetic field is capable of generating similar variations in the membrane conductivity and/or the membrane permittivity of chick embryo myoblasts. The results indicate that when the myogenic cells are exposed to a static magnetic field of either 1, 3 or 5 mT (values comparable with the previous extremely low frequency study), no changes in the membrane electrical parameters can be observed with respect to controls. Differences in the characteristics of static and extremely low frequency fields as well as the possible mechanisms underlying the contrasting results with these two types of magnetic fields are discussed.

Frequency domain electrical conductivity measurements of the passive electrical properties of human lymphocytes

An extensive set of electrical conductivity measurements of human lymphocyte suspensions has been carried out in the frequency range from 1 kHz to 100 MHz, where the surface polarization due to the Maxwell-Wagner effect occurs. The data have been analyzed according to well-established heterogeneous system theories and the passive electrical parameters of both the cytoplasmic and nuclear membranes have been obtained. Moreover, a further analysis to take into account the roughness of the membrane surface on the basis of a fractal model yields new estimates for the membrane conductivity and the membrane permittivity, independently of the surface architecture of the cell. These findings are confirmed by measurements carried out at higher frequencies, in the range from 1 MHz to 1 GHz, on lymphocytes dispersed in both hyperosmotic and hypoosmotic media, that influence the surface complexity of the membrane due to the microvillous protusions. The surface roughness of the cell is described by a macroscopic parameter (the fractal dimension) whose variations are associated to the progressive swelling of the cell, as the osmolality of the solution is changed.

On the dielectric method of monitoring cellular viability

Abstract

The Fusion Radiosensitivity of Differentiating Chick Embryo Myoblastsin VitroIs Not Determined by the Plasma Membrane

We have recently demonstrated by dielectric relaxation studies in the radiofrequency range that the sharp drop in the conductivity and permittivity of the membranes of chick embryo myoblasts in vitro, representative of fusion, is either delayed or completely blocked by sublethal doses of ionizing radiation (Santini et al. 1990a). The lowest of the doses investigated (3.25 Gy) caused a 10 h delay in myoblast membrane fusion when the cells were exposed at 24 h of culture, indicating that radiation-induced membrane injury had occurred. The purpose of this study was to determine if the myoblast system under investigation shows the same radiosensitive characteristics if irradiated with 3.25 Gy at various stages of differentiation. Consequently, the myoblasts were exposed to this dose at two different stages of differentiation (12 h or 48 h of culture). We show here that the time at which the myogenic cells are irradiated (state of differentiation) does not seem to affect the magnitude of the fusion delay (which was used as a measure of radiosensitivity of the myoblasts). In fact, the sharp drop in both membrane conductivity and membrane permittivity occurs with the same 10 h delay independent of the time of exposure. The role played by the plasma membrane in determining myoblast response to radiation damage is discussed.