Electrorotation Data Research Articles

DIELECTRIC CHARACTERIZATION OF THE YEAST CELL BUDDING CYCLE

We combine experimental electrorotation data and the numerical analysis of the electrorotation chamber and cell to electrically characterize the Saccharomyces cerevisiae yeast budding cell cycle and to obtain the electrical parameters of the cell. To model the yeast cell we use spherical and doublet-shaped geometries with a four layered structure: cytoplasm, membrane, inner and outer walls. To derive the geometrical and electrical parameters of the yeast model we use the flnite element method to calculate the yeast rotational velocity spectrum and apply the least-square method to flt the calculated values to experimental data. We show that the calculated yeast electrorotation spectra undergo signiflcant changes throughout its budding cycle and that the calculated spectra flt experimental data obtained for 0% (start) and 50% representative budding stages very well. The analysis also shows the small variation of the rotation crossover frequency within a full span of the yeast growth cycle. As an application of this work, we apply the Maxwell-Wagner formalism to obtain the dielectric spectra of truly synchronized yeast suspensions.

Interactions of DMPC and DMPC/gemini liposomes with the cell membrane investigated by electrorotation

The electrorotation technique was utilized to investigate the interactions between a mouse fibroblast cell line and zwitterionic liposomes formed by a natural phospholipid or cationic liposomes formulated with the same phospholipid and a cationic gemini surfactant. The application of this technique allowed an accurate characterization of the passive dielectric behavior of the plasma membrane by the determination of its specific capacitance and conductance. Changes of these parameters, upon interaction with the liposomes, are related to variations in the structure and or in the transport properties of the membrane. Cells were exposed to both types of liposomes for 1 or 4h. Electrorotation data show a dramatic reduction of the dielectric parameters of the plasma membrane after one hour treatment. After 4h of treatment the effects are still observed only in the case of the cationic liposomes. Surprisingly, these same treatments did not cause a relevant biological damage as assessed by standard viability tests. A detailed discussion to rationalize this phenomenon is presented.

Polarizability of shelled particles of arbitrary shape in lossy media with an application to hematic cells

We show that within the dipole approximation the complex polarizability of shelled particles of arbitrary shape can be written as the volume of the particle times a weighted average of the electric field in the particle, with weights determined by the differences in permittivities between the shells and the external, possibly lossy media. To calculate the electric field we use an adaptive-mesh finite-element method which is very effective in handling the irregular domains, material inhomogeneities, and complex boundary conditions usually found in biophysical applications. After extensive tests with exactly solvable models, we apply the method to four types of hematic cells: platelets, T-lymphocytes, erythrocytes, and stomatocytes. Realistic shapes of erythrocytes and stomatocytes are generated by a parametrization in terms of Jacobi elliptic functions. Our results show, for example, that if the average polarizability is the main concern, a confocal ellipsoid may be used as a model for a normal erythrocyte, but not for a stomatocyte. A comparison with experimental electrorotation data shows quantitatively the effect of an accurate geometry in the derivation of electrical cell parameters from fittings of theoretical models to the experimental data.

AC electrokinetic characterisation and separation of cells with high and low embryogenic potential in suspension cultures of carrot (Daucus carota)

Suspension cultures of Daucus carota L. were established, and cells with embryogenic potential were separated from those without by density gradient centrifugation in Ficoll at different stages in the growth curve. In order to obtain information about the electrical properties of individual cells, electrorotation spectra of single plant cells from different fractions were measured before and after induction of embryogenesis. The data were analysed using models based on Maxwell–Wagner's theories of interfacial polarisation. It was found that the denser cells had a higher embryogenic potential, a darker appearance and a higher internal conductivity (>1 S m−1) than the less dense cells, which had less or no embryogenic potential and a lower internal conductivity (<1 S m−1). Modelling the dielectrophoretic (DEP) response on the basis of the electrorotation data suggested that separation of cells with high embryogenic potential may be achievable in the frequency range 1–10 MHz. Actual dielectrophoretic separation of cells with high embryogenic potential from suspensions in which embryogenesis had not yet been induced was achieved using steric as well as hyperlayer dielectrophoretic Field-Flow Fractionation (DEP-FFF).

Measurements of the dielectric properties of peripheral blood mononuclear cells and trophoblast cells using AC electrokinetic techniques

The separation of trophoblast cells from the maternal circulation could provide a valuable diagnostic tool for prenatal diagnosis of genetic abnormalities. This has been attempted using antibody methods, but due to non-specificity of the antibodies, maternal cell contamination remains a problem. We have investigated the potential of dielectrophoretic separation methods as a means of isolating trophoblast cells from mixed peripheral blood mononuclear cells. To determine the potential of this method the dielectric properties of trophoblast cells and mixed peripheral blood mononuclear cells were measured using dielectrophoretic crossover and single cell electrorotation methods. Both dielectrophoretic crossover data and electrorotation data gave an average specific membrane capacitance of the peripheral blood mononuclear cells of 11.5 mF m −2. Trophoblast cells prepared using three different methods had a higher average specific membrane capacitance in the range 13–18 mF m −2. The differences in capacitance between the cell types could be exploited as the basis of an AC electrokinetic-based system for the separation of trophoblast cells from peripheral blood mononuclear cells.

Dielectric Properties of Human Leukocyte Subpopulations Determined by Electrorotation as a Cell Separation Criterion

The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (± 3.1), 12.6 (± 3.5), 15.3 (± 4.3), and 11.0 (± 3.2) mF/m 2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions.

Electrorotation of liposomes: verification of dielectric multi-shell model for cells

The reliability of multi-shell dielectric models, used to describe the ac electrokinetic behaviour of cells, has been tested by performing electrorotation and dielectrophoretic measurements on unilamellar, oligolamellar, and multilamellar liposomes of diameters ranging from 5 to 24 μm. Fluorescence microscopy, flow cytometry and electron spin resonance were used to characterise the morphology and membranes of the liposomes. The dielectric properties of the various types of liposomes, based on appropriate dielectric shell models, were then analysed using a general purpose, recursive, algorithm. Through simulations, the confidence levels that can be assigned to parameters derived through application of simple shell models are estimated. From this, we confirm that electrorotation data enable accurate determinations to be made of the dielectric properties of the outermost membrane of liposomes, and provide good indications of the level of complexity of the shells and internal compartments. We also demonstrate that, used with sufficient additional information, such as that provided by dielectrophoresis, electrorotation data yields unique solutions for the dielectric parameters of liposome-like particles.

Dynamic Light Scattering from Oriented, Rotating Particles: A Theoretical Study and Comparison to Electrorotation Data

Ces precedentes annees, l'electrorotation a evolue comme une technique de caracterisation des attributs dielectriques d'une cellule unique. Il n'y a que peu d'applications pour les particules colloidales, bien que la methode fournisse de l'information sur la structure electrique de l'interieur de la particule. Cette methode explore la difference entre la polarisabilite des particules et du moyen de suspension en utilisant des champs electriques tournants avec une frequence de 1 kHz a 200 MHz pour induire une rotation individuelle de la particule. Afin de permettre l'interpretation des spectres de l'electrorotation mesure a l'aide des techniques de lumiere diffusee, la fonction d'autocorrelation theorique de la lumiere diffusee par des particules tournant d'une symetrie cylindrique a ete calculee. Nous supposons que toutes les particules ne possedent qu'une cote diffusante et que les axes de rotation sont paralleles. Il en derive une expression appropriee pour la diffusion rotationelle autour de l'angle longitudinal. La diffusion des particules autour de l'angle azimutal est negligee. Le resultat theorique consiste en une fonction d'autocorrelation bien structuree. Les limites theoriques de la grandeur de particule ainsi que la vitesse de rotation, qui permettent la detection de l'electrorotation, ont ete explorees. On peut expliquer les fonctions d'autocorrelation experimentales de l'electrorotation des cellules rouges du sang humain quand on presuppose des parametres raisonnables pour les attributs cellulaires.

Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin

When placed in rotating electric fields red blood cells show a typical electrorotation spectrum with antifield rotation in the lower and cofield rotation in the higher frequency range. Assuming a spherical cell geometry, however, dielectrical parameters were obtained that differ from those measured by independent methods. Dielectrophoresis and, in particular, electrorotation yielded lower membrane capacitance values than expected. Introduction of an ellipsoidal model with an axis ratio of 1:2 allowed a description that proved to be consistent with dielectrophoresis and electrorotation data. For control cells an internal conductivity of 0.535 S/m, a specific membrane capacitance of 0.82 x 10(-2) F/m2, and a specific conductance of 480 S/m2 were obtained. The first characteristic frequency (frequency of fastest antifield rotation) and the related rotation speed can be measured quite quickly by means of a compensation method. Thus it was possible to follow changes of dielectric properties on individual cells after nystatin application. Ionophore-membrane interaction caused cell shrinkage in parallel to a decrease of the first characteristic frequency and rotation speed. Analysis of data revealed a decrease of the internal conductivity that is not only caused by ion loss but also, to a large extent, by a strong increase of hindrance because of shrinkage. Ionophore-induced membrane permeabilities can be calculated from volume decrease as well as from electrorotational data. In no case can these permeabilities count for the high membrane-AC conductivity that is attributed to the band-3 anion exchanging protein. The membrane-AC conductance was found not to be decreased for cells in Donnan equilibrium, which had leaked out almost completely.

Dielectric behavior of the anion-exchange protein of human red blood cells: Theoretical analysis and comparison to electrorotation data

An electrical model for a membrane incorporating anion exchange protein is proposed and the admittance as a function of frequency is calculated theoretically. Expressions for the specific capacitance and conductance as functions of frequency are given. Both depend on the membrane potential, ion binding constants, and velocity of membrane transport. The theoretical predictions thus obtained were compared to the properties of red cell band-3 protein determined from electrorotation measurements. Measurements were made on control cells and on cells treated with DIDS and diamide. Both inhibitors shift the first characteristic frequency towards lower values, and both increase the electrorotation speed. This behavior is qualitatively consistent with the theoretical predictions. A quantitative comparison is difficult, since dispersion was predicted in a range of frequency where electrorotation measurements are insensitive. The membrane specific conductance and capacitance were estimated from the position of the peak of the control. A reasonable agreement with the theoretical estimates was observed. It is demonstrated that more informtion is available from admittance measurements than is available from flux measurements.

Dielectric behavior of the anion-exchange protein of human red blood cells: Theoretical analysis and comparison to electrorotation data

An electrical model for a membrane incorporating anion exchange protein is proposed and the admittance as a function of frequency is calculated theoretically. Expression for the specific capacitance and conductance as functions of frequency are given. Both depend on the membrane potential, ion binding constants, and velocity of membrane transport. The theoretical predictions thus obtained were compared to the properties of red cell band-3 protein determined from electrorotation measurements. Measurements were made on control cells and on cells treated with DIDS and diamide. Both inhibitors shift the first characteristic frequency towards lower values, and both increase the electrorotation speed. This behavior is qualitatively consistent with the theoretical predictions. A quantitative comparison is difficult, since dispersion was predicted in a range of frequency where electrorotation measurements are insensitive. The membrane specific conductance and capacitance were estimated from the position of the peak of the control. A reasonable agreement with the theoretical estimates was observed. It is demonstrated that more information is available from admittance measurements than is available from flux measurements.