Method and device for automatically determining the position of a microsystem for manipulating a spherical microobject

Abstract

In a method for automated determination of the relative position (x/y/z) between a first hole (27) on a first microsystem component (11), which is preferably provided with a first channel (44) opening in the first hole (27), and at least one second hole (29) on a second microsystem component (12), which is preferably provided with a second channel (45) opening in the second hole (29), the two microsystem components (11, 12) lie in a liquid medium (41) at least in the region (25, 26) of the holes (27, 29). Under the supervision of a control device (15) controlled by a computer (22), the first and second microsystem components (11, 12) are displaced relative to one another into different relative positions (x/y/z). Electrical signals (37) are delivered to one of the two microsystem components (12, 12) and are recorded on the other of the two microsystem components (11, 12) as measurement values (38) which depend on the relative position of the two microsystem components (11, 12) with respect to one another. For different relative positions (x/y/z) between the two microsystem components (11, 12), measurement values (38) are determined, from which the relative position (xn/yn/zn) in which the two microsystem components (11, 12) are to be positioned with respect to one another in such a way that their holes (27, 29) are mutually aligned is ascertained in the control device (15).

Claims

1. A device for an automated contacting of at least one microscopic test object, comprising: a first microsystem component having a first hole; a second microsystem component having a second hole; a reaction vessel holding a liquid medium, the first and the second microsystem components being at least partially immersed in the liquid medium; mechanism for displacing the two microsystem components relative to one another at least in two mutually orthogonal spatial directions, a control device for controlling the mechanism for displacing of the first and the second microsystem components relative to one another; a source of one of a voltage and a current signal supplied to one of the first and the second microsystem components; circuitry for recording one of the voltage and the current signal on the other of the first and the second microsystem components, measurement values of which depend on relative position of the first and the second microsystem components with respect to one another and the control device being adapted to ascertain, from recorded signals, a relative position in which the first and the second microsystem components are positioned so that their respective holes are mutually aligned.

2. The device of claim 1, wherein the first microsystem component is provided with a first channel opening in the first hole and the second microsystem component is provided with a second channel opening in the second hole.

3. The device of claim 1, wherein one of the voltage and the current signal is delivered into the liquid medium by a signal electrode arranged in the first channel, and one of the voltage and the current signal is recorded by a measurement electrode arranged in the second channel.

4. The device of claim 1, wherein one of the voltage and the current signals are voltage pulses delivered into the liquid medium, pulse amplitudes of which are acquired as the measurement values at the second microsystem component.

5. The device of claim 1, wherein the first and the second microsystem components are displaced with respect to one another along at least two mutually orthogonal spatial axes, a first spatial axis extending approximately parallel to the longitudinal axes of the two holes into the a plane spanned by said two mutually orthogonal spatial axes.

6. The device of claim 5, wherein a rough relative position is determined along the first spatial axis by displacing the first and the second microsystem components along the first spatial axis into different relative positions while maintaining a fixed relative position along the second spatial axis and taking a measurement value for each relative position.

7. The device of claim 6, wherein the fixed relative position along the second spatial axis is selected as a function of dimensions of the microsystem components in a direction of the second spatial axis, between the first and the second microsystem components in the direction of the second spatial axis.

8. The device of claim 5, wherein a rough relative position is determined along a second spatial axis by displacing the first and the second microsystem components along a second spatial axis into different relative positions while maintaining a fixed relative position along the first spatial axis and taking a measurement value for each relative position.

9. The device of claim 8, wherein the fixed relative position along the first spatial axis is determined as a function of the rough relative position along the first spatial direction, between the two holes in a direction of the first spatial axis.

10. The device of claim 8, wherein an initial finely resolved relative position is determined along the first spatial axis by displacing the first and the second microsystem components along the first spatial axis into different relative positions while maintaining a rough relative position along the second spatial axis and taking a measurement value for each relative position.

11. The device of claim 10, wherein a subsequent finely resolved relative position is determined along the second spatial axis by displacing the first and the second microsystem components along the second spatial axis into different relative positions while maintaining the initial finely resolved relative position along the first spatial axis and taking a measurement value for each relative position.

12. The device of claim 11, wherein the first and the second microsystem components are displaced relative to one another along a third spatial axis which extends orthogonally to the first and the second spatial axes, a relative position being determined in the third spatial axis by displacing the first and the second microsystem components along the third spatial axis into different relative positions while maintaining a fixed relative position along the first and the second spatial axes and taking a measurement value for each relative position.

13. The device of claim 1, wherein the first microsystem component is configured for immobilizing a biological cell and the second microsystem component is configured for contacting the immobilized cell.

14. The device of claim 1, wherein a third microsystem component having a third hole is provided, and a relative position between the second and third holes is determined.

15. A method for an automated contacting of at least one microscopic test object between two holes, one hole on each of a first microsystem component and a second microsystem component, the two microsystem components located in a liquid medium at least in the region of the holes , the method comprising: A) determining a relative position between the two holes, utilizing electrical resistance as a measurement parameter B) immobilizing a microscopic test object on a first of the holes, C) aligning the two holes along a first spatial axis with a start distance at least approximately greater than a maximum diameter of the microscopic test object to be contacted, D) measuring a resistance in front of a second of the holes with a sensor electrode, E) reducing distance between the two holes along the first spatial axis by an amount, and F) repeating steps D) and E) until a predetermined change in the measured resistance in front of the second hole is detected indicting that the microscopic test object has been contacted.

16. The method of claim 15, wherein the start distance is at least two times the maximum diameter of the microscopic test object to be contacted.

17. A device for an automated contacting of at least one microscopic test object which is immobilized on a first hole and is then contacted through a second hole, comprising: at least one first microsystem component, on which the first hole is provided; at least one second microsystem component, on which the second hole is provided; a reaction vessel for holding a liquid medium, the two microsystem components being immersed in the liquid medium at least in the region of the holes; a mechanism for displacing the two microsystem components relative to one another at least in two mutually orthogonal spatial directions; a control device for controlling the mechanism for displacing in an automated fashion; a source of one of a voltage and a current signal electrically coupled to one of the first and the second microsystem components; and circuitry for recording one of the voltage and the current signal on the other of the first and the second microsystem components, the control device being adapted to ascertain, from measurement values for different relative positions between the two microsystem components, a relative position in which the two microsystem components are positioned so that their respective holes are mutually aligned.

Description

(1) Embodiments of the invention are represented in the appended drawing and will be explained in more detail in the description below.

(2) FIG. 1 shows a schematic plan view of the new device in its basic position;

(3) FIG. 2 shows a front view of the reaction vessel which is fitted in the device of FIG. 1 and into which two microsystem components are immersed with their tips;

(4) FIG. 3 shows the tips of two microsystem components in a schematic plan view and diagrams of measurement values for different relative positions of the microsystem components with respect to one another;

(5) FIG. 4 shows two microsystem components, the holes of which are mutually aligned, in a representation as in FIG. 3;

(6) FIG. 5 shows two microsystem components, on one of which a microscopic test object is immobilized, in a representation as in FIG. 3, a diagram of the determination of the diameter of the test object furthermore being shown; and

(7) FIG. 6 shows a variant of the device shown in FIG. 1.

(8) In FIG. 1, a device for automated determination of the relative position between two microsystem components 11 and 12 is schematically shown by 10.

(9) The device 10 comprises a mechanism 14, by means of which the two microsystem components 11 and 12 can be displaced relative to one another under the supervision of a computer-controlled control device 15 in the two orthogonal spatial axes x and y.

(10) To this end, the mechanism 14 comprises a carriage 16, on which the second microsystem component 12 is fastened. The first microsystem component 11 is arranged spatially fixed.

(11) The mechanism 14 furthermore comprises an x guide 17, on which a y guide 18 which can be displaced in the x direction, and on which the carriage 16 is mounted displaceably in the y direction, is arranged.

(12) The y guide is displaced by means of an x motor 19 in the x direction, and the carriage 16 is displaced by means of a y motor 21 in the y direction.

(13) The movements of the carriage 16 take place under the supervision of a computer 22, into which the control device 15 may be integrated.

(14) A reference point 23, the position or location of which is predetermined by the computer 22 in a coordinate system indicated at 24, is indicated by way of example on the carriage 16.

(15) In the basic position shown in FIG. 1, the carriage 16, i.e. the reference point 23, lies at the position x1/y1, which is indicated in the coordinate system 24.

(16) The two microsystem components 11 and 12 shall now be displaced relative to one another in such a way that their tips 25 and 26 are mutually aligned. More precisely, this involves mutually aligning a hole 27 having a longitudinal axis 28 on the first microsystem component 11 with a hole 29 having a longitudinal axis 31 on the second microsystem component 12, in such a way that they lie directly in front of one another so that the tips 25 and 26 almost touch with their end sides and the longitudinal axis 28 lies in the hole 29, while the longitudinal axis 31 lies in the hole 27. Preferably, the longitudinal axes 28 and 31 are to coincide when the holes 27 and 29 are mutually aligned according to the invention.

(17) In order to achieve this alignment of the holes 27 and 29, the microsystem component 12 must be displaced in the x direction by an actuation distance 32 and in the y direction by an actuation distance 33, as is indicated in FIG. 1 both on the device 10 and in the coordinate system 24.

(18) A problem in this case is now that the exact position of the tip 25, or of the hole 27, is just as poorly known as the exact position of the hole 29 with respect to the reference point 23.

(19) This uncertainty is due to the variations, for reasons of production, in the length of the tips 25 and 26 as well as in the location of the holes 27 and 29 on the tips 25 and 26.

(20) The actuation distances 32 and 33 are therefore unknown, and first need to be ascertained so that the microsystem component 12 can be displaced into a relative position, with respect to the microsystem component 11, in which the holes 27 and 29 are mutually aligned.

(21) According to the invention, therefore, a measurement arrangement 34 is provided in the computer 22, which arrangement interacts with a signal electrode 35 at the hole 27 in the first microsystem component 11 and with a measurement electrode 36 at the hole 29 in the microsystem component 12.

(22) By means of the measurement arrangement 34, a chronological sequence of voltage pulses 37 is output at the signal electrode 35, which are measured as a chronological sequence of measurement values 38 at the measurement electrode 36.

(23) So that this measurement is possible, the tips 25 and 26 of the microsystem components 11 and 12 are immersed in a reaction vessel 39 which is filled with a liquid medium 41, as can be seen particularly in the side view of FIG. 2.

(24) The reaction vessel 39 and the medium 41 are also required in order to guide test objects to the microsystem components 11, 12 and to carry out measurements or manipulations on these test objects, as will be described in more detail below.

(25) It can be seen from FIG. 2 that the microsystem components 11 and 12 are arranged with their longitudinal axes 28 and 31 inclined in the x/z plane, i.e. they point with their tips 25 and 26 obliquely from above, i.e. as seen in the z direction, into the medium 41.

(26) The arrangement is in this case organized in such a way that the projection of the longitudinal axes 28 and 31 in the x/y plane, as shown in FIG. 1, extends parallel to the y axis and transversely with respect to the y axis.

(27) Returning to FIG. 1, it can be seen that the voltage pulses 37 have a pulse amplitude 42 which has a higher value than the pulse amplitudes 43 of the measurement values 38 that are acquired at the measurement electrode 36 and are forwarded to the measurement arrangement 34.

(28) The difference in the pulse amplitudes 42 and 43 is a measure of the distance between the holes 27 and 29. This difference is now used according to the invention in order to ascertain both the actuation distance 32 and the actuation distance 33 in a search method, as will now be explained with the aid of FIG. 3.

(29) In FIG. 3, two microsystem components 11 and 12 are firstly shown in the region of their tips 25 and 26, respectively, on which the holes 27 and 29 are provided. These holes 27 and 29 are openings of channels 44 and 45, respectively, which extend in the microsystem components 11 and 12 and open on the end sides 25a and 26a, respectively, of the tips 25 and 26.

(30) The first channel 44 in the microsystem component 11 is, for example, configured in order to suck a test object onto the hole 27 and hold it by negative pressure, or eject it again by positive pressure.

(31) The second channel 45 is, for example, configured in order to carry out electrical measurements on the membrane of a test object, or from the interior of a test object, with the aid of the measurement electrode 36. The tip 26 may also be configured as a cannula, so that substances can be delivered onto a test object or into a test object, or substances can be sucked out of a test object, through the second channel 45, as will be explained in more detail below.

(32) The relative positioning of the microsystem components 11 and 12 with respect to one another corresponds to the starting situation as shown in FIG. 1. As represented in FIGS. 1 and 3, the first microsystem component 11 and the second microsystem component 12 are mounted in the device 10, the carriage 16 being in the starting position in which the reference point 23 occupies the relative position x1/y1, as shown in the coordinate system 24 in FIG. 1.

(33) With this arrangement, the end sides 25a and 26a have a safety distance 46 between them as seen in the x direction, while the tips 25 and 26 have a safety distance 47 from one another with their longitudinal sides 25b and 26b in the y direction.

(34) These safety distances 46 and 47 are selected as a function of the macroscopic geometrical dimensions of the microsystem components 11 and 12 being used, in such a way that there is no risk of collision when the microsystem component 12 is displaced starting from the basic position shown in FIG. 3 either only in the x direction or else only in the y direction.

(35) The search algorithm now proceeds in such a way that it initially displaces the microsystem component 12 stepwise in the x direction, measurement values 38 being taken for each relative position in the x direction (i.e. with a constant fixed position in the y direction).

(36) FIG. 3 shows a diagram 48 which represents the pulse amplitudes A of the measurement values 38 for different x values.

(37) For this given safety distance 47 in the y direction, there is a maximum of the measurement values 38 at the coordinate xn.

(38) The microsystem component 12 is now displaced into the starting position of FIG. 3 or into the proximity of the roughly estimated relative position xm for the x direction, and is then correspondingly moved stepwise in the y direction, in which case again a measurement value 38 is taken for each relative position with a constantly fixed relative position in the x direction.

(39) The profile of the pulse amplitudes A for different y values is represented in the diagram 49. At yn, the measurement values 38 have a maximum.

(40) From these two diagrams, it can now be seen that the holes 27 and 29 are at least roughly aligned with one another when the microsystem component 12 is displaced into the relative position xn/yn.

(41) Starting from the rough values xn/yn, the search method described above can be repeated, the safety distances 46 and 47 being reduced each time, but the microsystem component 12 not being displaced as far as the relative position xm/ym.

(42) In this way, the relative position xm/ym of the second microsystem component 12 with respect to the first microsystem component 11 is iteratively refined in such a way that the holes 27 and 29 are increasingly more accurately aligned with one another when the microsystem component 12 is actually displaced into the relative position xn/yn.

(43) This alignment of the two holes 27 and 29 with respect to one another is represented in FIG. 4.

(44) In FIG. 4, it can firstly be seen that neither the hole 27 nor the hole 29 lies centrally in the end side 25a or 25b, respectively, so that in the case of mutually aligned holes 27 and 29 the tips 25 and 26 may be arranged offset with respect to one another.

(45) In the alignment shown in FIG. 4, the holes 27 and 29 are mutually aligned in the sense of the present application since the midaxis 28 of the hole 27 extends in the hole 29, and the midaxis 31 of the hole 29 extends in the hole 27. In the ideal case, the midaxes 29 and 31 coincide exactly with one another when the holes 27 and 29 have been mutually aligned, that is to say when the second microsystem component 12 has been displaced into the relative position xn/yn, determined by the iteration method, with respect to the first microsystem component 11.

(46) It should also be mentioned here that the distance of the end sides 25a and 25b from one another lies in the micrometer distance, and merely a residual safety distance 50 which prevents contact of the end sides 25a and 26a remains.

(47) The channel 44 of the first microsystem component 11 moreover has a diameter 51 which lies in the range of from 3 to 5 μm, while the channel 45 in the second microsystem component 12 has a diameter 52 which is less than 1 μm.

(48) The positioning method has been described so far only for the x/y plane, although a comparable iteration method is also carried out in order to align the holes 27 and 29 with respect to one another in the direction of the z spatial axis.

(49) The method correspondingly proceeds in such a way that the two microsystem components 11 and 12 are arranged at a safety distance from one another in the x and/or y direction, whereupon the microsystem component 11 is then displaced along the z axis past the microsystem component 11 and a diagram corresponding to the diagrams 48 and 49 is recorded for the measured pulse amplitudes.

(50) In this way, the position zn in which the holes 27 and 29 are mutually aligned in the direction of the z spatial axis is also determined.

(51) After the method described above with the aid of FIGS. 1 to 4 has been carried out, a relative position xn/yn/zn in which the holes 27 and 29 are mutually aligned as shown with the aid of FIGS. 1 to 4 is stored in the computer 22.

(52) The device 10 is now ready for use, in order to immobilize a test object 53 on the microsystem component 11 and then contact it with the microsystem component 12, as will now be explained with the aid of FIG. 5.

(53) To this end, test objects 53 suspended in the medium 41 are introduced into the reaction vessel 39. In the first channel 44, a negative pressure is then generated so that test objects 53 diffusing past the first hole 27 are sucked onto the hole 27 and immobilized, i.e. held, there by negative pressure. If the test objects 53 are intended to be released again, then a positive pressure is generated in the channel 44.

(54) The fact that a test object 53 has been positioned on the hole 27 is recognized by the computer 22, for example by the fact that the negative pressure in the channel 44 increases.

(55) The second microsystem component 12 has now been aligned with the first microsystem component 11 in such a way that the midaxes 28 and 31 coincide, or deviate from one another so slightly that they pass through the other respective hole. In the direction of the x spatial axis, a distance 54 which is at least two times the usual diameter of an object of the same type as the test object 53 is provided between the end sides 25a and 26a.

(56) As already described, the test object is a microscopic test object whose usual diameter lies between 5 and 50 μm. The test object 53 may, for example, be a biological cell which is now contacted according to the patch clamp method.

(57) In order now to be able to position the tip 26 of the second microsystem component 12 on the test object 53, or inside the test object 53, the diameter of the test object 53 must first be determined.

(58) To this end, a sensor electrode 55 is arranged in the channel 45 at the hole 29 in the second microsystem component 12 and is connected to a circuit 56 which may in turn be integrated in the computer 22.

(59) By means of the circuit 56, the sensor electrode 55 can be operated in a manner known per se in the voltage clamp mode or in the current clamp mode, so that the resistance indicated at 57 in front of the microsystem component 12 can be calculated with the aid of the varying voltage or the varying current, respectively.

(60) When the end side 26a now approaches the test object 53, the resistance 57 initially remains constant, and the resistance value does not change until immediately before contact with the test object 53, for example increasing as shown in the diagram 58.

(61) The relative x position of the microsystem component 12 is in this case at xm. In this relative position, the end side 26a is still separated from the end side 25a by the difference xn-xm, this difference thus corresponding to the diameter of the test object 53.

(62) After the diameter of the test object 53 has now been ascertained, the computer 22 may initially check whether an appropriate test object 53 has actually been immobilized, or whether it is cell debris or excessively large or small particles which have been “picked out” from the medium 41.

(63) If the test object 53 is not suitable, it is released back into the medium 41 by positive pressure in the channel 44, and the next test object 53 is sucked on by negative pressure in the channel 44.

(64) If it is a suitable test object 53, however, the test object 53 may now be contacted on its membrane 59 by means of the second microsystem component 12.

(65) All the information required so that the tip 26 can be pressed with its end side 26a onto the membrane 59 is stored in the computer 22.

(66) The supervision of the immobilization of the test object 53 on the hole 27 may also be carried out with the aid of a sensor electrode 61 which is arranged in the first channel 44 of the first microsystem component 1 and is connected to a circuit 62, which may in turn be integrated in the computer 22.

(67) By means of the circuit 62, the sensor electrode 61 can be operated in a manner known per se in the voltage clamp mode or in the current clamp mode, so that the resistance indicated at 63 in front of the microsystem component 11 can be calculated with the aid of the varying voltage or the varying current, respectively.

(68) The time profile of the resistance value of the resistance 63 corresponds to the profile according to diagram 58, except that the time rather than the measured distance is now plotted on the x axis.

(69) After the test object 53 has been recognized as immobilized, the diameter of the test object 53 can then be determined in the described way, and from this it can initially be ascertained whether it is a suitable test object 53.

(70) Alternatively, however, the immobilized test object 53 may also be checked for its suitability for subsequent examinations by other methods, for example in an optical or electrical way.

(71) When the test object 53 has been qualified as suitable, the method is continued, otherwise it is discarded and a new test object is sucked on and checked for suitability.

(72) Depending on whether a measurement is then intended to be carried out on the membrane patch enclosed by the tip 26, or whether measurements are intended to be carried out on the entire cell, the membrane 59 is now perforated in the region of the hole 29 by applying a negative pressure in the channel 45, or the measurements which are carried out by means of the sensor electrode 55 are immediately started.

(73) Likewise, it is now possible to insert the tip 26 partially into the test object 53 so as to introduce substances into the test object 53 or suck substances out of the test object 53.

(74) Furthermore, it is possible to arrange the microsystem component 12 with a predetermined distance between the membrane 59 of the test object 53 and the end side 26a of the tip 26, in order then to deliver substances, which are intended to influence the behavior of the test object 53, onto the membrane 59 in pulses.

(75) Furthermore, after the production of a gigaseal, it is possible to introduce test substances into the medium 51 in order to measure the reaction of the test object 53 to the test substances.

(76) For these measurements, the sensor electrode 55 which is identical to the measurement electrode 36 is used. Likewise, the sensor electrode 61 is identical to the signal electrode 35.

(77) After/before or during this measurement, the test object 53 may be transferred onto the second hole 29 and moved away from the first hole 27. By virtue of the known relative geometrical conditions, the test object 53 can be positioned at a defined distance from the first hole 27, from which test substances are then delivered onto the test object 53. With the electrode 36/55, the reactions of the test object 53 to these test substances can then be measured.

(78) Of course, it is also possible to use not just one second microsystem component 12 but a plurality of microsystem components 12, for the holes of which the relative position with respect to the hole in the first microsystem component 11 is respectively determined.

(79) In this case, it is also possible to arrange the microsystem component provided for holding the test object 53 on the carriage 16 and to arrange a plurality of microsystem components in the reaction vessel 39, which are used for contacting in the patch clamp mode and for flushing with test substances.

(80) In such a case, for the microsystem component arranged on the carriage 16, in each individual case it is initially necessary to determine the relative position in which the hole of this microsystem component is aligned with each of the holes of the other microsystem components. The diameter of a captured test object, however, only needs to be determined once, and it can then respectively be taken into account when approaching the other microsystem components since the diameter is determined absolutely, in contrast to the relative positions of the various holes with respect to one another.

(81) In this way, the device according to the invention provides the possibility of carrying out the method according to the invention, and therefore of successively carrying out measurements and/or manipulations on different test objects in an entirely automated way.

(82) The precise determination of the relative positions of the individual microsystem components with respect to one another, into which they need to be brought so that the corresponding manipulations and/or measurements can be carried out, is then respectively carried out again and in an automated way when new microsystem components 11, 12 have been mounted in the device 10.

(83) Furthermore, a third microsystem component 65 having a third hole 66 may be connected in a mechanically fixed way to the first microsystem component 11 on a carrier 64, as schematically shown in FIG. 6.

(84) An electrode 67, which is connected via a cable 68 to the computer 22 of FIG. 1, is arranged in the hole 66. The electrode 67 may be used like the signal electrode 35 and/or like the sensor electrode 61, in order to determine the relative position between the hole 66 and the hole 29 in the described way, and/or in order to recognize whether a test object has been immobilized on the hole 66.

(85) The second microsystem component 12 can be displaced relative to the two other microsystem components 11, 65, which is indicated by an arrow 69. In this way, a test object immobilized on the first hole 27 can be taken and transported to the third microsystem component 65, where test substances can then be delivered onto the test object from the third hole 66. With the electrode 36/55, the reactions of the test object to the test substances are then recorded.

(86) In all these applications, the first microsystem component 11 is used in order to be able to immobilize a test object from a suspension and check whether it is suitable for the planned examinations. If the test object is qualified as suitable, then it can be contacted by the microsystem component 12 and optionally moved away from the first microsystem component 11. Test substances may then be delivered onto the test object from the first and/or a third microsystem component 11, 65.

(87) In this way, from a suspension containing only a few suitable test objects, it is possible rapidly to find a suitable test object in a reproducible and reliable way and then subject it to the measurements.

(88) These methods are only possible because, according to the new method, the relative positions of the holes 27, 29 and 66 with respect to one another can be determined automatically and with high accuracy.