Evaluation of process-induced dimensional changes in the membrane structure of biological cells using impedance measurement

The impact of high intensity electric field pulses, high hydrostatic pressure, and freezing-thawing on local structural changes of the membrane was determined for potato, sugar beet tissue, and yeast suspensions. On the basis of the electrophysical model of cell systems in biological tissues and suspensions, a method was derived for determining the extent of local damage of cell membranes. The method was characterized by an accurate and rapid on-line determination of frequency-dependent electrical conductivity properties from which information on microscopic events on cellular level may be deduced. Evaluation was based on the measurement of the relative change in the sample's impedance at characteristically low (f(l)) and high (f(h)) frequencies within the beta-dispersion range. For plant and animal cells the characteristic frequencies were f(l) approximately 5 kHz and f(h) > 5 MHz and for yeast cells in the range f(l) approximately 50 kHz and f(h) > 25 MHz. The observed phenomena were complex. The identification of the underlying mechanisms required consideration of the time-dependent nature of the processing effects and stress reactions of the biological systems, which ranged from seconds to several hours. A very low but significantly detectable membrane damage (0.004% of the total area) was found after high hydrostatic pressure treatment of potato tissue at 200 MPa. The membrane rupture in plant tissue cells was higher after freezing and subsequent thawing (0.9% of total area for potato cells and 0.05-0.07% for sugar beet cells determined immediately after thawing), which increased substantially during the next 2 h.     

Dielectric analysis for estimation of oil content in the mycelia of Mortierella alpina.

The dielectric behavior of the filamentous fungi Mortierella alpina SAM2104 and 1S-4, which produce polyunsaturated fatty acid enriched oil in the mycelia, was investigated. During the cultivation carried out in a 10-kL fermentor for 12-15 days, the relative permittivity and conductivity of the broth were measured in the frequency range of 100 kHz to 30 MHz. The dielectric parameters, i.e., the amplitude of dielectric relaxation (Deltaepsilon) and the characteristic frequency (f(c)), were obtained by fitting the Cole-Cole equation to the observed dielectric relaxation, and the conductivity of the medium (kappa(a)) was also measured. The value of Deltaepsilon gradually increased from the second day through the end of cultivation, suggesting that volume fraction of the cell increased with oil accumulation. The conductivity of the cytoplasm (kappa(i)) was calculated from the experimental values of f(c) and kappa(a), using a theoretical equation based on an ellipsoidal cell model. As a result, good correlation between the calculated kappa(i) and the oil content was obtained. These findings indicate that dielectric analysis enables us to estimate the oil content in the mycelia of oleaginous fungi and also provides a useful tool for monitoring cell growth and for controlling the cultivation process.     

Theoretical and experimental determination of SAR patterns for spherical tissue models in a rectangular resonant cavity

Specific absorption rates (SARs) were determined theoretically and experimentally for several spherical models of tissue exposed to electrical fields of TE101 mode in a rectangular cavity of 57.3 MHz resonant frequency. The approximate theoretical SAR can be calculated according to the Mie theory by superposition of four plane waves representing the fields excited in the cavity. The theoretical and thermographically determined SAR patterns in spheres with radii of 5, 7.5, and 10 cm and with conductivities of 0.1, 1, and 10 S/m were compared. For a sphere with radius less than 7.5 cm and conductivity less than 1 S/m, the SAR was quite uniform. When conductivity was increased to 10 S/m, the SAR patterns showed higher absorption in the periphery of the largest sphere (10-cm radius). These characteristics are important in evaluating the scaling technique of exposing a model of a human to very-high-frequency fields to obtain power absorption data in humans exposed to high-frequency or very-low-frequency fields.     

Dielectric analysis of Escherichia coli suspensions in the light of the theory of interfacial polarization.

Dielectric measurements of Escherichia coli suspensions were carried out over a frequency range from 10 kHz to 100 MHz, and marked dielectric dispersions having characteristic frequency of approximately 1 MHz were observed. On the basis of the cell model that a spheroid is covered with two confocal shells, a dielectric theory was developed to determine accurately four electrical parameters for E. coli cells such as the conductivity of the cell wall, the dielectric constant of the cell membrane, and the dielectric constant and the conductivity of the protoplasm. The observed data were analyzed by means of the procedure based on the dielectric theory to yield a set of plausible electrical parameters for the cells. By taking account of the size distribution of the cells and a dielectric relaxation of the protoplasm, the observed dispersion curves were successfully reconstituted by the present theory.     

Alteration of the passive electrical properties of lymphocyte membranes induced by GM1 and GM3 glycolipids.

The electrical conductivity of normal human lymphocyte suspensions has been measured in the frequency range from 10 kHz to 100 MHz, where a well-pronounced conductivity dispersion occurs, caused by the surface polarization at the interface between the cell membrane and the extracellular solution. We have investigated the alteration of the passive electrical properties of the cytoplasmatic cell membrane induced by two different gangliosides (GM1 and GM3) inserted, at various concentrations, into the outer leaflet of membrane double layer. The alterations observed in the dielectric parameters (the membrane conductivity and the membrane permittivity) derived on the basis of a 'double-shell' model, result in an overall increase of the ion permeation across the membrane and an enhanced polarizability of its hydrophilic region for both gangliosides investigated. The relevance of these alterations is discussed.     

Dielectrophoretic spectra of single cells determined by feedback-controlled levitation.

In this paper we have utilized the principle of dielectrophoresis (DEP) to develop an apparatus to stably levitate single biological cells using a digital feedback control scheme. Using this apparatus, the positive DEP spectra of both Canola plant protoplast and ligament fibroblast cells have been measured over a wide range of frequencies (1 kHz to 50 MHz) and suspending medium conductivities (11-800 muS/cm). The experimental data thus obtained have been interpreted in terms of a simple spherical cell model. Furthermore, utilizing such a model, we have shown that various cellular parameters of interest can be readily obtained from the measured DEP levitation spectrum. Specifically, the effective membrane capacitance of single cells has been determined. Values of 0.47 +/- 0.03 muF/cm2 for Canola protoplasts and 1.52 +/- 0.26 muF/cm2 for ligament fibroblasts thus obtained are consistent with those determined by other existing electrical methods.     

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.     

Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures.

High-frequency electric fields can be used to induce deformation of red blood cells. In the temperature domain T = 0 degrees to -15 degrees C (supercooled suspension) and for 25 degrees C this paper examines for human erythrocytes (discocytes, young cell population suspended in a low ionic strength solution with conductivity sigma(25 degrees) = 154 microS/cm) in a sinusoidal electric field (nu = 1 MHz, E0 = 0-18 kV/cm) the following properties and effects as a function of field strength and temperature: 1) viscoelastic response, 2) (shear) deformation (steady-state value obtained from the viscoelastic response time), 3) stability (by experimentally observed breakdown of cell polarization and hemolysis), 4) electrical membrane breakdown and field-induced hemolysis (theoretical calculations for ellipsoidal particles), and 5) mechanical hemolysis. The items 2-4 were also examined for the frequency nu = 100 kHz and for a nonionic solution of very low conductivity (sigma(25 degrees) = 10 microS/cm) to support our interpretations of the results for 1 MHz. Below 0 degrees C with decreasing temperature the viscoelastic response time tau(res)(T) for the cells to reach steady-state deformation values d(infinity,E) increases and the deformation d(infinity,E)(T) decreases strongly. Both effects are especially high for low field strengths. The longest response time of approximately 30 s was obtained for -15 degrees C and small deformations. For 1 MHz the cells can be highly elongated up to 2.3 times their initial diameter a0 for 25 degrees and 0 degrees C, 2.1a0 for -10 degrees C and still 1.95a0 for -15 degrees C. For T > or = 0 degrees C the deformation is limited by hemolysis of the cells, which sets in for E0(lysis)(25 degrees) approximately 8 kV/cm and E0(lysis)(0 degrees) approximately 14 kV/cm. These values are approximately three times higher than the corresponding calculated critical field strengths for electrically induced pore formation. Nevertheless, the observed depolarization and hemolysis of the cells is provoked by electrical membrane breakdown rather than by mechanical forces due to the high deformation. For the nonionic solution, where no electrical breakdown is expected in the whole range for E0, the cells can indeed be deformed to even higher values with a low hemolytic rate. Below 0 degrees C we observe no hemolysis at all, not even for the frequency 100 kHz, where the cells hemolyze at 25 degrees C for the much lower field strength E0(lysis) approximately 2.5 kV/cm. Obviously, pore formation and growth are weak for subzero temperatures.     

Ultrashort electric pulse induced changes in cellular dielectric properties

The interaction of nanosecond duration pulsed electric fields (nsPEFs) with biological cells, and the models describing this behavior, depend critically on the electrical properties of the cells being pulsed. Here, we used time domain dielectric spectroscopy to measure the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after exposure to five 10ns, 150kV/cm electrical pulses. The cytoplasm and nucleoplasm conductivities decreased dramatically following pulsing, corresponding to previously observed rises in cell suspension conductivity. This suggests that electropermeabilization occurred, resulting in ion transport from the cell's interior to the exterior. A delayed decrease in cell membrane conductivity after the nsPEFs possibly suggests long-term ion channel damage or use dependence due to repeated membrane charging and discharging. This data could be used in models describing the phenomena at work.     

Conductivities of pig dermis and subcutaneous fat measured with rectangular pulse electrical current

We examined experimentally the relationship between perpendicular and tangential electrical conductivities, σ, and peak current density J, in pig skin dermis and subcutaneous fat specimens by using a four-electrode measuring system with rectangular pulse electrical current (RPEC). We also investigated the relationship of the conductivity, σ, vs. pulse rate, f. The rates were selected at 8, 32, 64, and 128 pulses per second (pps), and the pulse width was fixed at 140 μs. These values are often used in vivo to enhance cutaneous regeneration with RPEC stimulation. It was found that the conductivities may be approximated to be for the skin dermis and for the subcutaneous fat in the conditions of this experiment. These findings implies that the conductivities of pig skin dermis and subcutaneous fat are anisotropic, i.e., σ_x = σ_y ≠ σ_z. It was also found that the conductivities are independent of current density and pulse rate in the current range from 20 μA/cm² to 120 mA/cm². © 1996 Wiley-Liss, Inc.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

Passive Electrical Properties of Microorganisms: I. Conductivity of Escherichia coli and Micrococcus lysodeikticus

Effective conductivities are reported for the bacteria Escherichia coli and Micrococcus lysodeikticus over a range of environmental conductivity. The apparent conductivities of the organisms can be explained in terms of the properties of the cell wall. At low conductivities of the environment, the conductivity of the cell appears to be dominated by the counterions of the fixed charge of the cell wall. At higher conductivities of the suspending medium, evidence suggests that ions from the environment invade the cell wall causing an increase in the effective conductivity of the cell so that it takes on values roughly proportional to that of the environment. The model points to the usefulness of dielectric techniques in studies of the properties of intact cell walls.     

Electro-rotation of mouse oocytes: single-cell measurements of zona-intact and zona-free cells and of the isolated zona pellucida

Passive electrical properties of oocytes and of zonae pellucidae, and the mechanical coupling between them, can be elucidated by means of rotating-field-induced rotation. In low-conductivity media (25-100 microS/cm) rotation of mouse oocytes (with or without their zonae) requires fields in the 1-100 kHz frequency range. However, an isolated zona shows weak rotation in the opposite direction to that of a cell, and in response to much higher field frequencies (approx. 1 MHz). In zona-intact mouse oocytes, the rotation of cell and zona are not rigidly coupled: thus rotation of the cell can still be induced when the zona is held stationary. However, rotation of freely suspended zona-intact cells is much slower than that of zona-free cells and requires an optimum field frequency that is approximately 1.5 kHz higher. These observations show that the electrical properties of the oocyte that are measured by rotation are altered by the presence of the zona pellucida, even though no such influence has been detected using micro-electrodes. The data are consistent with the zona acting as a porous shell with a conductivity of 40 microS/cm (preliminary estimate made at a single medium conductivity of 26 microS/cm). Measurements on cells from which the zonae had been removed gave values for the membrane capacity and resistivity of 1.2-1.3 microF/cm2 and 400 omega.cm2, respectively. These values may reflect the presence of plasmalemma microvilli. The results strongly suggest that the technique may be useful for studies of cell maturation and for in vitro fertilization, because the cells may be further cultured after measurement.     

Continuous dielectrophoretic separation of cell mixtures.

Use of stream-centered dielectrophoresis (1-4) produced continuous separations on three cell mixtures (1) Chorella vulgaris with Netrium digitus, (2) Ankistrodesmus falcatus with Staurastrum gracile, and (3) Saccharomyces cerevisiae with Netrium digitus. Maximal separations were obtained for these mixtures of live cells at 100 kHz, 600 kHz, and 2.0 MHz, respectively. The technique was restricted to a frequency range of 0.01-32 MHz, and to suspensions of low conductivity in which microorganisms such as these algae and yeast are tolerant. Extension, however, to cellular organisms requiring higher osmolarity is readily feasible through the use of nonionic solutes such as sucrose, mannose, glycine, etc.     

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.     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Electric Conductivity and Internal Osmolality of Intact Bacterial Cells

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.     

Experimental investigation on neural cell survival after dielectrophoretic trapping

Negative dielectrophoretic forces can effectively be used to trap cortical rat neurons. The creation of dielectrophoretic forces requires electric fields of high non-uniformity. High electric field strengths, however, can cause excessive membrane potentials by which cells may unrecoverably be changed or it may lead to cell death. In a previous study it was found that cells trapped at 3 Vtt/14 MHz did not change morphologically as compared to cells that were not exposed to the electric field. This study investigates the viability of fetal cortical rat neurons after being trapped by negative dielectrophoretic forces at frequencies up to 1 MHz. A planar quadrupole micro-electrode structure was used for the creation of a non-uniform electric field. The sinusoidal input signal was varied in amplitude (3 and 5 Vtt) and frequency (10 kHz-1 MHz). The results presented in this paper show that the viability of dielectrophoretically trapped postnatal cortical rat cells was greatly frequency dependent. To preserve viability frequencies above 100 kHz (at 3 Vtt) or 1 MHz (5 Vtt) must be used.     

Dielectric properties of mouse MCA1 fibrosarcoma at different stages of development

The in vitro bulk electrical properties of MCA1 fibrosarcoma induced in C57B1/6 male mice were measured at frequencies of 10 kHz to 100 MHz, with some tissues measured to 2 GHz. The properties of normal surrounding tissue also were measured. A comparison of the dielectric properties between three different stages of tumor development as well as that between various locations within the tumor is reported. Statistical analysis of the experimental results revealed statistically significant differences in the dielectric constant and conductivity of the tumor tissues at various stages of development as measured at frequencies below 10 MHz. Conductivity values at different stages also differ at a frequency of 100 MHz. At other frequencies these differences were found to be statistically insignificant.     

Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel.

When illuminated, oriented purple membranes isolated from Halobacterium halobium give a photoelectric effect. The frequency response of a photocurrent measuring system for purple membranes oriented and immobilized in a polyacrylamide gel is analyzed from DC to 100 MHz. The waveform of the photocurrent can depend on both the sample conditions (including bathing solution) and the measuring system (electrode and ammeter) at both the low and high frequency ends. In the DC-1 kHz range (millisecond signals), the apparent lifetime of the photocurrent component is distorted if the electrode is not platinized and if the conductivity of the bathing solution is not low. In the 1 kHz to 1 MHz range (microsecond signals), the frequency response is flat under most conditions. In the MHz range (nanosecond signals), the apparent lifetime of the photocurrent component will be distorted if the conductivity of the bathing solution is not high and if the input impedance of the ammeter is not low and constant throughout the frequency range. With our optimized apparatus, we could measure the photocurrent components from oriented purple membrane with lifetimes from 70 ms to 32 ns without distortion by the measuring system.