EP1682868A1

EP1682868A1 - Methods and devices for analysing a deformable object

Info

Publication number: EP1682868A1
Application number: EP04797789A
Authority: EP
Inventors: Thomas Schnelle; Torsten Müller; André HÖMKE
Original assignee: Evotec Technologies GmbH
Current assignee: Revvity Cellular Technologies GmbH
Priority date: 2003-11-10
Filing date: 2004-11-10
Publication date: 2006-07-26
Also published as: DE10352416A1; US20070119714A1; DE10352416B4; WO2005045400A1

Abstract

The invention relates to methods for analysing at least one deformable object (O) in a liquid suspension, comprising the steps: generation of an electric positioning field and positioning of the object (O) in a minimum potential of the positioning field; generation of an electric deformation field in such a way that a deformation force is exerted on the object (O); and detection of at least one dielectric, geometric or optical characteristic of said object (O). According to the invention, the positioning field is generated in a compartment (12) of a fluidic microsystem (10) and the positioning of the object (O) in a freely suspended state takes place in a contactless manner. The invention also relates to measuring apparatus that is used to carry out said method.

Description

Methods and devices for examining a deformable object

The invention relates to methods for examining a deformable object suspended in a liquid, in particular for examining deformation properties of biological particles, such as biological cells, with the features of the preamble of claim 1. The invention also relates to devices for implementing such methods and Applications of high-frequency field cages in fluidic microsystems.

It is known that damaged, transformed or degenerate biological cells often have mechanical properties that differ from healthy cells, whereby they are usually softer than healthy cells (see B. Alberts et al. In "Textbook of Molecular Cell Biology", Wiley VCH, Weinheim, 1998; and JM Vasiliev et al. In "BBA" Vol. 780, 1985, pp. 21-65 "Spreading of non-transformed and transformed cells"). In addition, different cell types differ, such as white and red blood cells according to their deformability (see R. Glaser in "Biophysics", Spektrum Akademischer Verlag, Heidelberg, 1996). It was also found that cancer cells between 2- and 10- times softer than healthy cells and can deform significantly more than healthy cells under the influence of forces (see J. Guck et al. in "Biophysical Journal", Vol. 81, 2001, pp. 767-784). It is known that the cytoskeleton and thus the viscoelastic properties of cells can be changed by adding certain agents, for example cytochalasin or colchezine (see B. Alberts et al.) B. Alberts et al. also describe that the cytoskeleton changes during cell ontogenesis / differentiation and the cell cycle.

An example of damaged biological cells whose mechanical properties change as a result of the damage are red blood cells damaged by parasite attack in malaria (see FK Glenister et al. In “Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells ", BLOOD, Vol. 99, No. 3, 2002, pp. 1060 to 1063).

To differentiate between cancer cells and healthy cells, J. Guck et al. and in US 6 067 859 an optical micromanipulator is proposed which acts as a so-called optical stretcher (or: laser stretcher). The optical stretcher uses two counter-rotating, barely focused laser beams to capture cells suspended in a liquid in the flow at low light output (10 - 100 mW). If the light output is increased (100 mW - 1.5 W), the cells are distorted (deformed) to different extents depending on the cell type. Healthy cells hardly change, while tumor cells deform significantly.

The use of the optical stretcher to detect cancer cells has several disadvantages. A major disadvantage is that the optical stretcher can only function reliably with individual cells, but there is no possibility of selecting individual cells from the suspension liquid. Several cells caught between the laser beams interact with each other so that the deformation to be examined is influenced. To avoid this problem, extremely diluted samples must be used. This limits the sample throughput. Another disadvantage is that the de- tection of the deformation cannot be reliably automated. Finally, there is a disadvantage of the laser stretcher that the capture area due to the small diameter of the light guide for coupling the laser beams is only a small size of z. B. 5 microns.

In the publication "Reversible Electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of sub-microsecond duration" by KJ Müller et al. ("J. Membrane Biol." Vol. 184, 2001, pp. 161-170) describes that cells suspended in a liquid can be deformed in DC or AC electric fields (E). Depending on the electrical conductivities σ of the suspension liquid (index 1) and the cell cytosol (index c), both elongating and compressive pressures P _D can be exerted (see FIG. 6). For high-frequency fields, the pressure (stress) is P _D (ε ₀ : absolute dielectric constant, 8χ: relative dielectric constant of the suspension liquid, Θ: angle between the electric field and the considered direction of action of the pressure):

P _D = (1)

The by KJ Müller et al. The described deformation of cells serves to influence the permeability of the cell membrane during the so-called electropermeabilization. This deformation technique is unsuitable for the above-mentioned detection of healthy or sick cells for the following reason. Depending on the mechanical and dielectric properties of the cells, field strengths of a few 10 kV / m up to the MV / m range are required for the deformation. Due to the high field strengths, only low conductivity solutions are used to avoid ohmic losses. The Cells are additionally drawn to the electrodes via positive dielectrophoresis to generate the DC or AC voltage fields, so that interactions occur between the cells and the electrodes, which make reproducible and quantitative observation of the deformation difficult. Electrode contact in the cell affects its vitality and the cell can often not be detached from the electrodes, or not at all, without destruction.

The use of high-frequency electric fields to investigate the viscoelastic properties of erythrocytes is described by H. Engelhardt et al. in: "Nature", vol. 307, 1984, p.

378-380, "Viscoelastic properties of erythrocyte embranes in high-frequency electric fields". Sharp-edged electrodes are arranged at a distance of 50 μm in a cuvette. When the electrodes are exposed to a high-frequency electrical voltage, one or more erythrocytes are arranged between the The erythrocytes are drawn to the electrodes. A brief increase in field strength causes a deformation that can be optically observed and quantitatively evaluated. The technique described by H. Engelhardt has several disadvantages. A major problem is that, as with In the technique described above by KJ Müller et al., the erythrocytes touch the electrodes. This distorts the observation of the deformation. In addition, the erythrocytes cannot be deformed in different directions in a defined manner. Another problem is that in the case of H. Engelhardt e t al. proposed experimental conditions must be worked with an extremely low conductivity of the buffer solution that surrounds the erythrocytes. The conductivity of the buffer solution is in the range from 1 mS / m to 10 S / m. However, these conductivities are considerably lower than the conductivities of physiological solutions, so that the investigated erythrocytes are exposed to additional stress or can be destroyed.

Another disadvantage of H. Engelhardt et al. The proposed measurement is that only an integral light measurement is provided. Topographic deformation images cannot be recorded using conventional technology. Finally, the one by H. Engelhardt et al. The technology described cannot be implemented in the flow system and is unsuitable for automation.

Furthermore, it is known to capture and hold individual cells under the action of high-frequency electrical fields in field cages by means of negative dielectrophoresis. Up to now, the use of high-frequency field cages has been aimed at manipulating the cells as gently as possible, with one-sided force effects or deformations of the cells being undesirable. For example, H. Wissel et al. in "American Journal of Physiology Lung Cell Mol. Physiol." (Vol. 281, 2001, L345-L360 "Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes") that cells can be kept gently even at high field strengths because they are in one hand a field minimum (zero field) and secondly micro-electrodes that minimize heating are used.

T. Schnell et al. describe in "J. Electrostat." (Vol. 50, 2000, pp. 17-29, "Trapping in ac octode field cages") various phase drives of high-frequency dielectric field cages. With a suitable control, objects can be kept stable or released from the field cage in a specific direction, or conditions can be found under which several objects in the cage are brought into contact with one another. The object of the invention is to provide improved methods for examining deformation properties of objects, in particular biological cells, with which the disadvantages of the conventional methods are overcome and which in particular enable characterization of deformation properties with increased accuracy and reproducibility. Methods according to the invention are also intended to enable quantitative characterization of the deformation properties and with reduced device technology

Effort can be automated. Another object of the invention is to provide improved devices for examining deformation properties of objects, in particular for implementing the method according to the invention.

These objects are achieved with methods and devices with the features of claims 1 and 25. Advantageous embodiments and applications of the invention result from the dependent claims.

In relation to the method, the invention is based on the general technical teaching, for the examination of an object suspended in a liquid after its positioning in a potential minimum of a high-frequency electrical positioning field in an examination area of a fluid microsystem on the object with a deformation field to exercise and to detect a reaction of the object to the deformation force by detecting at least one property from the group of the electrical, geometric and optical properties of the object. The use of the high-frequency electrical positioning field advantageously enables contactless, dielectric positioning of individual objects with high stability and location accuracy. The non-contact Positioning includes holding individual objects, such as. B. individual biological cells in a freely suspended state, ie floating in the suspension or a treatment liquid without direct mechanical contact (without direct contact) with components of the fluid microsystem. The object to be examined is in free solution during positioning and deformation, ie it is surrounded on all sides by the liquid, and there are distances from all neighboring wall surfaces or electrodes of the microsystem. The stability of the positioning allows a detector to be precisely adjusted to the object and to be set up to record the desired properties.

According to a preferred embodiment of the method according to the invention, the deformation field acts on the basis of negative dielectrophoresis. The combination of the holder by means of negative dielectrophoresis with the effect of the deformation field, which was proposed for the first time with the present invention, has the advantage of enabling a particularly gentle examination of biological objects in fluidic microsystems, as they are per se for manipulation, treatment, sorting and analysis for example of biological cells are already available.

A capture field is preferably generated by negative dielectrophoresis and alternately or at the same time a deformation field is generated using positive or negative dielectrophoresis, this combination of capture field and deformation field preventing the objects from contacting the electrodes.

Holding objects by negative dielectrophoresis has particular advantages when studying biological ones Objects. The conductivity of the surrounding suspension or treatment liquid can, for example, be considerably increased compared to the technique described by H. Engelhardt, in particular in the range of physiological conditions. The conductivity can e.g. B. greater than 0.3 S / m and in particular corresponding to the physiological value 1.5 S / m. In particular when measuring on biological cells in which the conductivity inside the cell is lower than in the external medium, the negative dielectrophoresis advantageously occurs in the entire frequency range of interest, in particular above 1 kHz up to the GHz range. In comparison to conventional techniques, an enlarged frequency range is available for the deformation field, wherein the deformation field can be generated for negative or positive dielectrophoretic conditions and different deformation effects can be set at different frequencies. Another important advantage of using a suspension or treatment liquid with an increased external conductivity is the reduction of ohmic heating effects, e.g. B. up to a factor of 5, so that the cell physiology is hardly influenced during the measurement.

According to an alternative variant, the deformation field acts on the basis of positive dielectrophoresis, which can be advantageous for certain objects for contactless holding.

Electrode contact is advantageously avoided in general by the method and the device according to the invention. This avoids mechanical damage to the cells, particularly when treating cells. A suitable temporal and geometric field formation can cause a deformation in both homogeneous and also take place in inhomogeneous electric fields, corresponding to the parallel or anti-parallel polarization in the external electric field.

Which of the electrical, geometric and / or optical properties of the object is detected depends on the specific application of the invention. For example, an impedance measurement in the examination area can determine whether and at what speed the object deforms and, if necessary, relaxes into the undeformed state. This variant can be advantageous for the operation of automated microsystems without optical process monitoring. A detection of the geometric properties of the object accordingly means that the outer shape of the object, for example, with a camera during the deformation and / or

Relaxation is recorded and then evaluated. The detection of optical properties here denotes the detection of the interaction of the object with light, such as a fluorescence measurement or a scattered light measurement. If the examination according to the invention is carried out, for example, on biological cells which react to mechanical stimuli by changing the membrane structure and can accordingly activate fluorescence markers, the optical detection comprises a fluorescence measurement during the deformation and / or relaxation.

Further important advantages of the methods according to the invention are that they allow a reproducible, quantitative evaluation of the detected properties to determine elastic properties of the object, such as, for B. enable the viscoelastic properties of biological cells. The process can be fully automated. The detection of properties that are characteristic of the deformation or relaxation can be carried out in real time or subsequently via stored data (for example video recordings). Drawing) using algorithms known per se for image evaluation. Static or dynamic elastic properties of the objects to be examined can be determined.

The method according to the invention advantageously has a high degree of flexibility with regard to the time of the deformation measurement. According to preferred embodiments of the invention, the detection can be carried out once or several times at times selected in the entire time range during and after the deformation of the object. Correspondingly, the detection can include detection of deformation properties or, when the deformation forces are briefly exerted, relaxation properties of the object. Advantages with regard to an expanded information content of the detection can result if a time dependency of the respectively measured electrical, geometrical and / or optical quantity is recorded.

Particular advantages, in particular when examining biological samples, can result if the positioning field is generated with a cage electrode arrangement as a high-frequency field cage, since experience with the design and control of known high-frequency field cages already exists.

According to a variant of the invention, the radio-frequency field cage is operated as a field cage which is closed on all sides and has an essentially point-shaped potential minimum which is fixed in the microsystem. The deformation and detection can advantageously be carried out on the stationary object. According to an alternative variant of the invention, the high-frequency field cage is operated as an open field cage with a line-shaped potential minimum which is in longitudinal extends direction of a channel in the fluidic microsystem. The object moves with the suspension fluid through the cage electrode arrangement, the field cage merely ensuring that the object is positioned on a specific trajectory through the channel. The deformation and detection can be carried out dynamically on the moving object, so that the invention can also be implemented during the operation of high-throughput systems.

Advantageously, the technical effort involved in implementing the invention can be reduced if the cage electrode arrangement provided for forming the positioning field is also used to generate the deformation field. By switching from a catching or positioning mode and to a stretching or deformation mode, dielectric and / or optical properties and additionally the desired mechanically elastic properties of the object can be examined in a manner known per se. This variant is particularly advantageous for screening tasks in which the mechanical-elastic properties of the object are to be related to other measurement parameters. Alternatively, a separate deformation electrode arrangement can be used to generate the deformation field, which may result in advantages for the control of the positioning and deformation fields.

If, according to a preferred embodiment of the invention, the deformation field is set for a duration of 1 ms to 500 ms, there can be advantages with regard to a relatively low mechanical, low electrical and low thermal load on the object. It is particularly advantageous if the deformation field is generated in a pulsed manner, since in this case the time behavior of the Relaxation of the object deformation can be detected with increased accuracy.

If a treatment liquid is passed into the examination area before the deformation field is generated, a temporary solution exchange can advantageously take place, for example to examine the deformation behavior of the object in different media or to carry out an object treatment with a specific treatment liquid between examinations with different deformation directions , In this case, the object is preferably positioned at a channel junction or crossing in the fluidic microsystem, to which the respective treatment liquid is supplied.

According to a further preferred embodiment of the invention, a multiple measurement is carried out on a specific object, such as, for example, on a biological cell to be examined. For this purpose, the steps of generating the deformation field and detection are carried out several times in succession. The multiple detection can be directed, for example, to repeat the deformation with identical process conditions in order to increase the accuracy of the measurement. Alternatively or additionally, the process conditions, such as, for example, the forces exerted, the field strengths, phases and / or frequencies of the high-frequency electrical fields, the duration of the force application or the addition of the treatment liquid can be varied in order to obtain additional information about the object. In particular, a control loop can advantageously be implemented in which the positioning field and / or the deformation field is set as a function of the result of the previous detection. Furthermore, in the case of successive examinations, the deformation field can be set and / or the object is rotated so that the object is deformed in different directions. This variant of the invention can have advantages for the examination of objects with anisotropic elastic properties.

If the multiple measurement takes place over a longer period of time, plastic deformations or the deformation depending on the force exerted can advantageously be examined. For this purpose, the duration of the multiple measurement is preferably at least one second.

When putting the invention into practice, in particular when examining biological cells, it is preferred to determine elastic properties of the object from the detected electrical, geometric and / or optical properties. For example, one or more of the following variables can be recorded as deformation properties as integral or structure-selective parameters: elastic modulus, shear modulus, viscosity, spring constant, stiffness constant and relaxation time. Furthermore, an adjustment can be made on the basis of models known per se, as described, for example, by H. Engelhardt et al. (see above). The invention is not limited to the measurement of elastic properties. Plastic deformation properties or intermediate forms between elastic and plastic behavior can also be determined.

Further advantages when examining samples with a large number of objects, of which one or more objects are to be examined individually, result from the combination of the method according to the invention with a dielectric cell sorter, which is set up to line up and, if necessary, sort the objects. If a specific object is selected from the sample before the positioning step, which has been subjected to a dielectric alignment, the selectivity of the deformation study according to the invention can be increased.

The method according to the invention can generally be carried out with any flexible, in particular elastically or plastically deformable, object that is smaller than the respective examination area. For the preferred application in fluidic microsystems, objects to be examined typically have sizes in the range from 1 μm to 50 μm. The object can have a regular or irregular shape and in particular be spherical. The object can be a synthetic object made of a deformable, compact or hollow material. According to the invention, for example, membrane vesicles filled with a liquid can be examined in order to examine the structure of the membrane envelope. Preferred applications of the invention are given when the object to be examined comprises at least one biological cell, a cell group, a cell component or such an object in combination with a synthetic particle. The object to be examined can also be porous and the objects themselves can be cells, cell pairs, cell aggregates / cell groups or cell components. It is also possible for synthetic particles, for example several beads, to also aggregate.

It is also possible, through a standard driving a dielectrophoretic ^'field cage n a symmetrical deformation of objects to reach, which are not small in the ratio to the cage (size ratio of for example approximately 1/2), and are relatively easily deformable. Cells that are trapped centrally in a cage and are small in relation to the electrode dimensions of the cage (e.g. 1/8) cannot normally be deformed. The cell does not drive dipole polarization at the capture point. In order to nevertheless generate a deformation by multipoles, it is preferred that the capture field is not closed, as shown, for example, in FIGS. 3A and 3B.

The preferred application of the invention in biotechnology and pharmacy is based on the idea of capturing individual cells in dielectric field cages and changing the field at the electrodes for a short time in such a way that sufficiently high deformation forces are generated. The electric field can then be put back into capture mode and the relaxation of the cell deformation can be observed in the area of low field strength. Alternatively, the field between the modes can also be temporarily switched off. This process can advantageously be repeated several times on a cell. According to a preferred variant of the invention, depending on the detected dielectric, geometric and / or optical properties, a distinction is made between normal and modified cells or between normal cells with different physiological properties, for example during their cell cycle. Furthermore, the method according to the invention can be used to differentiate between normal differentiated cells and stem cells. The method according to the invention can be used in particular for the detection and sorting of stem cells from a large number of cells. Additional information about the cells examined can be obtained if the dielectric, geometric and / or optical properties of the cell are detected as a function of the frequency and / or voltage of the positioning field and / or the deformation field. Furthermore, dependencies on the ambient temperature, on the material composition, the ambient fluid and / or the duration of individual deformations or the duration of multiple measurements can be carried out. If, according to the invention, cell pairs or cell aggregates are measured, which may only be brought together or connected in the positioning field, there are advantages compared to the laser stretcher, with which only individual particles can be deformed

In terms of the device, the above-mentioned object of the invention is achieved with a measuring apparatus for examining at least one object, which has a fluidic microsystem with an examination area with at least one electrode arrangement, a detector device for electrical and / or optical measurement of object properties, and a field shaping device with at least one contains a high frequency generator and a switching device with which between the

Capture mode, in which a high-frequency positioning field is generated in the examination area with the at least one electrode arrangement, and the deformation mode can be switched over, in which a deformation field is generated in the examination area with the at least one electrode arrangement.

If the detector device has a microscope with a camera, there may be advantages with regard to the accuracy of the detection on microscopic objects, the diameter of which is typically less than 25 μm.

According to a preferred embodiment of the invention, the measuring apparatus is equipped with a control device which is connected to the detector device and the switching device. This connection advantageously enables the implementation of a control loop in which parameters of the positioning and / or deformation fields and / or the suspension fields tion liquid depending on the result of a detection can be adjusted or changed.

The fluidic microsystem of the measuring apparatus is preferably equipped with a fluidic device with which the suspension and / or an additional treatment liquid can be moved through the examination area and which is also connected to the control device.

Another independent object of the invention is the use of a fluidic microsystem with a high-frequency field cage for the investigation of deformation and / or relaxation properties of biological cells and in particular for the separation or sorting of healthy cells and diseased cells, for example cancer cells, or the sorting of Stem cells from a cell sample.

The invention has the following further advantages. For the first time, an examination method is created with which the deformation and / or relaxation of biological cells can be detected in a fully automated manner. The inventors have found that in cells held dielectrically in high-frequency field cages with high-frequency electrical fields, such high deformation forces can be exerted that the deformation fields only have to be generated for short times and with local limitation. This reduces the strain on the cells. In addition, undesired heating of the electrode arrangement in the examination area is avoided or significantly reduced.

The deformation of particles, in particular cells, can be supported or achieved by cells with _

Beads or metal balls (Au) can be occupied. These beads or metal balls show a at a suitable capture frequency positive dielectrophoresis and can deform the particles or cells. Furthermore, it is also possible to have cells beads or metal spheres phagocytized.

A further advantageous possibility for deforming cells or particles is to use magnetic beads with which the cells are coated or which are phagocytized by the cells in order to deform the magnetic by an arrangement of switchable magnetic elements, for example electromagnets arranged outside the channel Beads and thus to reach the cells.

Another advantageous possibility of achieving a deformation of a cell or a particle is to excite the object to oscillate at a resonance frequency by quickly switching the deformation field on and off. This results in a repeated deformation with the resonance frequency, a measurement of the decay of the vibration allowing conclusions to be drawn about the mechanical properties of the cell.

The method according to the invention or the device according to the invention can also be used advantageously for pair separation. Under pair separation _(the separation of two or else understood more each of attached particles may. If such a pair caught in an inventive electrode arrangement, and then separated by switching to the stretching mode, so as a function of the forces exerted by the dielectrophoresis separation of the objects can in Different flow paths are made possible by variation of the frequencies and / or deformation stress and / or deformation time further characterization of the bond between objects. Further details and advantages of the invention will become apparent from the description of the accompanying drawings. Show it:

FIG. 1: a schematic representation of an embodiment of a measuring apparatus according to the invention,

FIGS. 2, 3: schematic, enlarged illustrations of electrode arrangements used according to the invention,

FIG. 4 shows a flow chart to illustrate an embodiment of a method according to the invention,

FIG. 5 measurement and simulation results to illustrate the deformation of cells according to the invention,

FIG. 6 shows a schematic illustration of the object deformation in an external electrical field, and

FIG. 7: schematic, enlarged illustrations of electrode arrangements used according to the invention.

The invention is illustrated below with reference to the examination of biological cells in a fluidic

Microsystem described. It is emphasized that the implementation of the invention is not limited to the examination of biological cells, but is possible accordingly, in particular with the object types mentioned above. Fluidic micro Systems with electrode arrangements for the dielectric manipulation of suspended particles are known per se, so that only the details required for implementing the method according to the invention are explained here.

FIG. 1 shows an embodiment of the measuring apparatus according to the invention with the fluidic microsystem 10, the detector device 20 and the field shaping device 30. The fluidic microsystem 10 is only shown in part with an electrode arrangement of cage electrodes 1-4 and optionally provided deformation or impedance measurement electrodes 5, 6. The electrodes 1-6 are microelectrodes known per se, which are attached to the bottom, side and / or top walls of a compartment, e.g. B. a channel 12 of the fluidic microsystem. A suspension liquid flows through the channel 12 in the direction of the arrow A. The examination area 11 is formed between the electrode ends (electrode tips) of the electrode arrangement 1-6, in which the positioning and deformation of the object 0 to be examined, which is explained below, is carried out. The examination area 11 can be formed at a point of intersection of the channel 12 with another channel (not shown) of the microsystem in order to expose the object 0 in the held state, if necessary, to a treatment liquid which is supplied through the further channel.

The detector device 20 comprises a microscope 21, a camera 22 and an image data memory 23, which interact in a known manner. The microscope 21 is, for example, a 1X70, manufacturer: Olympus with a CCD camera 22 of the Sensicam Vision type, manufacturer: Photonics.

The field shaping device 30 contains a high-frequency generator 31 and a switching device 32. Both components can be integrated in a common circuit. The high-frequency generator is a voltage source for generating high-frequency electrical voltages, typically in the voltage range from 0.1 to 10 Vrms and in the frequency range from 1 kHz to 100 MHz. The output voltage values, phases and frequencies of the voltages generated by the high-frequency generator 31 can preferably be set manually or with the control device 40.

For manipulation, especially deformation, biological

Cells by means of negative dielectrophoresis, the frequency of the high-frequency voltage can advantageously be set as a function of the external conductivity so that the membrane of the cell under investigation is fully charged. For this purpose, the frequency is chosen to be significantly lower than the reciprocal value of the membrane relaxation time τ _m (f «f _m ). The membrane relaxation time is related to the conductivities according to equation (2):

In this case, the cell can only be elongated according to equation (3).

P _D = ^ ε ₀ ε _m E ² cos ² [®] (3) λ n

At high frequencies f »f _m , the cell membrane is capacitively bridged, so that the cell is either elongated or compressed depending on the external and internal conductivities (see equation (1), above). The high-frequency generator 31 is set up to generate voltage profiles in accordance with various operating modes of the measuring apparatus, which are illustrated below by way of example. A first operating mode is the hold or capture mode, in which the object O (e.g. the biological cell) in the

Potential minimum of the dielectric field cage generated by the cage electrodes 1-4 is held. Cell 0 is at rest despite the flowing suspension liquid (arrow A). The control protocols of the cage electrodes (voltages, frequencies, phases) are known per se from fluidic microsystem technology. In the second operating mode, namely the deformation mode, voltages are generated such that deformation forces directed onto the held cell 0 are exerted (see below), the liquid flow being stopped in this state.

The schematically shown switching device 32 is used to select the currently desired operating mode in which the respective voltage is applied to the respective electrodes. The switching device 32 can comprise a changeover switch or a phase shifter and / or can be integrated in the control of the high-frequency generator 31. In both variants, actuation with the control device 40 can be provided.

The reference numeral 50 refers to a fluidic device with which the suspension and / or treatment liquid is moved in the channel 12 of the fluidic microsystem 10. The fluidic device 50 includes, for example, a pump that can be actuated with the control device 40, if necessary.

Figure 2 shows the cage electrodes 1-4, l'-4 'in an enlarged perspective view. The lower four electrodes 1'-4 'are on the bottom 13 of the fluidic microsystem 10, while the electrodes 1-4 are arranged on the top surface (not shown). The channel direction corresponds to the flow direction A of the suspension liquid. When the cage electrodes are subjected to high-frequency electrical voltages in accordance with one of the control types "trap red", "trap ac I" or "trap ac II" specified in the following table, a dielectric field cage with a point-like potential minimum is formed in the middle between the ends of the electrodes on which the cell to be examined is located.

Electrode / 1 2 3 4 r 2 '3' 4 '

Trap red mode 0 ° 90 ° 180 ° 270 ° 180 ° 270 ° 90 ° 180 ° trap ac 1 0 ° 180 ° 0 ° 180 ° 180 ° 0 ° 180 ° 0 ° trap ac II 0 ° 180 ° 0 ° 180 ° 0 ° 180 ° 0 ° 180 °

Stretch ac 1 0 ° 0 ° 180 ° 180 ° 0 ° 0 ° 180 ° 180 °

Stretch ac II 0 ° 0 ° 0 ° 0 ° 180 ° 180 ° 180 ° 180 °

Stretch ac III 0 ° Mass 180 ° mass 0 ° Mass 180 ° mass trap-stretch F ^ O ⁰ F _2/0 ° F - / 180 ⁰ F _2/180 ° Fi / 0 ° F _2/180 ° Fι / 180 ₂ ° F / 0 °

The control mode "trap red" is used to capture the cells in the field cage and to rotate the cell in a predetermined orientation relative to the surrounding microsystem. In this case, deformations in certain directions can advantageously be examined. The control modes "trap ac I" and "trap ac II "serve to capture the cell in the field cage without a specific orientation. By switching the relative phase position between the high-frequency voltages at the cage electrodes in accordance with the control types "stretch ac I" or "stretch ac II", a switch is made from the capture mode to the deformation mode. The polarization of the cells to be examined makes deformation forces parallel to the direction of flow in the example of the control types specified A formed so that the cell deforms (see Figure 5). Another stretch mode "stretch ac III" generates a deformation field in an octode cage with two opposite electrode pairs, the other electrodes being at ground. In addition, a "trap-stretch" mode is specified, which demonstrates how a particle is simultaneously across different electrodes can be deformed at a frequency Fl and can be dielectrically focused with another frequency F2 in the direction weakened by the deformation field. It is particularly advantageous to use this type of control in connection with an electrode field cage with twelve or more electrodes, as shown for example in FIG. 7C. For example, the deformation with the narrow, central two pairs of electrodes can take place with simultaneous dielectric focusing of the particle by the outer electrodes of the remaining octode cage.

FIG. 3A shows cage electrodes 1, 2, 1 'and 2' which are used to form a dielectric open in the direction of flow A.

Field cage are set up. In the following table, the control types for the trap ac trapping mode, in which the cells are focused in the center of the channel, and the deformation mode "stretch ac I" or "stretch ac II" are compiled accordingly.

Electrode / 1 2 r 2 'mode trap ac 0 ° 180 ° 180 ° 0 ° stretch ac 1 180 ° 0 ° 180 ° 0 ° stretch ac II 180 ° 180 ° 0 ° 0 °

FIGS. 3B and 3C show the electrical potentials for the "trap ac" mode (FIG. 3B) and for the "stretch ac I" Mode (Figure 3C) for the cage electrode arrangement of Figure 3A shown. FIGS. 3B and 3C are a sectional view of the channel with the electrodes shown in section, the direction of flow of the channel being perpendicular to the image plane. 3B for the “trap ac I” mode the centered cell is drawn with polarization charges. It can clearly be seen that cells with this electrode arrangement can be deformed more easily than in the eight-electrode cage even without stretching fields because of the cell No forces act in the direction perpendicular to the plane, ie in the direction of flow .. Advantageously, a further homogenization of the stretching field (FIG. 3C) can be achieved by means of additional electrodes which are arranged in the plane between the existing electrodes.

The sequence of steps for carrying out the method according to the invention is illustrated schematically in FIG. After a line-up (step 100), in which a sample from a multiplicity of cells is dielectrically strung upstream of the examination region 11 in a manner known per se, in step 200 the positioning of a particle in the dielectric field cage of the cage electrodes takes place, for example according to FIG by changing the electrode control, the cell deformation (step 300), the deformation (step 400) or the relaxation (step 500) being detected electrically or optically while the deformation forces are exerted and / or after the deformation forces have been switched off. Subsequently or at the same time (online), the evaluation and quantitative analysis of the deformation follows in step 600. Depending on the result of step 600, it can be provided that a further deformation with associated detection is carried out. Before the further deformation, the object can optionally be rotated in step 200. Finally, at step 700, a decision is made as to whether another object is to be examined or the measurement is to be ended. If necessary, at step 800 the object is placed in a suitable container or on a substrate, e.g. B. provided in a microtiter plate.

FIG. 5 shows an example of the deformation of an erythrocyte in a field cage by switching from capture mode to deformation mode. The examination area has a diameter of approx. 40 μm. The frequency of the positioning and deformation _fields is 700 kHz (3V _rms ). The conductivity of the suspension liquid is 0.3 S / m. FIGS. 5A and B show microscopic images of erythrocyte 0 in the capture and deformation mode. FIGS. 5C to D illustrate the field distribution in the horizontal central plane of the electrode arrangement according to FIG. 2 in the capture mode for the control protocols “trap red” or “trap ac II”. The courses of the time-averaged values of the square of the electric field (E ² ) in the central horizontal plane between the electrodes at a certain point in time are shown as equipotential lines. The polarization forces acting on the object are proportional to the quantity E ² . FIG. 5E shows the course of E ² in the stretching mode. FIG. 5F shows the electrical potential f in the stretch mode when the “stretch ac I” type of control is implemented.

As can be seen from the field simulations, a cell that was initially trapped centrally in the cage (FIG. 5A, zero field) is in a strong, relatively homogeneous (saddle-shaped) electrical field when switching to the stretching mode. Without external disturbances, the cell remains in the central area, where it is deformed (FIG. 5B). With well-equilibrated fluidics, the cell remains experimentally in the central area for up to a few seconds, where relaxation can be observed after switching over or off. These investigations can be carried out in the river (see FIG. 3) and possibly on several z. B. over cells aligned with funnel-shaped field barriers (by "funnel electrodes" or so-called funnel).

During the measurement of the deformation and / or relaxation (steps 400, 500), for example, images of the object 0, which were recorded with the detector device 20, are measured. For example, the object diameter is recorded before and during the deformation. A corresponding time dependency is included to determine relaxation times. If, for example, a spherical cell is deformed into an ellipsoid, the semiaxes of the ellipsoid are measured. The desired elastic properties are determined from the freshly measured values and / or the determined time function, depending on the model used.

Deviating from the procedure described above by way of example, the following modifications can be provided when realizing the invention.

A parameter optimization can be provided, for example in order to keep the object 0 as well as possible in the center of the examination area. For this purpose, the image data of

Detector device derived control signals with which the parameters of the output voltages of the high-frequency generator are set until the desired centering is given. A corresponding control loop can be directed to the change in the field strength or frequency of the deformation fields in order to achieve a specific deformation result. Field strength and / or frequency-dependent deformation measurements can be carried out. Alternatively or in addition to the above-described stretching mode in a horizontal plane, a deformation in another direction, e.g. B. be carried out in a vertical plane. Furthermore, a directional stretching of the object 0 in the field cage to a needle shape can be provided.

To optimize the positioning or deformation fields, their frequencies can differ. The deformation fields can be generated simultaneously to form a permanently formed positioning field. According to equation (1), elongating or compressing fields can be formed.

In general, it is possible to couple both the capture and the stretching field simultaneously and not alternately (for example, "trap-stretch" mode). This can advantageously be done, for example, in that the capture field and the stretching field operate at different frequencies.

Instead of the eight or four-electrode field cages, other electrode geometries can be realized, as are known per se from fluidic microsystem technology. For example, six-pole electrode arrangements can be implemented.

It can also be advantageous to use multi-electrode arrangements, in particular electrode arrangements with twelve electrodes, to generate homogeneous stretching fields with a virtually unchanged capture field. 7 shows the square of the averaged field strength E ² in the horizontal plane between the electrodes. FIGS. 7A and 7B show electrode arrangements which correspond to those described above in connection with FIGS. 5A to 5F. The direction of flow of the channel is marked with the arrow. In FIGS. 7C and 7D show electrode arrangements with a total of twelve electrodes, of which only the top six electrodes are shown. The additional electrodes are preferably attached centrally on a plane between the eight electrodes, the additional electrodes cutting the channel perpendicular to the direction of flow. In the stretching mode, the additional electrodes make the field near the capture point more homogeneous, so that a particle arranged there experiences lower dielectrophoretic forces (see FIGS. 7C and 7D). Especially when the flow in the channel direction has not completely calmed down, the middle additional electrodes can be particularly advantageous since they focus in the flow direction. The control is particularly easy to implement in the “stretch ac I” mode since no additional phases are required. The additional electrodes are switched to either 0 ° or 180 ° in accordance with the two adjacent electrodes.

Both in the case of the eight electrodes and in the case of the twelve electrodes, a saddle results in the middle of the capture field in the “stretch ac I” mode (see FIGS. 7B and 7D). To avoid this, it can be advantageous if the outer boundaries of the electrode tips of the obliquely arranged electrodes run parallel to one another, as shown in Figures 7E and 7F, Figures 7E and 7F show the parallel outer boundaries of the electrode tips in connection with a twelve-electrode arrangement However, outer boundaries of the electrode tips can also be used advantageously in the case of an eight-electrode arrangement, as shown in Figures 7A and 7. B. In general, parallel outer boundaries of the electrode tips are suitable for producing more homogeneous stretching fields Stretching field in the case of parallel outer boundaries the electrode tips are shown in FIG. 7F for the “stretch ac I” mode. Another possibility of achieving greater homogeneity is that the electrodes are not arranged equidistantly (not shown).

The features of the invention disclosed in the forthcoming description, the drawings and the claims can be of significance both individually and in combination for the implementation of the invention in its various configurations.

Claims

claims

1. A method for examining at least one deformable object (0) in a suspension liquid, comprising the steps:

Generation of an electrical positioning field and positioning of the object (0) in a potential minimum of the positioning field,

- Generation of an electrical deformation field such that a deformation force is exerted on the object (0), and

- Detection of at least one property from the group of the dielectric, geometric and optical properties of the object (0), characterized in that

- The positioning field is generated in a compartment (12) of a fluidic microsystem (10), and - The positioning of the object (0) takes place without contact with electrodes or in a freely suspended state without contact.

2. The method according to claim 1, wherein the positioning of the object (0) takes place under the effect of negative dielectrophoresis or under the effect of positive dielectrophoresis.

3. The method according to claim 1 or 2, in which the generation of the deformation force takes place under the action of negative dielectrophoresis or under the action of positive dielectrophoresis.

4. The method according to claim 1, 2 or 3, wherein the detection takes place during or after the deformation of the object (0) and correspondingly includes a detection of deformation or relaxation properties of the object (0).

5. The method according to at least one of claims 1 to 4, wherein the positioning field is generated as a high-frequency field cage with a cage electrode arrangement (1-4, l'-4 ').

6. The method according to claim 5, wherein the high-frequency field cage is operated as a closed field cage with a punctiform potential minimum in which the object (0) rests.

7. The method according to claim 5, wherein the high-frequency field cage is operated as an open field cage with a linear potential minimum through which the object (0) moves with the suspension liquid.

8. The method according to any one of claims 5 to 7, wherein the cage electrode arrangement (1-4, l'-4 ') is used to generate the deformation field.

9. The method according to any one of claims 5 to 7, in which a separate deformation electrode arrangement (5, 6) is used to generate the deformation field.

10. The method according to at least one of the preceding claims, wherein the deformation field is set for a duration of 1 ms to 500 ms.

11. The method according to at least one of the preceding claims, in which the generation of the deformation field is pulse-shaped.

12. The method according to at least one of the preceding claims, wherein the object (0) is exposed to a treatment liquid before or during the generation of the deformation field.

13. The method according to at least one of the preceding claims, with a multiple measurement, in which the steps of generating the deformation field with the detection and generating the positioning field are carried out alternately several times in succession.

14. The method according to claim 13, wherein the multiple measurement is carried out for a period of at least one second.

15. The method according to claim 13 or 14, wherein the positioning field and / or the deformation field is set or changed depending on the result of the previous detection.

16. The method according to at least one of claims 13 to 15, wherein the deformation field and / or the positioning field is set several times so that the object (0) is deformed in each case in different directions.

17. The method according to at least one of the preceding claims, in which viscoelastic properties of the object (0) are determined from the detected dielectric, geometric and / or optical properties.

18. The method according to at least one of the preceding claims, in which, prior to the positioning step, the object (0) is selected from a sample which has been subjected to a dielectric alignment.

19. The method according to at least one of the preceding claims, wherein the object (0) comprises at least one biological cell, a cell group, a cell component or a synthetic particle.

20. The method according to claim 19, in which, depending on the detected dielectric, geometric and / or optical properties, a distinction is made between normal and modified cells or between normal cells with different physiological properties.

21. The method according to claim 19, in which stem cells are recognized as a function of the detected dielectric, geometric and / or optical properties.

22. The method according to claim 19, 20 or 21, in which the dielectric, geometric and / or optical properties of the cell are detected as a function of at least one of the following parameters: - Frequency of the positioning field

- frequency of the deformation field,

- Voltage of the positioning field

- stress of the deformation field,

- temperature of the suspension or treatment liquids, - material composition of the suspension or treatment liquids,

- duration of the individual deformation, and

- Duration of several deformations.

23. The method according to at least one of claims 19 to 22, in which cell pairs or cell aggregates are measured and / or cell pairs are separated.

24. The method according to claim 22, in which the cell pairs or cell aggregates are brought together in the positioning field.

25. Measuring apparatus for examining at least one object, which comprises:

- A fluidic microsystem (10), which has a compartment (12) with at least one electrode arrangement (1-4, 1'-4 '), - A detector device (20), which is used for the electrical, geometric and / or optical measurement of object properties is set up, and

- A field shaping device (30) with at least one high-frequency generator (31), characterized in that

the field shaping device (30) can be switched between an operating state in which a high-frequency positioning field is generated in the compartment (12) with the at least one electrode arrangement (1-4, 1'-4 '), and an operating state, in which a deformation field is generated in the examination area (11) with the at least one electrode arrangement.

26. Measuring apparatus according to claim 25, in which the field shaping device (30) contains a switching device (32) with which it is possible to switch between the operating states.

27. Measuring apparatus according to claim 25 or 26, wherein the detector device (20) has a microscope (21) with a camera (22).

28. Measuring apparatus according to at least one of the preceding claims 25 to 27, in which the fluidic microsystem (10) with a fluidic device (40) for moving a suspension Sions- and / or a treatment liquid through the examination area (11) is equipped.

29. Measuring apparatus according to at least one of the preceding claims 25 to 28, in which a control device (50) is provided which is connected to the detector device (20) and the switching device (32).

30. Measuring apparatus according to claim 29, in which a control circuit is formed with the control device (50), in which the positioning field and / or the deformation field can be set or changed depending on the result of the previous detection.

31. Measuring apparatus according to at least one of the preceding

Claims 25 to 30, in which the electrode arrangement comprises electrodes with electrode tips, wherein the electrode tips of adjacent electrodes have edges running parallel at least in sections.

32. Measuring apparatus according to claim 31, in which the parallel edges are aligned parallel or perpendicular to a longitudinal direction of the compartment of the fluidic microsystem.

33. Measuring apparatus according to at least one of the preceding claims 25 to 32, in which the positioning field and the deformation field are switched on simultaneously in the second operating state.

34. Use of a fluidic microsystem with a high-frequency field cage for the investigation of deformation and / or relaxation properties of biological cells.