Electrorotation of axolotl embryos

The frequency dependent dielectric properties of individual axolotl embryos (Ambystoma mexicanum) were investigated experimentally utilizing the technique of electrorotation. Individual axolotl embryos, immersed in low conductivity media, were subjected to a known frequency and fixed amplitude rotating AC electric field and the ensuing rotational motion of the embryo was monitored using a conventional optical microscope. None of the embryos in the pregastrulation or neurulation stages of development exhibited any rotational motion over the field frequency range (10 Hz-5 MHz). Over the same frequency range, the embryos in the gastrulation stage of development exhibited both co-field and counterfield rotation over different ranges of the applied field frequency. Typically, the counterfield rotation exhibited a peak in the rotation spectrum at similar 1 KHz while the co-field peak was located at similar 1-2 MHz. The rotational spectral data was analyzed using a multishelled spherical embryo model to determine the electrical character of embryos during the early development stages (Stages 5-16; i.e., 16 cell through open neural plate stages).     

Electrorotation of colloidal particles and cells depends on surface charge.

The importance of surface conductivity to the frequency-dependent polarizability and the rotation of particles in circular electric fields (electrorotation) is emphasized by various theoretical and experimental investigations. Although surface conductivity seems to be naturally related to the ionic double layer, there is rare experimental evidence of a direct relationship. To highlight the role of surface charges in electrorotation, an apparatus was developed with a symmetrical three-electrode arrangement for field frequencies between 25 Hz and 80 MHz. The three-dimensional electrostatic field distribution between the electrodes was evaluated numerically. With this device, rotating, gradient, and homogeneous electric fields of defined precision and homogeneity could be applied to slightly conducting suspensions. Surface properties of monodisperse latex particles (O 9.67 microm), carrying weak acid groups, were characterized by suspension conductometric titration. This procedure determined the amount of carboxyl groups and showed that strong acid groups were missing on the surface of these particles. To obtain the electrophoretic mobility, the spheres were separated by free-flow electrophoresis, and the zeta-potential was calculated from these data. Single-particle rotation experiments on fractions of specified electrophoretic mobility were carried out at frequencies between 25 Hz and 20 MHz. By analyzing the pH dependence of the rotation velocity, it could be shown that the rotation rate is determined by surface charges, both at the peak in rotation rate near the Maxwell-Wagner frequency (MWF) and at low frequencies. The inversion of the rotation direction at the MWF peak for vanishing surface charges was demonstrated. An analytical model for the double layer and dissociation on a charged surface was developed that is valid for low and high zeta-potentials. This model could provide convincing evidence of the linear dependence of the MWF rotation velocity on surface charge.     

Cellular spin resonance in rotating electric fields

Cells and certain other electrically polarizable objects can be seen to spin when in a rotating electric field. When a rotating field (from four Pt electrodes) is applied over a frequency range of 500 to 75,000 Hz, living cells exhibit two or three response peaks, whereas dead cells exhibit only one response peak. Yeast (Saccharomyces cerevisiae) exhibit two peaks. The nature of these cellular spin resonances is under active study.     

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.     

中频交变磁场对大鼠F98胶质瘤细胞的体外生物效应

目的:观察中频交变磁场对大鼠F98胶质瘤细胞增殖能力的影响,初步探讨中频交变磁场抑制肿瘤细胞增殖的生物学机制。方法:体外培养大鼠F98胶质瘤细胞,使其暴露于场强为5 m T的恒定磁场中,并分别加载频率为0、50、100、200、300、400、500、600、700 KHz的不同磁场。磁场暴露24至72小时后,采用MTT法检测细胞增殖、计算抑制率并筛选出抑制胶质瘤细胞生长的最佳磁场频率,应用染色技术和流式细胞术对细胞形态、细胞凋亡、细胞周期进行验证。结果:MTT检测数据表明,100、200、300KHz组细胞增殖抑制率明显高于其他组(P0.05),并且200 KHz组细胞增殖抑制最为显著,抑制率为17%;HE染色显示磁场干预后细胞生长欠佳;Hoechst染色显示细胞核染色质浓缩聚集、核碎裂、核边集,表现出细胞凋亡的典型变化。流式细胞术显示:200 KHz组细胞凋亡率高于对照组(P0.05);各组细胞周期无显著差异。结论:200 KHz的中频交变磁场有抑制大鼠F98胶质瘤细胞增殖的作用,促使肿瘤细胞凋亡,但其具体机制仍待继续研究。     

Low frequency electrorotation of fixed red blood cells.

Electrorotation of fixed red blood cells has been investigated in the frequency range between 16 Hz and 30 MHz. The rotation was studied as a function of electrolyte conductivity and surface charge density. Between 16 Hz and 1 kHz, fixed red blood cells undergo cofield rotation. The maximum of cofield rotation occurs between 30 and 70 Hz. The position of the maximum depends weakly on the bulk electrolyte conductivity and surface charge density. Below 3.5 mS/m, the cofield rotation peak is broadened and shifted to higher frequencies accompanied by a decrease of the rotation speed. Surface charge reduction leads to a decrease of the rotation speed in the low frequency range. These observations are consistent with the recently developed electroosmotic theory of low frequency electrorotation.     

Study of the torque of the bacterial flagellar motor using a rotating electric field.

Bacterial flagella are driven by a rotary motor that is energized by an electrochemical ion gradient across the cell membrane. In this study the torque generated by the flagellar motor was measured in tethered cells of a smooth-swimming Escherichia coli strain by using rotating electric fields to determine the relationship between the torque and speed over a wide range. By measuring the electric current applied to the sample cell and combining the data obtained at different viscosities, the torque of the flagellar motor was estimated up to 55 Hz, and also at negative rotation rates. By this method we have found that the torque of the flagellar motor linearly decreases with rotation rate from negative through positive rate of rotation. In addition, the dependence of torque upon temperature was also investigated. We showed that torque at the high speeds encountered in swimming cells had a much steeper dependence on temperature that at the low speeds encountered in tethered cells. From these results, the activation energy of the proton transfer reaction in the torque-generating unit was calculated to be about 7.0 x 10(-20) J.     

Towards high-resolution 1H-NMR in biological membranes: magic angle spinning of bicelles

Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment.     

Towards high-resolution H-NMR in biological membranes: magic angle spinning of bicelles

Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment.     

Effects of weak microwave fields amplitude modulated at ELF on EEG of symmetric brain areas in rats

Averaged electroencephalogram (EEG) frequency spectra were studied in eight unanesthetized and unmyorelaxed adult male rats with chronically implanted carbon electrodes in symmetrical somesthetic areas when a weak (0.1–0.2 mW/cm) microwave (MW, 945 MHz) field, amplitude-modulated at extremely low frequency (ELF) (4 Hz), was applied. Intermittent (1 min “On,” 1 min “Off”) field exposure (10-min duration) was used. Hemispheric asymmetry in frequency spectra (averaged data for 10 or 1 min) of an ongoing EEG was characterized by a power decrease in the 1.5–3 Hz range on the left hemisphere and by a power decrease in the 10–14 and 20–30 Hz ranges on the right hemisphere. No differences between control and exposure experiments were shown under these routines of data averaging. Significant elevations of EEG asymmetry in 10–14 Hz range were observed during the first 20 s after four from five onsets of the MW field, when averaged spectra were obtained for every 10 s. Under neither control nor pre- and postexposure conditions was this effect observed. These results are discussed with respect to interaction of MW fields with the EEG generators. Bioelectromagnetics 18:293–298, 1997. © 1997 Wiley-Liss, Inc.     

The sensitivity of chlamydomonas photoreceptor is optimized for the frequency of cell body rotation.

For phototactic migration, Chlamydomonas scans the surrounding light environment by rotating the cell body with an eyespot located on the equator. The intensity of the light signal received by the eyespot should therefore change cyclically at the frequency of the cell body rotation. In this study, the response of the photoreceptor to cyclically changing light stimuli was analyzed using immotile mutant cells. To simulate the light intensity change perceived by a rotating cell, light stimuli were applied that consisted of a light phase with the intensity changing similar to a half cycle of a sine wave and a dark phase of the same length. The fluence rate at the peak of the sine wave was of the order of 10(19) photons m(-2) s(-1), i.e. high intensity at which phototaxis is saturated. A photoreceptor current (PRC) was produced at the onset of each light phase. Interestingly, its amplitude varied depending on the frequency and was largest at 1-5 Hz, a frequency range similar to the frequency of cell body rotation. Experiments on the kinetics of the PRC indicate that the response was small at low frequency because of the inactivation of the PRC before full activation. In contrast, at high frequency the PRC was suppressed by adaptation to the repetitive stimuli. These characteristic kinetics of the PRC should be important for Chlamydomonas cells to extract information from the signals generated by the cell body rotation.     

Rotation of cells in an alternating electric field theory and experimental proof

Summary Protoplasts ofAvena sativa rotate in an alternating electric field provided that at least two cells are located close to each other. An optimum frequency range (20 to 30 kHz) exists where rotation of all cells exposed to the field is observed. Below and above this frequency range, rotation of some cells is only occasionally observed. The angular velocity of rotation depends on the square of the electric field strength. At field strengths above the value leading to electrical breakdown of the cell membrane, rotation is no longer observed due to deterioration of the cells. The absolute value of the angular velocity of rotation at a given field strength depends on the arrangement of the cells in the electric field. A maximum value is obtained if the angle between the field direction and the line connecting the two cells is 45^o. With increasing distance between the two cells the rotation speed decreases. Furthermore, if two cells of different radii are positioned close to each other the cell with the smaller radius will rotate with a higher speed than the larger one. Rotation of cells in an alternating electric field is described theoretically by interaction between induced dipoles is adjacent cells. The optimum frequency range for rotation is related to the relaxation of the polarization process in the cell. The quadratic dependence of the angular velocity of rotation on the field strength results from the fact that the torque is the product of the external field and the induced dipole moment which is itself proportional to the external field. The theoretical and experimental results may be relevant for cyclosis (rotational streaming of cytoplasm) in living cells.     

Calcium homeostasis of isolated heart muscle cells exposed to pulsed high-frequency electromagnetic fields

The intracellular calcium concentration ([Ca²⁺]_i) of isolated ventricular cardiac myocytes of the guinea pig was measured during the application of pulsed high-frequency electromagnetic fields. The high-frequency fields were applied in a transverse electromagnetic cell designed to allow microscopic observation of the myocytes during the presence of the high-frequency fields. The [Ca²⁺]_i was measured as fura-2 fluorescence by means of digital image analysis. Both the carrier frequency and the square-wave pulse-modulation pattern were varied during the experiments (carrier frequencies: 900, 1,300, and 1,800 MHz pulse modulated at 217 Hz with 14% duty cycle; pulsation pattern at 900 MHz: continuous wave, 16 Hz, and 50 Hz modulation with 50% duty cycle and 30 kHz modulation with 80% duty cycle). The mean specific absorption rate (SAR) values in the solution were within one order of magnitude of 1 mW/kg. They varied depending on the applied carrier frequency and pulse pattern. The experiments were designed in three phases: 500 s of sham exposure, followed by 500 s of field exposure, then chemical stimulation without field. The chemical stimulation (K⁺-depolarization) indicated the viability of the cells. The K⁺ depolarization yielded a significant increase in [Ca²⁺]_i. Significant differences between sham exposure and high-frequency field exposure were not found except when a very small but statistically significant difference was detected in the case of 900 MHz/50 Hz. However, this small difference was not regarded as a relevant effect of the exposure. © 1996 Wiley-Liss, Inc.     

Torque generated by the flagellar motor of Escherichia coli.

Cells of the bacterium Escherichia coli were tethered and spun in a high-frequency rotating electric field at a series of discrete field strengths. This was done first at low field strengths, then at field strengths generating speeds high enough to disrupt motor function, and finally at low field strengths. Comparison of the initial and final speed versus applied-torque plots yielded relative motor torque. For backward rotation, motor torque rose steeply at speeds close to zero, peaking, on average, at about 2.2 times the stall torque. For forward rotation, motor torque remained approximately constant up to speeds of about 60% of the zero-torque speed. Then the torque dropped linearly with speed, crossed zero, and reached a minimum, on average, at about -1.7 times the stall torque. The zero-torque speed increased with temperature (about 90 Hz at 11 degrees C, 140 Hz at 16 degrees C, and 290 Hz at 23 degrees C), while other parameters remained approximately constant. Sometimes the motor slipped at either extreme (delivered constant torque over a range of speeds), but eventually it broke. Similar results were obtained whether motors broke catastrophically (suddenly and completely) or progressively or were de-energized by brief treatment with an uncoupler. These results are consistent with a tightly coupled ratchet mechanism, provided that elastic deformation of force-generating elements is limited by a stop and that mechanical components yield at high applied torques.     

Microfabricated Inserts for Magic Angle Coil Spinning (MACS) Wireless NMR Spectroscopy

This article describes the development and testing of the first automatically microfabricated probes to be used in conjunction with the magic angle coil spinning (MACS) NMR technique. NMR spectroscopy is a versatile technique for a large range of applications, but its intrinsically low sensitivity poses significant difficulties in analyzing mass- and volume-limited samples. The combination of microfabrication technology and MACS addresses several well-known NMR issues in a concerted manner for the first time: (i) reproducible wafer-scale fabrication of the first-in-kind on-chip LC microresonator for inductive coupling of the NMR signal and reliable exploitation of MACS capabilities; (ii) improving the sensitivity and the spectral resolution by simultaneous spinning the detection microcoil together with the sample at the "magic angle" of 54.74° with respect to the direction of the magnetic field (magic angle spinning - MAS), accompanied by the wireless signal transmission between the microcoil and the primary circuit of the NMR spectrometer; (iii) given the high spinning rates (tens of kHz) involved in the MAS methodology, the microfabricated inserts exhibit a clear kinematic advantage over their previously demonstrated counterparts due to the inherent capability to produce small radius cylindrical geometries, thus tremendously reducing the mechanical stress and tearing forces on the sample. In order to demonstrate the versatility of the microfabrication technology, we have designed MACS probes for various Larmor frequencies (194, 500 and 700 MHz) testing several samples such as water, Drosophila pupae, adamantane solid and LiCl at different magic angle spinning speeds.     

Change of cholinesterase relative activity under modulated ultra high frequency electromagnetic radiation in experiments in vitro

Changes in the activity of enzyme cholinesterase (ChE) have been experimentally investigated under the influence of amplitude-modulated super-high-frequency electromagnetic radiation (carrier frequency of 2.375 MHz; power flux density of 8 mW/cm2, 20 mW/cm2 and 50 mW/cm2; modulation frequency range 10 to 210 Hz; exposure time 5 min). The appearance of peaks of the cholinesterase increased relative activity, as well as the changes in the direction and intensity of the reaction associated with the modulation frequency and power flux are observed at equal power flux densities and exposure times.     

Applications of a new optical technique for measuring the dielectrophoretic behaviour of micro-organisms

It is shown that the dielectrophoretic behaviour (motion in non-uniform a.c. electric fields) of micro-organisms can conveniently and reproducibly be measured by monitoring the decrease in optical absorbance of a cell suspension as the cells are collected at a micro-electrode array. The dielectrophoretic behaviour, as a function of the frequency of the applied electric field and conductivity of the supporting solution, can be determined more quantitatively and rapidly than by methods so far described in the literature. Results are presented for Micrococcus lysodeikticus, Bacillus subtilis and Escherichia coli for the frequency range 20 Hz to 4 MHz and theoretical considerations are presented for the effect of solution conductivity. A value of 0.2 S/m has been derived for the effective conductivity of M. lysodeikticus.     

Cellular spin resonance of aging yeast and mouse sarcoma cells

The way in which individual cells may be made to spin by the application of alternating electric fields is examined. The spinning of a given living cell is observed to respond rather sharply and in a resonant manner at several frequencies, hence the term "cellular spin resonance" (CSR). The frequencies of the applied field can be orders of magnitude higher than the actual spin rate. The CSR varies with the conductivity of the medium, with the square of the applied field, with the cell type and with the phase of the yeast cell life cycle. Living cells respond readily and individually are sharply resonant. Dead cells show little such response.From the behavior of the CRS in sinusoidal AC, as compared to pulsed DC, it appears likely that one cause of CSR, at least that in high frequency electric fields, is the presence of natural rf oscillations arising from the cells, and modulated by their high polarizability.     

No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.     

Real-time imaging of fluorescent flagellar filaments

Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.