Abstract:
A two-part coaxial needle for a pipetting device, in particular for use in microscopy of cell probes, makes it possible both to inject a liquid into a pipetting container and to remove by suction a liquid from the pipetting container. Both the drive for lowering the coaxial needle into the pipetting container, and injection and removal by suction of the liquid take place pneumatically by way of only one pressure source. The design of the coaxial needle and of the drive systems makes possible fast and reliable pipetting with high metering accuracy and a particularly compact pipetting unit which without mutual interference can be used with a multitude of widely-used microscope types.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This patent application is a United States national phase patent application of International Patent Application No. PCT/EP2009/008637, filed Dec. 3, 2009, which claims priority to European Patent Application No. 08170677.2, filed Dec. 4, 2008, which applications are incorporated herein by reference in their entirety for all purposes. 
     FIELD OF THE INVENTION 
     The present invention relates to a pipetting device for use in microscopy, in particular in microscopy relating to cell probes. 
     BACKGROUND OF THE INVENTION 
     Test series with living cells are of paramount importance in biological and medical research and development, and are used to a large extent in the pharmaceutical industry, for example in the development of new active substances and medicaments. In this context there is a requirement for observing a multitude of different cell probes quickly and as far as possible in an automated manner by means of transmission microscopy or fluorescence microscopy, and during observation or between subsequent observation steps to feed, in a controlled manner, to the individual probes liquid substances which can, for example, comprise an active medical substance or some molecular-biological modification (e.g. siRNA or immunostaining). 
     To this purpose cell probes are distributed to separate chambers of an object carrier or of a multiwell plate, and for observation said probes, on a stage, are sequentially fed into the optical path of a microscope. In this process inverse microscopes are frequently used in which the image recording device and often also at least part of the illumination device are arranged below the stage so that the space above the stage can predominantly be reserved for positioning and filling the probe. For quick automatic positioning of the probes, generally speaking probe positioners are provided on the stage, which probe positioners can accommodate a multitude of different probe containers, wherein the individual probe chambers of said probe positioners can quickly and very accurately be moved into the optical path of the microscope, and for focussing can also be displaced along the optical axis. Adding a liquid, for example an active substance, at a determined dilution ratio or mixing ratio to the individual probe chambers, or removing the liquid by suction from the probe chambers, in part still takes place manually, for example with the use of a microliter pipette. However, manual filling or removal by suction is not only time-consuming and labour-intensive, but also quite error-prone. 
     Furthermore, it is often necessary to carry out test series under controlled temperature conditions and atmospheric conditions so that the probe container, together with the stage and parts of the observation equipment and illumination device, is accommodated in a so-called climatic chamber, and consequently the probes are practically no longer accessible from the exterior. For this reason automated pipetting systems with movable pipettes were invented, which systems make it possible to quickly and reliably feed a liquid into selected pipetting containers in a controlled atmosphere. In this arrangement, the liquid infeed from a reservoir normally takes place with the use of pumps comprising micro motors; positioning and operation of the pipette also takes place with the use of electric motors. An inverse microscope with such an automated pipetting device within a climatic chamber is, for example, described in patent specification U.S. Pat. No. 7,092,151 B2. 
     The pipetting devices known from prior art are associated with a difficulty in that the pipette and the drive unit require comparatively large installation space, and as a result of this they could negatively affect operation of the microscope. This problem occurs in particular if the probes in transmission are to be illuminated from above the stage, because the illumination device and the pipetting device cause mutual interference. However, the same difficulties can also arise in test series in which the illumination can be exclusively from below the stage, while the microscope used normally provides for a second illumination device above the stage, which second illumination device, while not needed for the test series that is to be carried out, nonetheless for design reasons cannot be moved out of the optical path far enough to allow unrestricted operation of the automatic pipetting device. Many widely used microscope types are associated with such spatial restrictions and can therefore be used with automatic pipetting devices known from prior art only with functional limitations or only after possibly time-consuming and cost-intensive modifications. There is thus a requirement for a pipetting device that can be used without mutual interference with microscopes of a known and widely used design. 
     Furthermore, the probe chambers used are often sealed by means of a cover, for example a metal foil or a plastic film, in order to protect the probe from the ambient atmosphere. There is thus furthermore a requirement for an automatic pipetting device which despite such a sealing arrangement makes it possible to quickly, reliably and precisely feed a liquid into selected probe chambers. 
     Likewise, apart from allowing automated feed-in of a liquid into selected probe chambers, the pipetting device should also allow the quick and effective removal of a liquid from selected probe chambers. 
     These objects are met by the coaxial needle according to the invention according to claim  1 , or by the pipetting device according to the invention according to claim  5  and claim  11 . The invention also relates to the corresponding pipetting method according to claim  13 . The subordinate claims relate to preferred embodiments. 
     SUMMARY OF THE INVENTION 
     The coaxial needle according to the invention, for a pipetting device, comprises a hollow suction lance for drawing off a liquid from a pipetting container, as well as a hollow insertion lance that encloses the suction lance at least in part so that between an exterior wall of the suction lance and an interior wall of the insertion lance a liquids duct for feeding a liquid into a pipetting container is formed. 
     The liquids duct can, in particular, comprise the entire hollow space between the exterior wall of the suction lance and the interior wall of the insertion lance. 
     The coaxial needle according to the invention allows careful and precise metering of the quantity of liquid to be fed in, both with droplet injection and with injection in continuous flow. 
     In a preferred embodiment, the insertion lance coaxially encloses the suction lance. This makes it possible to achieve particularly even liquid feed-in. 
     In a further preferred embodiment the suction lance and/or the insertion lance are/is designed in the shape of a hollow cylinder or of a truncated hollow cone. 
     Furthermore, in a preferred embodiment the suction lance comprises a first open end for the uptake of a liquid from a pipetting container, and a second open end, which is arranged axially opposite the first open end, for the delivery of the taken-up liquid to a suction removal nozzle. 
     Furthermore, the insertion lance preferably comprises a first open end for the delivery of a liquid to a pipetting container, and a second open end, which is arranged axially opposite the first open end, for the uptake of a liquid from an insertion nozzle. 
     The liquid to be fed in can be fed to the liquids duct from the insertion nozzle by way of the second open end of the insertion lance, and can be fed into the pipetting container by way of the first open end of the insertion lance, which end is axially opposite the second open end. Likewise, a liquid from the pipetting container can be removed by suction through the first open end of the suction lance into the interior of the suction lance, from where it can be delivered to the suction removal nozzle by way of the second open end, which is situated axially opposite the first open end. By means of the coaxial needle according to the invention, liquids can quickly and in a metered manner be injected into the pipetting container, and they can also be removed from the pipetting container. 
     In a preferred embodiment, the first open end of the insertion lance comprises an insertion tip. In particular, the first open end of the insertion lance can be pointed so as to be bevelled. In this manner liquids can also be fed to pipette containers that are sealed by means of a cover, or they can be removed from such pipetting containers. 
     In a further preferred embodiment the first open end of the suction lance is arranged in the interior of the insertion lance. On the one hand, this provides an advantage in that the suction lance is effectively protected from damage when puncturing sealed pipetting containers. On the other hand, an arrangement of the suction lance in the interior of the insertion lance has an advantageous effect on liquid metering. The liquid entering the liquids duct flows around the suction lance and wets it, and prior to being fed into the pipetting container can collect at the open end of the suction lance to form a liquid droplet of a defined size. 
     In a preferred embodiment the second end of the insertion lance comprises an insertion funnel. This ensures simplified feed-in of the liquid from the insertion nozzle to the liquids duct. 
     In a further preferred embodiment the suction lance and the insertion lance can be moved along a common axial direction. In this way the lances can be lowered in order to feed in or remove by suction a liquid into the pipetting container. 
     The suction lance and the insertion lance can, in particular, be movable independently from each other along the axial direction. This makes it possible, for example, for the suction lance to be moved from the insertion lance and for the purpose of drawing off a liquid to be lowered down to the bottom of the pipetting container. 
     In a pipetting device according to the invention the second open end of the insertion lance is preferably connected to a first reservoir by way of a first connecting pipe, and furthermore the second open end of the suction lance is connected to a second reservoir by way of a second connecting pipe. 
     In this way the liquid to be fed into the pipetting container can be removed from the first reservoir by way of the first connecting pipe, whereas the liquid removed by suction from the pipetting container is fed to the second reservoir by way of the second connecting pipe. 
     The first connecting pipe can comprise a feed valve that is arranged between the insertion lance and the first reservoir and that is situated in close proximity to the second open end of the insertion lance, wherein the distance between the feed valve and the second open end is preferably smaller than ten times the internal diameter of the first connecting pipe, or no more than 2 cm. By means of a feed valve that is situated in close proximity to the second open end of the insertion lance, and thus to the liquids duct, the point in time of the injection and the injection dose can be determined with great accuracy. 
     In a preferred embodiment the second connecting pipe comprises a suction removal valve between the second open end of the suction lance and the second reservoir. 
     The feed valve and/or the suction removal valve are preferably electronically controllable 3/2 valves. 
     In a further preferred embodiment the first reservoir is connected to a first pressure source by way of a first pressure pipe. This makes it possible to feed a liquid to the liquids duct by way of the first connecting pipe in that the first reservoir is pressurised by means of the first pressure source by way of the first pressure pipe, wherein metering is controlled with the use of the feed valve. In this way a precisely metered quantity of liquid can be injected into the pipetting container by means of a pressure surge. 
     In a preferred embodiment the pressure source is a nitrogen pressure source. 
     In an advantageous embodiment the first pressure pipe comprises a pressure reducer and/or a filter between the first reservoir and the first pressure source. The pressure reducer makes it possible to set the working pressure range, while the filter protects the reservoir and the liquid stored therein from contamination. 
     In a preferred embodiment the second reservoir is connected to a negative-pressure source by means of a second pressure pipe. In this way the second reservoir and the second connecting pipe, which is connected to said second reservoir, can be subjected to negative pressure so that by means of controlling the suction removal valve a liquid can be drawn off from the pipetting container through the suction lance into the second connecting pipe and from there into the second reservoir. 
     In a further preferred embodiment the negative-pressure source comprises a vacuum pump. In an alternative embodiment the negative-pressure source comprises a venturi nozzle that is connected to the first pressure source, which venturi nozzle converts overpressure of the first pressure source to negative pressure. In this embodiment, liquids can be both injected into the pipetting container and removed by suction from the pipetting container with the use of a single pressure source. 
     In an advantageous embodiment the insertion lance is connected to a first drive unit, and the suction lance is connected to a second drive unit. The first and/or the second drive unit are preferably pneumatic drive units. 
     As a result of the drive of the insertion lance and of the suction lance being implemented pneumatically, the design space required by the drive unit can be significantly reduced. Therefore the coaxial needle with the two drive units can advantageously be taken together to form a movable pipetting unit whose design height along an axial direction of the coaxial needle does not exceed 4 cm. The low design height in axial direction makes it possible, in particular, to move such a pipetting unit into the space between a stage or pipetting container and an illumination device of a microscope without this impeding operation of the microscope or requiring any design modification of the microscope. 
     In a preferred embodiment the first drive unit comprises a first pressure piston as well as a first connecting element and a first fastening element or fastening means, wherein the first fastening means can be connected to the insertion lance, and by way of the first connecting element can be connected to the first pressure piston. The second drive unit comprises a second pressure piston as well as a second connecting element and a second fastening element, wherein the second fastening element or fastening means is connected to the suction lance, and by way of the second connecting element can be connected to the second pressure piston. 
     In a preferred embodiment the first fastening element furthermore comprises the insertion nozzle for the uptake of a liquid from the first reservoir, and the second fastening element comprises the suction removal nozzle for delivery of the taken-up liquid to the second reservoir by way of the second connecting pipe. In this way the design space required by the pipetting unit can be further reduced. 
     In a further preferred embodiment the first fastening element is connected to the second fastening element by way of a spring, and can be connected by way of a driving pin. In this manner the insertion lance and the suction lance can together be lowered into the pipetting container with the use of only a single drive unit, as will be explained below with reference to an exemplary embodiment. 
     In an advantageous embodiment the first drive unit is connected to a second pressure source by way of a third pressure pipe, and the second drive unit is connected to said second pressure source by way of a fourth pressure pipe. In a preferred embodiment the second pressure source is identical to the first pressure source. In such an arrangement both the lances of the coaxial needle, and the liquids, can be driven with the use of a single pressure source. In this way a particularly compact and efficient pipetting device is implemented. 
     In a preferred embodiment the third pressure pipe comprises a first quick-exhaust throttle valve as well as a first drive valve, wherein the first quick-exhaust throttle valve is arranged between the first pressure piston and the first drive valve. 
     The quick-exhaust throttle valve results in a slower pressure build-up at the first drive unit, thus making it possible to reduce the movement speed during lowering of the insertion lance along its axial direction. 
     Correspondingly the fourth pressure pipe can comprise a second quick-exhaust throttle valve as well as a second drive valve, wherein the second quick-exhaust throttle valve is arranged between the second pressure piston and the second drive valve. 
     In a preferred embodiment the first drive valve and the second drive valve are electronically controllable 3/2 valves. 
     In a preferred embodiment the pipetting device according to the invention comprises a pipetting unit that can be moved in a direction perpendicular to an axial direction of the coaxial needle, which pipetting unit comprises the coaxial needle together with the first drive unit and the second drive unit, wherein the design height of the pipetting unit along an axial direction does not exceed 4 cm. 
     The invention also relates to a pipetting device with a pipette for feeding a liquid into a pipetting container and with a drive unit for moving the pipette, wherein the drive unit is a pneumatic drive unit and is connected to a first pressure source by way of a third pressure pipe. 
     As explained above, the pneumatic drive makes it possible to implement a particularly compact pipetting device, in particular a pipetting unit of a particularly low design height along an axial direction of the pipette, which pipetting device comprises the pipette and the drive unit. 
     In an advantageous embodiment the pipetting device additionally comprises a first connecting pipe that connects the pipette to a first reservoir, wherein the first reservoir is connected to the first pressure source by way of a first pressure pipe. In this way both the drive unit for moving the pipette and the injection device for feeding-in the liquid can be operated by way of a shared pressure source so that again a particularly compact and efficient pipetting device results. 
     The pipette of the pipetting device according to the invention can furthermore be designed for drawing off a liquid from a pipetting container, and can be connected to a second reservoir by way of a second connecting pipe, wherein the second reservoir is connected to a negative-pressure source by way of a second pressure pipe. 
     The negative-pressure source in turn can comprise a venturi nozzle that is connected to the first pressure source, as a result of which the advantages described above arise. 
     The invention also relates to a microscope with a pipetting device with the characteristics described above. In particular, the microscope can be an inverse microscope. 
     Lastly, the invention relates to a method for pipetting in which method a coaxial needle with a hollow suction lance and with a hollow insertion lance that encloses the suction lance at least in part is positioned above a pipetting container, the suction lance and the insertion lance are together moved into the pipetting container, a liquid is fed from a first reservoir for liquid to a liquids duct that is arranged between an exterior wall of the suction lance and an interior wall of the insertion lance, and the liquid is fed from the liquids duct into the pipetting container. 
     In a preferred embodiment a first end of the suction lance is positioned in the interior of the insertion lance so that the distance between a first open end of the suction lance, which end is opposite the pipetting container, and a first open end of the insertion lance, which open end is opposite the pipetting container, during feed-in of the liquid into the pipetting container is at least 1 mm. As explained above, in this manner the droplet formation in the liquids duct is enhanced, and injection without any splashing becomes possible. 
     In a further preferred embodiment the liquid is fed droplet by droplet into the pipetting container, wherein the volume of the droplets is set by means of selecting the distance between a first open end of the suction lance, which end is opposite the pipetting container, and a first open end of the insertion lance, which end is opposite the pipetting container. During feed-in of the liquid into the pipetting container the distance is preferably at least 1 mm. 
     In a preferred embodiment the step of moving the suction lance and the insertion lance involves penetration of a cover of the pipetting container. 
     In a further preferred embodiment the liquid is injected into the pipetting container by means of a pressure surge. As explained above, in this way the injected quantity of liquid can be metered out effectively and the point in time of injection can be determined with precision. 
     Furthermore, in a preferred embodiment the method according to the invention additionally comprises the step of connecting a second open end of the suction lance to a first negative-pressure source, as well as the removal by suction of excess liquid from the liquids duct through the interior of the suction lance into a second reservoir for liquid. 
     Removing by suction any excess liquid from the liquids duct reduces the danger of unintended feed-in of excessive quantities of liquid into the pipetting container. Furthermore, with the use of the method according to the invention the liquids duct can be cleaned in a simple manner by means of removal by suction. This is advantageous in particular in those instances where during a test series or between subsequent test series the liquid to be fed in is to be changed, and contamination of a subsequently used liquid with remainders of the previously used liquid must be avoided. 
     In a further preferred embodiment the method according to the invention additionally comprises the step of moving the suction lance along a shared axial direction of the suction lance and the insertion lance until a first open end of the suction lance is immersed in a volume of liquid within the pipetting container, and further comprises connecting a second open end of the suction lance to a second negative-pressure source and removing by suction a liquid from the pipetting container through the interior of the suction lance into a third reservoir for liquid. 
     Because the suction lance can be lowered along a shared axial direction independently of the insertion lance, a liquid can be effectively and completely removed by suction from the interior of the pipetting container even if the liquid level is low. 
     In a preferred embodiment the second negative-pressure source and the first negative-pressure source are identical, and/or furthermore the third reservoir for liquid and the second reservoir for liquid are identical. 
     Due to the compact design and the low design height the coaxial needle according to the invention or the pipetting device according to the invention can be used together with many widely-used types of microscopes without mutual interference, and they additionally make it possible to implement fast and controlled automated injection of liquids into a pipetting container, as well as the removal by suction of liquids from a pipetting container. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
       The numerous advantages of the coaxial needle according to the invention as well as of the pipetting device according to the invention and of the pipetting method according to the invention are best understood with reference to the detailed description of the enclosed drawings which show the following: 
         FIG. 1   a  a diagrammatic overview drawing of a preferred embodiment of the pipetting device according to the invention; 
         FIG. 1   b  an enlarged view of the quick-exhaust throttle valves used in the pipetting device of  FIG. 1   a;    
         FIGS. 2   a - 2   d  a preferred embodiment of a coaxial needle according to the invention in various operating positions; 
         FIGS. 3   a - 3   c  a diagrammatic lateral view of the drive device for operating the coaxial needle according to the invention in various operating positions; 
         FIGS. 4   a - 4   c  a diagrammatic front view of the drive device in the various operating positions of  FIGS. 3   a - 3   c ; and 
         FIG. 5  an improvement of the pipetting device according to the invention with several reservoirs for liquid. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       FIG. 1   a  shows a diagrammatic overview of a pipetting device according to the invention with the components essential to its operation. In the drawing of  FIG. 1   a  the pressure pipes comprising supply pressure are shown in double solid lines, the pressure pipes comprising reduced overpressure or negative pressure are shown in single solid lines, while the control signal lines are shown in dashed lines. The centre of the pipetting device shows a coaxial needle  10  according to the invention, which is partly immersed in a pipetting container  12 . The pipetting container  12  can, for example, be a cylindrical probe chamber of a well plate or of a Petri dish, which probe chamber comprises a cell probe and if applicable a liquid that has been fed to the probe, and has been inserted into the optical path of an inverse optical microscope (not shown in  FIG. 1   a ). 
     Below, the design and function of the coaxial needle  10  are described in detail with reference to  FIGS. 2   a - 2   d.    
       FIG. 2   a  shows the coaxial needle  10  in a parked position above the pipetting container  12  sealed with a cover foil  30 . As shown in the diagram, the coaxial needle  10  comprises a suction lance  14  as well as an insertion lance  16 . Both lances are designed in the form of a metallic hollow cylinder, wherein the insertion lance  16  is shorter than the suction lance  14  and comprises an internal diameter that exceeds the external diameter of the suction lance. 
     In the embodiment described, both the suction lance  14  and the insertion lance  16  are in the shape of hollow regular cylinders. However, depending on the field of use of the needle it is possible to use hollow bodies of different shapes. In the context of the present invention the term “suction lance” refers to any hollow body that is suitable for drawing off a liquid. Correspondingly the term “insertion lance” refers to any hollow body that in cooperation with the suction lance is suitable for feeding a liquid into a pipetting container  12 . The insertion lance can, in particular, be designed to pierce a cover foil  30 . 
     As shown in  FIG. 2   a , the suction lance  14  comprises a first open end  18  that is opposite the pipetting container  12 , and a second open end  20  that is opposite the first open end  18  along an axial direction  26 . Correspondingly the insertion lance  16  comprises a first open end  22  that is opposite the pipetting container  12 , and a second open end  24  that is opposite the first open end  22 . The first open end  18  of the suction lance  14  is inserted into the insertion lance  16  and is slidable therein, wherein the insertion lance  16  encloses the suction lance  14  partly coaxially, but the suction lance  14  due to its longer length always at least on one side projects from the insertion lance  16 . Since the internal diameter of the insertion lance  16  is larger than the external diameter of the suction lance  14 , at the position where the insertion lance  16  coaxially encloses the suction lance  14  a liquids duct  28  is formed between an outer wall of the suction lance  14  and an inner wall of the insertion lance  16 . 
     In the arrangement shown in  FIG. 2   a , the first open end  22  of the insertion lance  16  is designed in the form of a tip bevelled relative to the axial direction  26 . This tip is used to penetrate the cover foil  30  when the coaxial needle  10  is lowered into the pipetting container  12 . 
     At its second open end  24  the insertion lance  16  comprises an insertion funnel  32  by way of which from an insertion nozzle  34  connected to the insertion lance  16  a liquid can be inserted into the liquids duct  28 . In contrast to this, at its second open end  20  the suction lance  14  is connected to a suction removal nozzle  36 , by way of which a liquid can be removed by suction from the interior of the suction lance  14 . 
     As shown in  FIG. 1 , by way of a first connecting pipe  38  and a feed valve  40 , the insertion nozzle  34  is connected to a first reservoir  42  in which a liquid that is to be injected into the pipetting container  12  is stored. The first connecting pipe  38  can be a flexible plastic hose. The first reservoir  42  is connected to a pressure source  48  by way of a first pressure pipe  44  and a pressure switch  46 . The pressure source  48  can be a pressure source that is operated with the use of nitrogen as a working gas, which pressure source provides a working pressure of approximately 5 bar. By way of a pressure reducer  50 , which is arranged between the pressure source  48  and the first reservoir  42 , this working pressure is converted to a reduced pressure of approximately 0.2 to 0.3 bar. A filter  52  arranged downstream of the pressure reducer  50  protects the first reservoir  42  and the liquid stored therein from contamination. 
     The feed valve  40  is a so-called 3/2-valve that is magnetically operated and that provides three connections with two switching states. The illustration in  FIG. 1  shows both possible switching states of the feed valve  40  side by side, namely on the left-hand side the feed valve  40  open for the infeed of a liquid from the first reservoir  42  to the coaxial needle  10 , in which feed valve  40  the connections  1  and  2  are connected in the direction of passage, and on the right-hand side the blocked valve, in which the connections  2  and  3  are connected and the infeed of liquid from the first reservoir  42  to the pipette  10  is blocked. 
     By way of a first control line  54 , which connects the feed valve  40  to a control unit  56 , it is possible to electronically change, in a preselected timing pattern, between the two switching states of the feed valve  40 , and in this way to control the infeed of liquid to the pipette. Typical switching times of such a valve range between 10 ms and 50 ms. An input/output unit  58  that is connected to the control unit  56  is used to select and enter suitable timing sequences and to control and monitor the pipetting device. 
     The first feed valve  40  is arranged so as to be in close proximity to the coaxial needle  10 , wherein the distance between the second open end  24  of the insertion lance  16  and the feed valve  40  is preferably smaller than ten times the diameter of the first connecting pipe  38 , or no more than 2 cm. In this way the quantity and the point in time of the infeed of liquid can be determined particularly accurately. 
     The second open end  20  of the suction lance  14  is connected to a second reservoir  62  by way of the suction removal nozzle  36  and a second connecting pipe  60   a ,  60   b , which reservoir  62  in turn is connected to a negative-pressure source  66  by way of a second pressure pipe  64 . The second connecting pipe  60   a ,  60   b  can also comprise a flexible plastic hose. 
     In a first embodiment the negative-pressure source  66  not only comprises a conventional vacuum pump  68 , for example a sliding-vane rotary pump, but also a buffer volume  70  as well as a needle-valve bypass  72 . 
     The drawing in  FIG. 1  also shows an alternative embodiment with a negative-pressure source  66 ′. In this alternative embodiment the second reservoir  62  is coupled to the low-pressure connection of a venturi nozzle  74  by way of a second pressure pipe  64 ′. The venturi nozzle  74  in turn is connected to the first pressure pipe  44  and thus to the pressure source  48  and in this way transforms overpressure in the first pressure pipe  44 , which overpressure is provided by the pressure source  48 , to negative pressure in the second pressure pipe  64 ′. 
     By way of a suction removal valve  76  in the second connecting pipe  60   a ,  60   b , the suction removal nozzle  36  and the second open end  20 , connected to it, of the suction lance  14  can be controlled with negative pressure (in the embodiment shown −50 mbar to −100 mbar) so that a liquid from the pipetting container  12  can be removed by suction, through the suction lance  14 , the second connecting pipe  60   a ,  60   b  and the suction removal valve  76 , into the second reservoir  62 . A filter  78  in the second pressure pipe  64  or  64 ′ prevents liquids or their outgassing products from being removed by suction from the second reservoir  62  into the negative-pressure source  66  or  66 ′. 
     As is the case in the feed valve  40  described above, the suction removal valve  76  can be an electronically controlled 3/2 valve, which is connected to the control unit  56  by way of a second control line  80 . The first switching state, shown on the left-hand side in the illustration of  FIG. 1 , in which switching state the connections  2  and  3  are connected, is the active switching state of the suction removal valve in which the coaxial needle  10  is connected to the negative-pressure source  66  or  66 ′ by way of the second connecting pipe  60   a ,  60   b . The inactive or closed switching state, in which the connections  1  and  2  of the suction valve  76  are interconnected, is shown on the right-hand side adjacent. 
     In the embodiment shown in  FIG. 1 , the feed valve  40  and the suction removal valve  76  can be coupled by way of an intermediate connection  82  which connects connection  3  of the feed valve with connection  2  of the suction valve. This intermediate connection  82  makes it possible to empty and evacuate the first connecting pipe  38  and the insertion nozzle  34  and thus the liquids duct  28  by means of the negative-pressure source  66  or  66 ′ when all the liquid is to be discharged from the pipetting device, for example for cleaning work or maintenance work. 
     The diagrammatic overview drawing of  FIG. 1  also shows the pneumatic drive unit for operating the coaxial needle  10 . The pipetting device shown provides for two separate drive units  84  and  86  for the insertion lance  16  and the suction lance  14  so that the insertion lance  16  and the suction lance  14  can be moved independently of each other along their common axial direction  26 . 
     Below, the drive units  84  and  86  are described in detail with reference to  FIG. 3   a.    
     The first drive unit  84  comprises a first pressure piston  88  as well as a first connecting element  90  and a first fastening element  92 . The first fastening element  92  is directly connected to the insertion lance  16  and also comprises the insertion nozzle  34  (not shown in the illustration of  FIG. 3   a ). The first fastening element  92  is connected to the first pressure piston  88  by way of the first connecting element  90 . In this manner, by way of the first connecting element  90 , the movement of the first pressure piston  88  is translated into a movement of the insertion lance  16  along the axial direction  26  (compare  FIG. 2 ). 
     The second drive unit  86  for moving the suction lance  14  is designed in a similar manner; it comprises a second pressure piston  94 , a second connecting element  96  that is connected to the second pressure piston  94  and the first connecting element  90 , as well as a second fastening element  98 . The second fastening element  98  is directly connected to the suction lance  14  and also comprises the suction removal nozzle  36  (not shown in the illustration of  FIG. 3   a ). When the second pressure piston  94  is activated, the second connecting element  96  acts on the second fastening element  98  and in this manner makes it possible for the suction lance  14  to move along the axial direction  26 . 
     The drive unit according to the invention as well as the design, according to the invention, of the coaxial needle  10  make it possible to implement a movable pipetting unit  126  which comprises both the coaxial needle  10  and the first drive device  84  and the second drive unit  86 , with the design height along the axial direction  26  of said pipetting unit  126  being low enough for the pipetting unit  126  to be able to be inserted between a pipetting container  12  and an illumination unit, arranged above the pipetting container  12 , of an inverse optical microscope of conventional design, without microscopy operation and pipetting operation interfering with each other. In particular, it is possible to achieve pipetting units  126  with design heights along the axial direction  26  of less than 4 cm. 
     As shown in the overview drawing of  FIG. 1 , the first pressure piston  88  of the first drive unit  84  is connected to the first pressure pipe  44  and thus to the pressure source  48  by way of a third pressure pipe  100 . By way of two drive valves  102  and  104  the third pressure pipe  100  is coupled to the first pressure pipe  44  so that depending on the switching state the first pressure piston  88  can be subjected to pressure on both ends, wherein pressurisation from one end is translated into a downward movement of the insertion lance  16  by way of the first connecting element  90 , whereas pressurisation from the opposite end is translated into an upward movement of the insertion lance  16  along the axial direction. 
     The drive valves  102  and  104  again are electronically controllable 3/2 valves whose switching states in the illustration of  FIG. 1  among each other are shown, with said valves again being connected to the control unit  56  by way of the control lines that are shown in dashed lines. 
     In both branches of the third pressure pipe  100 , quick-exhaust throttle valves  106  and  108  are arranged upstream of the first pressure piston  88 , which quick-exhaust throttle valves  106  and  108  delay pressure build-up at the first pressure piston  88  and in this manner make it possible to set the movement speed of the insertion lance  16 . 
     The second pressure piston  94  is correspondingly connected to the first pressure pipe  44 , which in turn comprises two electronically controllable 3/2 valves  112  and  114  that are connected to the control unit  56 . In this arrangement the drive of the second pressure piston  94  takes place analogously to the above-described drive of the first pressure piston  88 , wherein again quick-exhaust throttle valves  116  and  118  are provided in both branches of the fourth pressure pipe  110 . 
     The action and function of the quick-exhaust throttle valves  106 ,  108 ,  116  and  118  that are used is diagrammatically illustrated in the enlarged section of  FIG. 1   b . Such a valve comprises a nonreturn valve  132  that in the case of a flow from the end of the throttle valve, which end faces the pressure source  48  and in  FIG. 1   b  is designated A, to the end of the valve that faces the lifting cylinder of the pipetting unit and that in  FIG. 1   b  is designated B blocks said flow while enabling a flow in the opposite direction. Furthermore, the throttle valve comprises a bypass  134  which bypasses the nonreturn valve  132  and that comprises a reducing valve  136  that can be regulated. 
     The pressure build-up on the lifting cylinder takes place more slowly because the pressure medium can flow in the flow direction A→B only through the bypass  134  whose capacity is limited by the reducing valve  136 . In contrast to this, the pressure reduction on the lifting cylinder can take place suddenly because in the flow direction B→A both the nonreturn valve  132  and the reducing valve  136  are open to the pressure medium. With suitable selection of the flow-through capacity at the reduction valve  136 , the pressure build-up and thus the movement speed of the insertion lance  16  or the suction lance  14  can be set accordingly. 
     For pipetting, the coaxial needle  10  as well as the pipetting unit  126  comprising the first drive unit  84  and the second drive unit  86  are positioned above a selected pipetting container  12 . Such positioning can take place on the one hand in that, by means of a movable positioning device on the stage of a microscope, the pipetting container  12  is moved underneath the coaxial needle  10 . As already explained, as an alternative, the pipetting device according to the invention also makes it possible for the pipetting unit  126  to be designed so as to be movable. To this effect the pipetting unit  126  can then be connected to a drive device (not shown in the illustration of  FIG. 1 ), which drive device makes it possible for the pipetting unit  126  to move along the plane of the stage and if need be also perpendicularly to said plane. For example, for filling and emptying a selected chamber of the pipetting container, a movable pipetting unit  126  can be swung into the optical path of the microscope and during the subsequent microscopy process can be swung out of the optical path. This ensures trouble-free microscopy operation even for observation in transmission. 
     Because of the low design height of the pipetting unit  126  along the axial direction  26 , the pipetting device  126  according to the invention can be used together with a multitude of commonly used microscope models and designs without pipetting operation and microscopy operation causing mutual interference. In particular, the coaxial needle  10  can be inserted into the optical path between the stage with the pipetting container  12  and an illumination device of the microscope, which illumination device is arranged above the stage. The pipetting device according to the invention can thus be used irrespective of the microscope that is used for observation providing for illumination from above the object, from below the object, or, as is the case in the microscope described in patent specification U.S. Pat. No. 7,092,151 B2, selectively from above or below the stage. This is one of the special advantages of the coaxial needle according to the invention and of the pipetting device according to the invention. 
     Furthermore, due to its compact design the pipetting device according to the invention is particularly suitable for use in climatic chambers. 
     The method for pipetting is explained below with reference to the embodiments shown in  FIGS. 2 ,  3  and  4 . In this arrangement  FIGS. 2   a ,  3   a  and  4   a  show the coaxial needle  10  in a parked position;  FIGS. 2   b ,  2   c ,  3   b  and  4   b  show the coaxial needle  10  in an injection position, and  FIGS. 2   d ,  3   c  and  4   c  show the coaxial needle  10  in a suction removal position. 
     In the parked position shown in  FIGS. 2   a ,  3   a  and  4   a  the coaxial needle  10  is situated above a pipetting container  12  that is sealed by means of a cover foil  30 , which pipetting container  12  contains the cell probe to be investigated. 
     In order to insert the coaxial needle  10  into the pipetting container  12 , pressure is applied to the first pressure piston  88  by way of the third pressure pipe  100  so that the insertion lance that is connected to the first pressure piston  88  by way of the first fastening element  92  and the first connecting element  90  moves downwards along the axial direction  26 . At the same time the second connecting element  96 , which is coupled to the first connecting element  90 , is made to establish contact with the second fastening element  98 , as illustrated in the diagram of  FIG. 3   b . As shown in the diagram of  FIG. 3   a , the first fastening element  92  of the insertion lance  16  is coupled to the second fastening element  98  of the suction lance  14  by way of a spring  120  that is released in the parked position and by way of a driving pin  130 . As shown in  FIG. 4   a , the first fastening element  92  and the second fastening element  98  are additionally guided together in a guide rail  128  that extends in axial direction  26 . When the first fastening element  92  and with it the insertion lance  16  moves along the guide rail  128  into the pipetting container  12 , a pin  130 , which is connected to the first fastening element  92  and whose widened cover surface engages the second fastening element  98 , pulls the suction lance  14 , which is firmly connected to the second fastening element  98 , along. While maintaining their relative positions, both the insertion lance  16  and the suction lance  14  move into the pipetting container  12 , and the insertion tip of the insertion lance  16  penetrates the cover foil  30 . The coaxial needle is then in the injection position shown in  FIGS. 2   b  and  2   c , as well as  3   b  and  4   b.    
     As a result of activation of the feed valve  40 , the liquid to be fed-in is channeled under pressure from the first reservoir  42 , by way of the first connecting pipe  38 , the insertion nozzle  34  and the insertion funnel  32 , into the liquids duct  28  between the suction lance  14  and the insertion lance  16 . As shown in  FIG. 2   b , the liquid flows around the suction lance  14  and at its first open end  18  collects to form a droplet. In the embodiment shown the distance between the first open end  18  of the suction lance  14 , which open end is opposite the pipetting container  12 , and a first open end  22  of the insertion lance, which end  22  is opposite the pipetting container  12 , during feed-in of the liquid into the pipetting container  12  is approximately 1 mm. By means of suitable selection of this distance, which in the embodiment shown can be adjusted by displacement of the pin  130 , the droplet size and thus the volume of injected liquid can be set correspondingly. By means of a pressure surge of the feed valve  40  the droplet is detached from the coaxial needle  10  and falls into the pipetting container  12 . Apart from droplet injection, injection of the liquid in constant flow is also possible in that the feed valve  40  is kept open for an extended period of time by way of the control unit  56 . 
     By connecting the second open end  20  of the suction lance  14  to the negative-pressure source by way of the second connecting pipe  60   a ,  60   b  and the suction removal valve  76 , in a subsequent step, illustrated in  FIG. 2   c , if required after detachment of the droplet, any remaining liquid still in the liquids duct  28  can be removed by suction into the second reservoir  62  by way of the interior of the suction lance  14 . 
     Subsequently the coaxial needle  10  can be removed from the pipetting container  12  in that as a result of activation of the first drive unit  84  the insertion lance  16  is moved along the guide rail  128  back into the home position. Since the second fastening element  98  of the suction lance  14  is connected to the first fastening element  92  of the insertion lance  16  by way of the spring  120 , in this process at the same time the suction lance  14  is moved back from the pipetting container  12  while said suction lance  14  maintains its relative position to the insertion lance  16 . The coaxial needle  10  can then be positioned above a further pipetting container in order to repeat the injection process. 
     However, the pipetting device according to the invention can also be used for removing by suction a liquid from a pipetting container  12 . To this effect, according to the method described above, the insertion lance  16  and the suction lance  14  are first jointly inserted into a selected pipetting container  12 . Following the feed-in of such a liquid, or as an alternative also without such a preceding injection step, to this effect the coaxial needle  10  is moved to the suction removal position shown in  FIGS. 2   d ,  3   c  and  4   c . Starting from the injection position shown in  FIGS. 2   b ,  2   c ,  3   b  and  4   b , the second pressure piston  94  is activated by way of the fourth pressure pipe  110  so that the second connecting element  96 , which is connected to the second pressure piston  94 , engages the second fastening element  98 , and the suction lance  14  under tension of the spring  120  is lowered in axial direction  26  along the guide rail  128  until the first open end  18  of the suction lance  14  is immersed into the volume of liquid  124  that has collected in the pipetting container  12 . In this process the position of the insertion lance  16  remains unchanged. By connecting the second open end  20  of the suction lance  14 , by way of the suction removal nozzle  36  and the second connecting pipe  60   a ,  60   b , to the negative-pressure source  66  or  66 ′, the volume of liquid  124  collected in the pipetting container  12  can subsequently partly or completely be removed by suction through the interior of the suction lance  14  into the second reservoir  62 . After completion of the suction process, the spring  120  is released as a result of the return movement of the drive unit  86  or of the connecting element  96  so that the fastening element  98  is pushed upwards as a result of the spring force, and the suction lance  14  that is connected to the fastening element  98  bounces back along the axial direction  26  into its home position shown in  FIGS. 2   c ,  3   b  and  4   b . The method can now be continued as described above. 
     According to the method described above, with the pipetting device according to the invention liquids can quickly, reliably and in a precisely metered manner be fed into a sealed pipetting container or removed from a sealed pipetting container. However, in many test series sequential feed-in or removal of different liquids or of a liquid in different concentrations is desired. It is often necessary to prevent mutual contact among liquids so as to prevent any contamination or undesirable reaction. 
     To this purpose, the method according to the invention and the device according to the invention can be designed to comprise several separate liquids circulation systems, each comprising a coaxial needle while for the remainder corresponding to the embodiment described above. In order to inject one of the liquids into a selected pipetting container, or in order to remove by suction one of the liquids from a selected pipetting container the corresponding coaxial needle can then be selected in the revolver system. 
     As an alternative, instead of using several coaxial needles, a single coaxial needle can also be used, which is designed to be connected to different liquids circulation systems and which furthermore can be connected to a separate cleaning circulation system. Between the connection to different liquids circulation systems in this way cleaning of the coaxial needle can be carried out in order to effectively prevent any contamination. Cleaning can take place either by rinsing the pipe system with a separate rinsing liquid or by removal by suction of the remaining pipetting liquids from the liquids circulation system. 
       FIG. 5  shows a diagrammatic overview of a corresponding improvement of the pipetting device according to the invention. The pipetting device shown in  FIG. 5  is in essential parts identical to the pipetting device shown in  FIG. 1   a , with corresponding components having the same reference characters. However, instead of comprising the first reservoir  42 , the pipetting device shown in  FIG. 5  comprises two reservoirs: one reservoir  138  for a first pipetting liquid, and one reservoir  140  for a second pipetting liquid. Furthermore, the improvement according to the invention, instead of comprising one feed valve  40 , comprises three feed valves: a first feed valve  146 , a second feed valve  148  and a third feed valve  150 . The first, second and third feed valves in the embodiment shown are 3/2 valves that are electronically controllable by way of the control unit  56 , as explained above with reference to the illustration of  FIG. 1   a.    
     By way of the connecting pipe  142  or the connecting pipe  144  it is possible, selectively under pressure, to feed to the insertion lance  16  a defined quantity of the first pipetting liquid from the reservoir  138  or to the second pipetting liquid from the reservoir  140 . To this effect the connecting pipe  142  for the first pipetting fluid is connected to the input port  1  of the second feed valve  148 . The output port  2  of the second feed valve  148  in turn is in contact with the input port  1  of the third feed valve  150 , whose output port leads to the insertion lance  16  by way of the first connecting pipe  38 . By activation of the second feed valve  148  and of the third feed valve  150  liquid is therefore fed from the reservoir  138  for the first pipetting liquid to the pipette. The first feed valve  146 , which is connected to the reservoir  140  for the second pipetting liquid by way of the connecting pipe  144 , is decoupled at this point in time. 
     If instead of feeding the first pipetting liquid the second pipetting liquid is to be fed to the pipette, then instead of the second feed valve  148  and the third feed valve  150 , the first feed valve  146  and the third feed valve  150  are activated. In this manner the second pipetting liquid can be fed under pressure to the insertion lance  16  by way of the connecting pipe  144  and the first feed valve  146 , the inactive second feed valve  148  and the third feed valve  150 , while the first pipetting liquid, which is connected to the inactive input port  1  of the second feed valve  148  by way of the connecting pipe  142 , is decoupled. The input port  3  of the first feed valve  146  is connected to the second reservoir  62  by way of a connecting pipe  60   c , which reservoir  62  takes up any residues removed by suction from the pipe system of the pipetting device. 
     With the improvement described, selectively either the first or the second pipetting liquid can be injected. In order to prevent mutual contamination of the pipetting liquids, the pipe system can be cleaned by suction removal between injection of the first pipetting liquid and injection of the second pipetting liquid, with cleaning taking place, for example, in that the first connecting pipe  38  is connected to the suction removal valve  76  by way of connection  3  of the third feed valve  150 , as correspondingly illustrated in  FIG. 1   a  by the intermediate connection  82 , or for example by activation of the valve  150  and of the connection  2 - 3  by way of the inactive valves  148  and  146  and the connecting pipe  60   c  into the container  62 . 
     In the same manner the pipetting device according to the invention can be expanded, by the addition of further reservoirs and feed valves, to operate with more than two pipetting liquids. 
     The embodiments described above and the illustrating drawings only serve to explain the device according to the invention and the method according to the invention; they should in no way be misinterpreted as limitations. The scope of protection of the invention is based solely on the following claims. 
     LIST OF REFERENCE CHARACTERS 
     
         
         
           
               10  Coaxial needle 
               12  Pipetting container 
               14  Suction lance 
               16  Insertion lance 
               18  First open end of the suction lance  14   
               20  Second open end of the suction lance  14   
               22  First open end of the insertion lance  16   
               24  Second open end of the insertion lance  16   
               26  Axial direction 
               28  Liquids duct 
               30  Cover foil 
               32  Insertion funnel 
               34  Insertion nozzle 
               36  Purge nozzle 
               38  First connecting pipe 
               40  Feed valve 
               42  First reservoir 
               44  First pressure pipe 
               46  Pressure switch 
               48  Pressure source 
               50  Pressure reducer 
               52  Filter 
               54  First control line 
               56  Control unit 
               58  Input/output unit 
               60   a ,  60   b ,  60   c  Second connecting pipes 
               62  Second reservoir 
               64 ,  64 ′ Second pressure pipe 
               66 ,  66 ′ Negative-pressure source 
               68  Vacuum pump 
               70  Buffer volume 
               72  Needle-valve bypass 
               74  Venturi nozzle 
               76  Suction removal valve 
               78  Filter 
               80  Second control line 
               82  Intermediate connection 
               84  First drive unit 
               86  Second drive unit 
               88  First pressure piston 
               90  First connecting element 
               92  First fastening element 
               94  Second pressure piston 
               96  Second connecting element 
               98  Second fastening element 
               100  Third pressure pipe 
               102 ,  104  Drive valves for first drive unit  84   
               106 ,  108  Nonreturn reducing valves for first drive unit  84   
               110  Fourth pressure pipe 
               112 ,  114  Drive valves for second drive unit  86   
               116 ,  118  Nonreturn reducing valves for second drive unit  86   
               120  Spring 
               122  Excess liquid 
               124  Volume of liquid in the pipetting container  12   
               126  Pipetting unit 
               128  Guide rail 
               130  Driving pin 
               132  Nonreturn valve 
               134  Bypass 
               136  Reducing valve 
               138  Reservoir for first pipetting liquid 
               140  Reservoir for second pipetting liquid 
               142  Connecting pipe for first pipetting liquid 
               144  Connecting pipe for second pipetting liquid 
               146  First feed valve 
               148  Second feed valve 
               150  Third feed valve