Electrorotation of single yeast cells at frequencies between 100 Hz and 1.6 GHz.

The determination of complete electrorotation spectra of living cells has been made possible by the development of a quadrature generator and an electrode assembly that span the frequency range between 100 Hz and 1.6 GHz. Multiple spectra of single cells of the yeast Saccharomyces cerevisiae have been measured at different medium conductivities ranging from 0.7 to 550 microS cm-1. A spherical four-shell model was applied that simulated the experimental data well and disclosed the four-layer structure of the cell envelope attributed to the plasma membrane, the periplasmic space, and a thick inner and a thin outer wall region. Below 10 kHz an additional rotation effect was found, which changed its direction depending on the ionic strength of the medium. This is supposed to be connected with properties of the cell surface and its close vicinity. From the four-shell simulation the following physical properties of cell compartments could be derived: specific capacitance of plasma membrane (0.76 microF cm-2), periplasmic space (0.5 microF cm-2), and outer wall region (0.1 microF cm-2). The conductivity of cytoplasm, plasma membrane, and inner wall region were found to vary with medium ionic strength from 9 to 12 mS cm-1, 5.8 nS cm-1 to approximately 50 nS cm-1, and 6 microS cm-1 to 240 microS cm-1, respectively.     

Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. II. Effects of osmotic perturbation

The effects of osmotic perturbation on the dielectric behavior of cultured rat basophilic leukemia (RBL-1) cells were examined. Cells exposed to osmolalities (pi) of 145-650 mosmolal showed dielectric dispersions of the following characteristics: Permittivity increment delta epsilon(= epsilon l - epsilon h where epsilon l and epsilon h refer to the low- and high-frequency limit values) for a fixed volume concentration increased with pi; gross permittivity behavior was apparently of a typical Cole-Cole type; however, frequency dependence of conductivity was undulant and could be simulated by a superposition of two separate Cole-Cole type dispersions; separation of these subdispersions along the frequency axis was an increasing function of pi, and so was conductivity increment in the high-frequency region. As examined by light microscopy, the cells were spherical in spite of imposed anisotonic stresses and behaved as osmometers at 200-410 mosmolal. When normalized by dividing by number (not volume) concentration, delta epsilon remained relatively constant irrespective of pi. Apparent membrane capacities (Cm), analyzed by applying a single-shell model, increased systematically from a hypotonic value of approx. 1 microF/cm2 up to 5 microF/cm2 at 650 mosmolal. This increase was interpreted as due to increased cellular 'surface/volume' ratios that were confirmed by scanning electron microscopy. Cole-Cole's beta parameter, which culminated around 0.9 for isotonic cells and declined to approx. 0.8 for anisotonic cells, did not parallel the broadening of cell volume distribution but appeared to reflect changes in the intracellular conductivity caused by the anisotonic challenge. The results indicate that the dispersion method can probe changes in surface morphology as well as subcellular organelles' constitution of living cells.     

Dielectric behavior of the frog lens in the 100 Hz to 500 MHz range. Simulation with an allocated ellipsoidal-shells model.

In an attempt to correlate the passive electrical properties of the lens tissue with its structure, we measured ac admittances for isolated frog lenses, lens nuclei, and homogenate of cortical fiber cells, over the frequency range 10(2)-5.10(8) Hz. The whole lenses molded into discoid shape show a characteristic "two-step" dielectric dispersion with a huge permittivity increment of the order of 10(5) at 1 kHz. Of the two subdispersions disclosed, dispersion 1 has a permittivity increment (delta epsilon) of 2.10(5) with a characteristic frequency (fc) of 2 kHz, and dispersion 2 has a delta epsilon of 400 with an fc of 2 MHz. In terms of loss tangent, these dispersions are more clearly located as two separate peaks. Data are analyzed using an allocated ellipsoidal-shells model which has been developed by taking into account fiber orientation inside the lens tissue. Dispersion 1 is assigned to the equatorial cortex, where fiber cells run parallel to the applied electric field, and dispersion 2 to the nucleus with a complex fiber arrangement and also to the polar cortex, in which the fiber alignment is predominantly perpendicular. In addition, the model analysis reveals that, in the frog lens, the nucleus occupies approximately 30% in volume and that relative permittivity and conductivity for the cell interior are, respectively, 45 and 3 mS/cm for the cortical cells, and 28 and 0.3 mS/cm for the nuclear cells.     

Dielectric properties of E. coli cell as simulated by the three-shell spheroidal model

Dielectric properties of E. coli cell have been re-studied by means of the three-shell spheroidal model, where the three shells correspond to the outer membrane, the periplasmic space and the inner membrane, respectively. With the model, a curve-fitting procedure has been developed to analyze the dielectric spectra. Although E. coli cell has been studied before, its special morphological structure was taken into account more comprehensively than any previous model in the present work. Dielectric properties of various cell components have been estimated from the observed dielectric spectra, especially the permittivity of the outer membrane, which was evaluated quantitatively for the first time. The values of epsilon(om) were 12 for kappa(om) of 0 to 10(-4) S/m and 34 for kappa(om) of 10(-3) S/m. The specific capacitance of the inner membrane was 0.6-0.70 microF/cm(2). The relative permittivity and the conductivity of the cytoplasm were about 100 and 0.22 S/m, respectively, and the conductivity of the periplasmic space was 2.2-3.2 S/m.     

Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. I. Dielectric behavior of cell suspensions in 0.01-500 MHz and its simulation with a single-shell model

Frequency dependence of relative permittivity (dielectric constant) and conductivity, or the 'dielectric dispersion', of cultured cells (RBL-1 line) in suspension was measured using a fast impedance analyzer system capable of scanning 92 frequency points over a 10 kHz-500 MHz range within 80 s. Examination of the resulting dispersion curves of an improved reliability revealed that the dispersions consisted of at least two separate components. The low-frequency component (dispersion 1) had a permittivity increment (delta epsilon) of 10(3)-10(4) and a characteristic frequency (fc) at several hundred kHz; for the high-frequency component (dispersion 2), delta epsilon was smaller by a factor of 10(2) and fc = 10-30 MHz. Increments delta epsilon for both components increased with the volume fraction of cell suspension, while fc did not change appreciably as long as the conductivity of suspending medium was fixed. By fitting a model for shelled spheres (the 'single-shell' model) to the data of dispersion 1, the dielectric capacity of the plasma membrane phase (Cm) was estimated to be approx. 1.4 microF/cm2 for the cells in an isotonic medium. However, simulation by this particular shell model failed to reproduce the entire dispersion profile leaving a sizable discrepancy between theory and experiment especially at frequencies above 1 MHz where dispersion 2 took place. This discrepancy could not be filled up even by taking into consideration either the effect of cell size distribution actually determined or that of possible heterogeneity in the intracellular conductivity. The present data strongly indicate the need for a more penetrating model that effectively accounts for the behavior of dispersion 2.     

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.     

Electrorotation of Erythrocytes Treated with Dipicrylamine: Mobile Charges within the Membrane Show their ``Signature' in Rotational Spectra

In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2–5 mS m⁻¹). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values. Received: 14 February 1996/Revised: 29 May 1996     

Differences in membrane properties between unfertilised and fertilised single rabbit oocytes demonstrated by electro-rotation. Comparison with cells from early embryos

The apparent membrane capacity of tubular rabbit oocytes increases from 1.7-2.0 microF/cm2 before fertilisation to 3.7-4.0 microF/cm2 after fertilisation. The membrane conductivity measured on single cells was also increased by fertilisation from less than 1 mS/cm2 to 14 mS/cm2. Cells obtained from 2-, 4- or 8-cell embryos exhibited intermediate values of membrane capacity (2.3-2.8 microF/cm2) and conductivity (5-22 mS/cm2). The values quoted are those effective between 1 and 10 kHz, the frequency of the rotating field used. The large apparent capacities are probably due to the presence of structures such as microvilli which cause the actual membrane area to exceed the smooth sphere area. It must be assumed that these structures change in form or number on fertilisation, and that they persist in embryos, at least up to the 8-cell stage. No difference was apparent between cells fertilised in vitro or in vivo. Comparison of the above zona-free data with measurements on zona-complete oocytes indicate how fertilised and unfertilised rabbit eggs may be distinguished from one another, even in the presence of the zona pellucida.     

Dielectric properties of yeast cells as determined by electrorotation.

Electrorotational spectra of yeast cells, Saccharomyces cerevisiae strain R XII, were measured over a frequency range of nearly 7 decades. The physical properties of distinct cell parts were simultaneously determined for individual cells by comparison with an electrical two-shell model: The conductivity of the cytoplasm, cell wall and cytoplasmic membrane of living cells were found to be 5.5 mS/cm, 0.1 to more than 0.5 mS/cm and less than 0.25 nS/cm to 4.5 microS/cm, respectively. The conductivity of the cytoplasmic membrane was dependent on the conductivity of the medium. Membrane behaviour is interpreted as an opening of membrane channels when the environment becomes more physiological. The specific membrane capacitance was determined to be 1.1 microF/cm2 and the thickness of the cell wall was calculated as 0.11 micron. Heat treated cells showed an increased membrane conductivity of more than 0.1 microS/cm (at 25 microS/cm medium conductivity) and a drop in cytoplasmic conductivity to between 0.1 and 0.8 mS/cm, depending on the length of time the cells were suspended in low conductivity water (25 microS/cm), indicating a perforation of the membrane. A slightly decreased spinning speed scaling factor for dead cells suggests a modification to the cellular surface, while the principal structure of the cell wall appears to be uneffected. It can be demonstrated by these observations, that cellular electrorotation permits the simultaneous investigation of the different cellular compartments of individual cells in vivo under various environmental conditions.     

Electrorotation studies of baby hamster kidney fibroblasts infected with herpes simplex virus type 1

The dielectric properties of baby hamster kidney fibroblast (BHK(C-13)) cells have been measured using electrorotation before and after infection with herpes simplex virus type 1 (HSV-1). The dielectric properties and morphology of the cells were investigated as a function of time after infection. The mean specific capacitance of the uninfected cells was 2.0 microF/cm2, reducing to a value of 1. 5 microF/cm2 at 12 h after infection. This change was interpreted as arising from changes in the cell membrane morphology coupled with alterations in the composition of the cell membrane as infection progressed. The measured changes in the cell capacitance were correlated with alterations in cellular morphology determined from scanning electron microscope (SEM) images. Between 9 and 12 h after infection the internal permittivity of the cell exhibited a rapid change, reducing in value from 75epsilono to 58epsilono, which can be correlated with the generation of large numbers of Golgi-derived membrane vesicles and enveloped viral capsids. The data are discussed in relation to the known life cycle of HSV-1 and indicate that electrorotation can be used to observe dynamic changes in both the dielectric and morphological properties of virus-infected cells. Calculations of the dielectrophoretic spectrum of uninfected and infected cells have been performed, and the results show that cells in the two states could be separated using appropriate frequencies and electrode arrays.     

Dielectric cytometry with three-dimensional cellular modeling

We have developed what we believe is an efficient method to determine the electric parameters (the specific membrane capacitance C(m) and the cytoplasm conductivity kappa(i)) of cells from their dielectric dispersion. First, a limited number of dispersion curves are numerically calculated for a three-dimensional cell model by changing C(m) and kappa(i), and their amplitudes Deltaepsilon and relaxation times tau are determined by assuming a Cole-Cole function. Second, regression formulas are obtained from the values of Deltaepsilon and tau and then used for the determination of C(m) and kappa(i) from the experimental Deltaepsilon and tau. This method was applied to the dielectric dispersion measured for rabbit erythrocytes (discocytes and echinocytes) and human erythrocytes (normocytes), and provided reasonable C(m) and kappa(i) of the erythrocytes and excellent agreement between the theoretical and experimental dispersion curves.     

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.     

Dielectric properties of alginate beads and bound water relaxation studied by electrorotation

The electrical and dielectric properties of Ba²⁺ and Ca²⁺ cross‐linked alginate hydrogel beads were studied by means of single‐particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well‐resolved co‐ and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 227–237, 1999     

Changes in myoblast membrane electrical properties during cell-cell adhesion and fusion in vitro

Characteristic of the process of myogenesis are the changes in the composition and organization of the cell membrane. While poorly understood, these changes have biochemical and biophysical relevance. Recently, changes in molecular order of the myoblast membrane which accompany differentiation in vitro have been observed (Santini, M.T., Indovina, P.L. and Hausman, R.E. (1987) Biochim. Biophys. Acta 896, 19–25). To further investigate these cell fusion processes we have examined additional physical parameters: conductivity and permittivity of the myoblast membrane during differentiation which reflect the molecular arrangement of the membrane. The determination of these parameters is possible because in the radio frequency range suspensions of cells in an electrolyte buffer show a characteristic conductivity dispersion due to the interfacial polarization. An analysis of our experimental data based on a ‘single-shell’ model showed that conductivity and permittivity of the membrane of pre- and post-fusion myoblasts varied significantly and abruptly. The conductivity of the cell interior (cytosol) remained constant. We discuss the significance of the observed changes in these membrane parameters for myogenesis.     

有限元法计算各向异性介质球头模型电位分布

在脑电正问题研究中,脑神经元所产生的电活动可用电流偶极子来模拟.本文提出把大脑看作各向异性介质球,即同时考虑电容效应、电导效应对脑内电流偶极子产生的电位的影响,并用有限元法推导出偶极子在各向异性介质球模型中的电位分布计算公式.结果表明介质的电容效应对电位分布是有影响的,反映了大脑活组织电特性,对外来不同频率的信号刺激有不同的响应.同时有限元法对大脑内某一区域内电位分布求解表明,测量较深层组织的电特性变化敏感的特点,可获得更多的测量信息.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

Electro-orientation of ellipsoidal erythrocytes. Theory and experiment.

The frequency-dependent orientation of human and llama erythrocytes suspended in isotonic solutions and subjected to linearly polarized electric fields is examined. Human erythrocytes may be represented as oblate spheroids (3.9:3.9:1.1 microns) with two distinguishable orientations, while the llama cells are approximated as ellipsoids with three distinct axes (4.0:2.0:1.1 microns). Under appropriate experimental conditions, both orientations of the human cells and all three orientations of the llama cells are observed. A theoretical cell model which accounts for the membrane as a thin confocal layer of ideal capacitance is used to predict the orientational spectra. The predicted spectra compare favorably in frequency range and orientational sequence with experimental data. Estimates for cell internal conductivity and permittivity are obtained by adjusting the values of these important parameters to achieve the closet fit of the theoretical curves to the data. By the use of this method, the internal conductivity of llama erythrocytes is estimated to be 0.26 S/m (+/- 20%), while the effective internal dielectric constant and conductivity of Euglena gracilis are estimated to be 120 (+/- 10%) and 0.43 S/m (+/- 20%), respectively.     

Quantitative modeling of viable cell density,cell size,intracellular conductivity,and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Dielectric spectroscopy was used to analyze typical batch and fed‐batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole–Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The β‐dispersion was analyzed using the Cole–Cole distribution parameters Δε (magnitude of the permittivity drop), f_c (critical frequency), and α (Cole–Cole parameter). Furthermore, the dielectric parameters static internal conductivity (σ_i) and membrane capacitance per area (C_m) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010