Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes.

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.

Trapping, deformation, and rotation of giant unilamellar vesicles in octode dielectrophoretic field cages.

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy.

Microdevices for separation, accumulation, and analysis of biological micro- and nanoparticles.

Microfabrication and performance of a novel microsystem for separation, accumulation and analysis of biological micro- and nanoparticles is reported. Versatile chip functions based on dielectrophoresis and microfluidics were integrated to isolate particles from complex sample solutions such as serum. A bead-based assay for virus detection is proposed. Separation of micro- and sub-mum beads employing dielectrophoretic deflector and bandpass structures is demonstrated. Individual antibody coated beads with hepatitis A virus bound to their surface were trapped by negative dielectrophoresis in a field cage and analysed by fluorescence microscopy.

Combined laser tweezers and dielectric field cage for the analysis of receptor-ligand interactions on single cells.

A new technique based on the combination of optical and chip-based dielectrophoretical trapping was developed and employed to manipulate cells and beads with micrometer precision. The beads were trapped with optical tweezers (OT) and brought into contact for defined times with cells held in the dielectrophoretic field cage (DFC). The well-defined ligand-receptor system biotin-streptavidin was used to study the multiple interaction between biotinylated live cells and streptavidin-coated beads. The biotin density on the cell surface was varied down to a few single bonds (3 +/- 2 bonds/microm2) to control the valency of the binding. The quantitative relationship between the contact area, ligand density and its diffusion rate in the outer membrane of the cell could be demonstrated. The increase of the strength of the cell-bead adhesion was strictly dependent on the increase of individual bond numbers in the contact area. This is in part due to accumulation of ligands (D approxiamtely (0.5 +/- 0.1) 10(-8) cm2/s) in the contact area as seen by confocal laser scanning microscopy. Individual receptor-ligand rupture forces were evaluated and are compatible with values obtained by biomembrane force probe techniques. To summarize, the combination leads to a new powerful microsystem for cell handling and pN-force measurements on the single-cell level.

A new microsystem for automated electrorotation measurements using laser tweezers.

We have developed a new microsystem for fast, automated studies of reactions and kinetics of single cells with biochemical or pharmacological agents. A cell spins in an external rotating electric field and the frequency dependence characterises the passive dielectric properties of membrane and cytoplasm. We use a planar microelectrode chip with microchannel (easily covered with a removable slip) for the application of frequencies exceeding 250 MHz to determine cytoplasmic properties in low and high conductivity electrolyte solutions. The laser tweezers serve as a bearing system, rotation is induced by microelectrodes and rotation speed is recorded automatically. This opens up new possibilities in biotechnology, e.g. for drug screening as demonstrated by measuring the influence of ionomycin on the passive dielectric properties of T-lymphoma cells. Additionally, a possible infrared-induced long-term cell damage could be observed by electrorotation and is discussed.

Dielectrophoretic manipulation of suspended submicron particles.

Planar and three-dimensiònal multi-electrode systems with dimensions of 2 - 40 microm were fabricated by IC technology and used for trapping and aggregation of microparticles. To achieve negative dielectrophoresis (repelling forces) in aqueous solution, radiofrequency (RF) electric fields were used. Experimentally, particles down to 100 nm in diameter were enriched and trapped as aggregates in field cages and dielectrophoretic microfilters and observed using confocal fluorimetry. Theoretically, single particles with an effective diameter down to about 35 nm should be trappable in micron field cages. Due to the unavoidable Ohmic heating, RF electric fields can induce liquid streaming in extremely small channels (12 microm in height). This can be used for pumping and particle enrichment but it enhances Brownian motion and counteracts dielectrophoretic trapping. Combining Brownian motion with ratchet-like dielectrophoretic forces enables the creation of Brownian pumps that could be used as sensitive separation devices for submicron particles if liquid pumping is avoided in smaller structures.

Dielectric single particle spectroscopy for measurement of dispersion.

Measuring the frequency-dependent behaviour of single particles or biological cells in inhomogeneous and/or rotating electric fields is a sensitive method for characterising their dielectric properties. This technique is able to detect broad dispersion in the megahertz range of homogeneous artificial Sephadex G15 spheres. Recent progress has opened up the possibility of carrying out dielectric spectroscopy in cell culture media. Dielectrophoretic and electrorotational spectra of different cells in media of varying conductivity can only be explained by the introduction of dispersive cell compartments. The cytoplasm of animal cells typically exhibits a broad dispersion around 15 MHz and there is evidence for membrane dispersion around 50 MHz.

Single micro electrode dielectrophoretic tweezers for manipulation of suspended cells and particles.

Cells or particles in aqueous suspension close to a single capacitively coupled micro electrode (CCME) driven with high frequency electric fields experience dielectrophoretic forces. The effects near the CCME can be used for trapping and manipulation of single cells using externally metallised glass pipettes and might be used to develop a microscope based on force or capacitance measurements in conductive media.

Dielectrophoretic sorting of particles and cells in a microsystem.

There are highly sensitive analytical techniques for probing cellular and molecular events in very small volumes. The development of microtools for effective sample handling and separation in such volumes remains a challenge. Most devices developed so far use electrophoretic and chromatographic separation methods. We show that forces generated by ac fields under conditions of negative dielectrophoresis (DEP) can also be used. Miniaturized electrode arrays are housed in a microchannel and driven with high-frequency ac. A laminar liquid flow carries particles past the electrodes. Modification of the ac drive changes the particle trajectories. We have handled latex particles of micrometer size and living mammalian cells in a device which consists of the following four elements: a planar funnel which concentrates particles from a 1-mm-wide stream to a beam of about 50-micron width, an aligner which narrows the beam further and acts to break up particle aggregates, a field cage which can be used to trap particles, and a switch which can direct particles into one of two output channels. The electrodes are made from platinum/titanium and indium tin oxide (ITO) on glass substrates. Particle concentration and switching could be achieved for linear flow velocities up to about 10 mm s-1. The combination of this new method with high-performance optical detection offers prospects for miniaturized flow cytometry.

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.

Cell manipulation and cultivation under a.c. electric field influence in highly conductive culture media.

Extreme miniaturisation of electrodes enabled us to apply high-frequency electric fields (between 100 kHz and several hundred MHz) of field strengths up to 50 kV/m into cell suspensions of high conductivity (several S/m), such as original cell culture media. The active electrode areas were additionally decreased and modified by insulating the terminals and/or coating of the electrodes with thin dielectric layers. Micro scaled electrode structures were fabricated on glass or silicon wafers in semiconductor technology. It could theoretically and experimentally be shown that cells exhibit exclusively negative dielectrophoresis if suspended in highly conductive media. Therefore, they can be repulsed from surfaces by appropriate arrangements of electrodes and easily be manipulated in free solution. Adherently growing animal cells, like mouse fibroblasts (3T3, L929), were cultivated in Dulbecco's Modification of Eagle's Medium (DMEM) or RPMI 1640 under permanent field application (frequency: 10 MHz, field strength: 50-100 kV/m).

Radio-frequency microtools for particle and liver cell manipulation.

Single particles can be manipulated by applying high frequencies to ultramicro electrode arrays fabricated on planar structures. Heat production can be reduced to the extent that intense electric fields can be applied even to unmodified cell culture media. Animal cells grow normally in the high field (up to 100 kV/m) between such continuously energized multielectrodes. As with laser tweezers [1-3], this technique can capture particles and cells in field traps, generate linear movement, and permit cell cultivation. It can also produce micropatterns of pH gradients, field-cast objects, and control cell adhesion. These microtools may be combined to develop cell separators, microsensors, and controlled-biocompatibility surfaces.

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.

A traveling-wave micropump for aqueous solutions: comparison of 1 g and microgram results.

The operation of a micro-chip-based fluid circulator based on the interaction of a high-frequency traveling wave with a thermally generated inhomogeneity in an aqueous medium is investigated. The profiles of the electric field, the thermal gradient and the driving force within the device are derived numerically, and the dependence upon applied voltage under different experimental conditions is derived. The importance of convection in the operation of the device is assessed by operating it in various orientations, as well as under microgravity, and also by investigating the reversal and turn-on characteristics. The pump showed the unexpected ability to trap microparticles present in the driven fluid.

Three-dimensional electric field traps for manipulation of cells--calculation and experimental verification.

The forces acting on dielectric particles and living cells exposed to alternating and rotating fields generated by three-dimensional multi-electrode arrangements are investigated. Numerical procedures are described for the calculation of the electric field distribution and forces. The physical treatment considers electrodes of any shape and dielectric particles of complex structure. Particle and cell trapping are based on negative dielectrophoretic forces produced by high-frequency a.c. or rotating electric fields up to 400 MHz. Various multi-electrode systems were realised in commercially fabricated microelectrode systems, and tested for their ability to move and assemble microparticles or living cells without contact with the electrodes. The field distribution and accuracy of phase-controlled power application was tested using individual artificial particles trapped in the electric field cage. Position and trajectories of particle motion were measured. The paper gives an overview of electrode and field cage design in the microscale range.

Direct clean-up and analysis of ribonucleosides in physiological fluids.

We describe the group-selective separation and quantification of unmodified, modified and hypermodified ribonucleosides in physiological fluids (urine, serum) by on-line multidimensional high-performance affinity chromatography (HPAC)-reversed-phase liquid chromatography (RPLC). The excretion levels and patterns of ribonucleosides such as N1-methyladenosine, N1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoylthreonyladenosine and 2-pyridone-5-carboxamido-N-ribofuranoside were determined in urines from a control group and from patients with different diseases. The HPAC-RPLC method applied represents a powerful tool, e.g. as a non-invasive screening test, a method to investigate disorders in ribonucleoside and/or RNA metabolism, a method for drug monitoring during nucleoside chemotherapy, and a method to study renal ribonucleoside reutilization.