EP0912720A1 - Device for separating micro objects - Google Patents

Device for separating micro objects

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Publication number
EP0912720A1
EP0912720A1 EP97931772A EP97931772A EP0912720A1 EP 0912720 A1 EP0912720 A1 EP 0912720A1 EP 97931772 A EP97931772 A EP 97931772A EP 97931772 A EP97931772 A EP 97931772A EP 0912720 A1 EP0912720 A1 EP 0912720A1
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EP
European Patent Office
Prior art keywords
objects
microcapillary
substrate
target substrate
biological objects
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97931772A
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German (de)
French (fr)
Inventor
Klaus Luttermann
Edgar Diessel
Markus Weidauer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LUTTERMANN, KLAUS
Original Assignee
Bayer AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Publication of EP0912720A1 publication Critical patent/EP0912720A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion

Definitions

  • the invention relates to a device for separating individual biological micro-objects, especially biological objects
  • the objects are in this case on a solid planar transformers arranged side by side with this method can spatially from a very large number of micro-objects (e.g. B lO 3 to 10 6) individual objects must be separated and rejected
  • a prerequisite for this separation process is the prior detection and selection of the objects in question on the basis of significant analytical properties (e.g. by
  • microobject transverse dimension ⁇ 50 ⁇ m means "biological objects” in the context of the present application primarily means (living) biological cells.
  • objects with optical ones can be used
  • Cultivation can only be achieved with additional effort.
  • these cells must be separated using a different method, such as, for example, using needles. Needles moved with micromanipulators, to which the cells adhere, are also used as the sole method. Here, the cells are touched directly and could therefore be mechanically loaded. Here too, the manipulation is limited to weakly adhering objects
  • Separating or sorting apparatuses suitable for separating a large number (> 10 5 ) of biological objects dispersed in a liquid are commercially available.
  • Fluorescence-activated cell sorting Fluorescence activated cell sorter
  • MCS Magnetic activated cell sorter
  • ablative photodecomposition processes in which pulsed UV lasers, in particular excimer lasers, are used to deliberately remove material from polymers. In the broadest sense, these processes can be regarded as etching processes.
  • a similar method, but using a continuously operated UV laser, is described in U.S. Patent 5,211,805.
  • This process is said to be suitable for the industrial processing of technical polymers and for the biomedical treatment of biological tissue.
  • This is related to a sorting principle that uses laser beams to destroy the undesired biological objects on a carrier with high radiation doses, while the selected (desired) objects remain (US Pat. No. 4,624,915). This process is relatively complex in order to select individual objects from large populations.
  • the object on which the invention is based is the spatial separation of individual micro-objects, in particular of known biological cells, which are applied next to one another with a high occupancy density on a planar carrier and adhere to this carrier.
  • the survivability of the biological objects should remain guaranteed; that is, the biological objects are not damaged by the separation process or affect trächti " ⁇ gt V be.
  • this object is achieved according to the invention in that the pipette system is arranged in a micromanipulator and an essentially vertically arranged microcapillary with a clear width of
  • a pump or piston syringe serves as the pressure generating device
  • the microcapillary preferably consists of a cylindrical glass tube and is advantageously bent at right angles. A deviation from the right angle by approximately ⁇ 20 ° can also be accepted
  • capillaries made of different materials such as borosilicate glass, aluminum silicate glass, or hematocrit glass are used
  • a particularly preferred embodiment of the invention is characterized in that the micromanipulator and the pump are controlled in such a way that, when the vacuum is adjusted in one operation, several biological objects are sucked in one after the other by the microcapillary and then rinsed out again in the next operation with an overpressure adjustment.
  • the current substrate and the target substrate expediently consist of a carrier coated with agar or agarose
  • microtiter plate can also be used as the target substrate
  • the drawing shows the basic structure of the transfer device at a microscope work station.
  • the device according to the invention can be used for the separation of micro-objects such as polymer beads in the context of combinatorial chemistry or bacteria in the context of molecular biology
  • Microcapillaries made of a special glass are used for the transfer of the bacteria, which are produced by a drawing process in the molten state.
  • borosilicate glass tubes from Hilgenberg, Malsfeld, GER
  • GER borosilicate glass tubes
  • GER pipette puller
  • capillaries with a cylindrical pipette shape in the end area of the capillary were opened with an opening diameter of approx. 6 ⁇ m at the melted end
  • the capillary can also be made from aluminum silicate glass or hematocrit glass
  • the microcapillary 1 is held in a collet 2, which is mounted on the micro-manipulator 3, which enables three-dimensional positioning in the ⁇ m range.
  • manipulators are commercially available.
  • the microcapillary 1 is connected via a hose 4 to a commercially available piston syringe 5, with the aid of which the internal capillary pressure is adjusted. The pressure is determined using a pressure gauge 6.
  • Both the micromanipulator 3 and the syringe 5 are operated remotely with stepper motors, which are not shown here.
  • the entire process of aspirating and separating a bacterium (picking process) is subject to visual control, which is made possible by observation with a 40-fold magnification in phase contrast.
  • the microcapillary 1 is heated above the softening point and bent over in such a way that it is almost a 90 ° angle forms and can thus be positioned to save space below the condenser 8 on this
  • the object to be picked or the bacterium 10 to be separated from the substrate 9 is now positioned below the capillary 1 by means of a coordinate-controlled movement of the microscope displacement table.
  • the internal capillary pressure is set to -300 mbar compared to the ambient pressure. With simultaneous microscope observation, the capillary 1 is placed directly over the one to be picked
  • the microcapillary 1 is raised via the height adjustment on the micromanipulator 3 and as a result the empty substrate area remains in the microscope image.
  • the microcapillary 1 or the displacement table is moved to a corresponding location on the target substrate.
  • the internal pressure is increased to + 100 mbar.
  • the capillary 1 is placed on the target substrate (here the edge zone on the substrate 9), the rinsing process takes place. After the capillary 1 has been lifted off the target substrate again, the rinsed-out bacterium becomes visible again in the phase contrast image of the microscope.
  • 10 bacteria in each case were transferred from a starting substrate to a target substrate which contained nutrient. After a growth period of a few days, colonies developed from the individual bacteria in 50 - 60% of the cases. A value of 60% was determined in a test of the vitality rate of the initial population, so that the transfer process can be regarded as very gentle.
  • the bacteria can be placed in a liquid e.g. PBS buffer are applied. This liquid can be found in the wells of commercially available 96 or 384 microtiter plates.
  • the micromanipulator 3 and the syringe 5 are controlled so that individual bacteria are picked one after the other with a constant negative pressure setting.
  • the bacteria are sucked up by successively lowering the capillary 1 onto the substrate locations that carry the bacteria to be picked.
  • the bacteria are then rinsed out with a constant overpressure setting by successively lowering the capillary to different locations on the substrate
  • PCR polymerase chain reaction

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention concerns a device for transferring micro objects, in particular biological objects, from their present substrate (9) to a target substrate. To that end, a movable pipette system is used which is connected to a pressure-generating device for drawing in and expelling the biological objects. The pipette system is disposed in a micromanipulator (3) and in principle comprises a substantially vertical microcapillary tube (1) with an inside width of between 1 νm and 50 νm.

Description

Vorrichtung zum Separieren von MikroobjektenDevice for separating micro objects
Die Erfindung betrifft eine Vorrichtung zum Separieren von einzelnen biologischen Mikroobjekten, insbesondere von biologischen Objekten Die Objekte sind hierbei auf einem festen planaren Trager nebeneinander angeordnet Mit diesem Verfahren können aus einer sehr großen Zahl von Mikroobjekten (z B lO3 bis 106) einzelne Objekte raumlich abgetrennt und ausgesondert werden Voraussetzung für dieses Separierverfahren ist die vorherige Erkennung und Selektion der betreffenden Objekte aufgrund signifikanter analytischer Eigenschaften (z B durchThe invention relates to a device for separating individual biological micro-objects, especially biological objects The objects are in this case on a solid planar transformers arranged side by side with this method can spatially from a very large number of micro-objects (e.g. B lO 3 to 10 6) individual objects must be separated and rejected A prerequisite for this separation process is the prior detection and selection of the objects in question on the basis of significant analytical properties (e.g. by
Fiuoreszenzspektroskopie oder durch radioaktive Markierung) Unter "Mikroobjek Querabmessung <50 μm zu verstehen Unter "biologischen Objekten" werden im Rahmen der vorhegenden Anmeldung vor allem (lebende) biologische Zellen verstanden.Fluorescence spectroscopy or by radioactive labeling) The term "microobject transverse dimension <50 μm" means "biological objects" in the context of the present application primarily means (living) biological cells.
Die Separation von biologischen Zellen mit Pipetten ist grundsatzlich bekanntThe separation of biological cells with pipettes is fundamentally known
Hierbei handelt es sich um Verfahren, die von Pasteurpipetten Gebrauch machen In J. A. Benson, J. Exp. Biol. 170, 203 (1992) wird die Separation von Zellen im Grόßenbereich zwischen 50 und 100 μm beschrieben, die von einer Pipette mit einem Durchmesser von 0,5 mm räumlich aus einer Population getrennt werdenThese are methods that make use of Pasteur pipettes. JA Benson, J. Exp. Biol. 170, 203 (1992) describes the separation of cells in the size range between 50 and 100 μm, which are carried out by a pipette with a diameter of 0.5 mm spatially separated from a population
Zur Separation einzelner biologischer Objekte lassen sich Objekte mit optischenTo separate individual biological objects, objects with optical ones can be used
Methoden, wie der optischen Pinzette (Optical Tweezer) in einer wäßrigen Losung bewegen (K Schütze, A Ciement-Sengewald, Nature, 667 (Vol 368) 1994) Aufgrund der geringen Kraftübertragung ist diese Methode auf Objekte beschrankt, die sich frei in der Losung bewegen können Da sich die sortierten wie die unsortierten Objekte in der gleichen Losung befinden, ist eine getrennteMethods such as moving optical tweezers (Optical Tweezer) in an aqueous solution (K Schütze, A Ciement-Sengewald, Nature, 667 (Vol 368) 1994) Due to the low power transmission, this method is limited to objects that are free in the solution Can move Since the sorted as well as the unsorted objects are in the same solution, there is a separate one
Kultivierung nur mit zusätzlichem Aufwand erzielbar Für eine getrennte Kultivierung müssen diese Zellen mit einer anderen Methode wie z B mit Nadeln abgetrennt werden. Mit Mikromanipulatoren bewegte Nadeln, an denen die Zellen adherieren, werden auch als alleinige Methode eingesetzt Hierbei werden die Zellen direkt berührt und konnten somit mechanisch belastet werden Auch hier ist die Manipulation auf schwach adherierende Objekte begrenztCultivation can only be achieved with additional effort. For a separate cultivation, these cells must be separated using a different method, such as, for example, using needles. Needles moved with micromanipulators, to which the cells adhere, are also used as the sole method. Here, the cells are touched directly and could therefore be mechanically loaded. Here too, the manipulation is limited to weakly adhering objects
Zur Separierung einer großen Zahl (> 105), in einer Flüssigkeit dispergierter, biologischer Objekte geeignete Trenn- bzw Sortierapparate sind kommerziell erhaltlich Wahrend bei der fluoreszenzaktiverten Zellsortierung (FACS = Fluorescence activated Cell Sorter) elektrostatische Prinzipien zur räumlichen Separation zum Einsatz kommen, arbeitet der magnetisch aktivierte Zellsortierer (MACS = Magnetic activated Cell Sorter) mit magnetischen Kräften. Hierbei liegen die Zellen jedoch nicht auf einem planaren Träger nebeneinander. Überdies haben beide Methoden den Nachteil, daß sich einzelne Objekte nur eingeschränktSeparating or sorting apparatuses suitable for separating a large number (> 10 5 ) of biological objects dispersed in a liquid are commercially available. In the case of fluorescence-activated cell sorting (FACS = Fluorescence activated cell sorter) electrostatic principles are used for spatial separation, the magnetically activated cell sorter (MACS = Magnetic activated cell sorter) works with magnetic forces. However, the cells are not next to each other on a planar carrier. Furthermore, both methods have the disadvantage that individual objects are only restricted
(FACS) oder überhaupt nicht getrennt voneinander absondern lassen (MACS).(FACS) or not separated at all separately (MACS).
Ferner sind unter dem Namen "Ablative Photodecomposition" Verfahren bekannt, bei denen mit gepulsten UV-Lasern, insbesondere mit Excimer-Lasern ein gezielter Materialabtrag bei Polymeren erfolgt. Diese Verfahren können im weitesten Sinne als Ätzverfahren angesehen werden. Ein ähnliches Verfahren, bei dem jedoch ein kontinuierlich betriebener UV-Laser verwendet wird, wird in dem US-Patent 5 21 1 805 beschrieben. Dieses Verfahren soll sich zur industriellen Bearbeitung von technischen Polymeren und zur biomedizinischen Behandlung von biologischem Gewebe eignen. Hiermit ist ein Sortierprinzip verwandt, das mit Laser- strahlen die auf einem Träger befindlichen unerwünschten biologischen Objekte mit hohen Strahlungsdosen zerstört, während die selektierten (erwünschten) Objekte zurück bleiben (US 4 624 915). Dieser Prozeß ist verhältnismäßig aufwendig, um einzelne Objekte aus großen Populationen zu selektieren.Also known under the name "ablative photodecomposition" are processes in which pulsed UV lasers, in particular excimer lasers, are used to deliberately remove material from polymers. In the broadest sense, these processes can be regarded as etching processes. A similar method, but using a continuously operated UV laser, is described in U.S. Patent 5,211,805. This process is said to be suitable for the industrial processing of technical polymers and for the biomedical treatment of biological tissue. This is related to a sorting principle that uses laser beams to destroy the undesired biological objects on a carrier with high radiation doses, while the selected (desired) objects remain (US Pat. No. 4,624,915). This process is relatively complex in order to select individual objects from large populations.
Die der Erfindung zugrundeliegende Aufgabe besteht in der räumlichen Separation einzelner Mikroobjekte, insbesondere von bekannten biologischen Zellen, die nebeneinander mit einer hohen Belegungsdichte auf einem planaren Träger ausgebracht sind und auf diesem Träger adherieren. Dabei soll die Überlebensfähigkeit der biologischen Objekte in der Regel gewährt bleiben; d.h. die biologischen Objekte sollen durch den Abtrennprozeß nicht geschädigt bzw. beein- trächti "ΌgtV werden.The object on which the invention is based is the spatial separation of individual micro-objects, in particular of known biological cells, which are applied next to one another with a high occupancy density on a planar carrier and adhere to this carrier. As a rule, the survivability of the biological objects should remain guaranteed; that is, the biological objects are not damaged by the separation process or affect trächti "Όgt V be.
Diese Aufgabe wird, ausgehend von einer Vorrichtung mit einem verfahrbaren Pipettensystem, das mit einer Druckerzeugungsvorrichtung zum Ansaugen und Ausspülen der biologischen Objekte verbunden ist, erfindungsgemäß dadurch gelöst, daß das Pipettensystem in einem Mikromanipulator angeordnet ist und eine im wesentlichen vertikal angeordnete Mikrokapillare mit einer lichten Weite vonStarting from a device with a movable pipette system, which is connected to a pressure generating device for aspirating and rinsing the biological objects, this object is achieved according to the invention in that the pipette system is arranged in a micromanipulator and an essentially vertically arranged microcapillary with a clear width of
1 μm bis 50 μm, vorzugsweise 5 μm bis 20 μm aufweist. "Im wesentlichen vertikal" bedeutet dabei, daß eine Abweichung von ± 20 ° zugelassen werden kann Als Druckerzeugungsvorrichtung dient hier im einfachsten Fall eine Pumpe oder Kolbenspritze1 μm to 50 μm, preferably 5 μm to 20 μm. "Essentially vertical" means that a deviation of ± 20 ° can be permitted. In the simplest case, a pump or piston syringe serves as the pressure generating device
Vorzugsweise besteht die Mikrokapillare aus einem zylindrischen Glasrohr und ist vorteilhaft rechtwinklig abgebogen. Dabei kann ebenfalls eine Abweichung vom rechten Winkel um ca ± 20° akzeptiert werdenThe microcapillary preferably consists of a cylindrical glass tube and is advantageously bent at right angles. A deviation from the right angle by approximately ± 20 ° can also be accepted
Um problemlos verschiedene Offnungsdurchmesser bei gleichem Ausgangsdurchmesser der Kapillaren im Herstellungsprozess zu realisieren, werden Kapillaren aus unterschiedlichen Materialien, wie Borosilikatglas, Aluminiumsilikatglas, oder Hamatokritglas eingesetztIn order to easily realize different opening diameters with the same starting diameter of the capillaries in the manufacturing process, capillaries made of different materials, such as borosilicate glass, aluminum silicate glass, or hematocrit glass are used
Eine besonders bevorzugte Ausführungsform der Erfindung ist dadurch gekennzeichnet, daß der Mikromanipulator und die Pumpe so gesteuert sind, daß bei einer Unterdruckeinstellung in einem Arbeitsgang mehrere biologische Objekte nacheinander von der Mikrokapillare angesaugt und anschließend im nächsten Arbeitsgang mit einer Überdruckeinstellung nacheinander wieder ausgepült werden.A particularly preferred embodiment of the invention is characterized in that the micromanipulator and the pump are controlled in such a way that, when the vacuum is adjusted in one operation, several biological objects are sucked in one after the other by the microcapillary and then rinsed out again in the next operation with an overpressure adjustment.
Im Hinblick auf den Transfer von Bakterien besteht das aktuelle Substrat und das Zielsubstrat zweckmäßig aus einem mit Agar oder Agarose beschichteten TragerWith regard to the transfer of bacteria, the current substrate and the target substrate expediently consist of a carrier coated with agar or agarose
Alternativ kann aber als Zielsubstrat auch eine Mikrotiterplatte eingesetzt werdenAlternatively, a microtiter plate can also be used as the target substrate
Im folgenden wird die Erfindung an Hand eines in der Zeichnung dargestelltenIn the following the invention is illustrated by means of one in the drawing
Ausfuhrungsbeispiels naher erläutert Die Zeichnung zeigt den prinzipiellen Aufbau der Transfervorrichtung an einem Mikroskoparbeitsplatz Die erfindungsge- maße Vorrichtung kann für die Separation von Mikroobjekten wie etwa von Polymerbeads im Rahmen der kombinatorischen Chemie oder von Bakterien im Rahmen der Molekularbiologie eingesetzt werden Exemplarisch wird hier derThe drawing shows the basic structure of the transfer device at a microscope work station. The device according to the invention can be used for the separation of micro-objects such as polymer beads in the context of combinatorial chemistry or bacteria in the context of molecular biology
Einsatz der Vorrichtun Όg zur Sortierung von Bakterien beschriebenUse of the device for sorting bacteria described
Fur den Transfer der Bakterien werden Mikrokapillaren aus einem Spezialglas verwendet, die durch einen Ziehprozeß im geschmolzenen Zustand hergestellt werden Für die zu beschreibenden Experimente wurden Borosilikatglasrohrchen (Fa Hilgenberg, Malsfeld, GER) mit einem Ausgangsdurchmesser von 1 6 mm einge- setzt In einem dreistufigen Ziehprozeß mit einem handelsüblichen Pipetten- ziehgerat (DMZ Universalpuller, Fa Zeitz-Instrumente, München, GER) wurden Kapillaren mit einer im Endbereich der Kapillare zylindrischen Pipettenform (d h nicht wie sonst üblich zu einer sich verjungenden Spitze ausgezogeni) mit einem Offnungsdurchmesser von ca 6 μm an dem aufgeschmolzenen Ende hergestelltMicrocapillaries made of a special glass are used for the transfer of the bacteria, which are produced by a drawing process in the molten state. For the experiments to be described, borosilicate glass tubes (from Hilgenberg, Malsfeld, GER) with an initial diameter of 16 mm were inserted. In a three-stage drawing process using a commercially available pipette puller (DMZ Universalpuller, Zeitz-Instrumente, Munich, GER), capillaries with a cylindrical pipette shape in the end area of the capillary (ie not pulled out to a tapering tip as usual) were opened with an opening diameter of approx. 6 μm at the melted end
Auf der anderen Seite bleibt der Ausgangsdurchmesser unverändert erhalten Für die Reproduzierbarkeit des Transferprozesses insbesondere des Ausspulprozesses hat sich diese Pipettenform bewahrt Alternativ kann die Kapillare auch aus einem Aluminiumsilikatglas oder Hamatokritglas hergestellt werdenOn the other hand, the initial diameter remains unchanged. This pipette shape has been retained for the reproducibility of the transfer process, in particular the unwinding process. Alternatively, the capillary can also be made from aluminum silicate glass or hematocrit glass
In der Zeichnung ist die Apparatur für den Transferprozeß schematisch dargestelltIn the drawing, the apparatus for the transfer process is shown schematically
Die Mikrokapillare 1 wird in einer Spannzange 2 gehaltert, die an dem Mikro- manipulator 3 montiert ist, der eine dreidimensionale Positionierung im μm-Be- reich ermöglicht. Solche Manipulatoren sind kommerziell erhältlich Die Mikrokapillare 1 ist über einen Schlauch 4 mit einer handelsüblichen Kolbenspritze 5 verbunden, mit deren Hilfe der Kapillareninnendruck eingestellt wird. Der Druck wird mit einem Druckmesser 6 bestimmt. Sowohl der Mikromanipulator 3 als auch die Spritze 5 werden mit Schrittmotoren, die hier nicht dargestellt sind, fernbedient. Der gesamte Vorgang des Ansaugens und der Separation einer Bakterie (Pickvorgang) unterliegt der visuellen Kontrolle, die durch die Beobachtung mit einer 40-fachen Vergrößerung im Phasenkontrast ermöglicht wird. Von dem Mikroskop sind hier nur das Objektiv 7 und der Beleuchtungskondensor 8 dargestellt Im Hinblick auf den erforderlichen Arbeitsabstands des Kondensors 8 von ca 22 mm zum Objekt wird die Mikrokapillare 1 über den Erweichungspunkt erhitzt und in der Weise umgebogen, das sie nahezu einen 90° Winkel bildet und damit platzsparend unterhalb des Kondensors 8 positioniert werden kann Auf dieseThe microcapillary 1 is held in a collet 2, which is mounted on the micro-manipulator 3, which enables three-dimensional positioning in the μm range. Such manipulators are commercially available. The microcapillary 1 is connected via a hose 4 to a commercially available piston syringe 5, with the aid of which the internal capillary pressure is adjusted. The pressure is determined using a pressure gauge 6. Both the micromanipulator 3 and the syringe 5 are operated remotely with stepper motors, which are not shown here. The entire process of aspirating and separating a bacterium (picking process) is subject to visual control, which is made possible by observation with a 40-fold magnification in phase contrast. Of the microscope, only the objective 7 and the illumination condenser 8 are shown here. With regard to the required working distance of the condenser 8 of approx. 22 mm to the object, the microcapillary 1 is heated above the softening point and bent over in such a way that it is almost a 90 ° angle forms and can thus be positioned to save space below the condenser 8 on this
Weise kann die Beobachtung des Transferprozesses ungestört stattfinden. Für die hohe raumliche Auflösung des Transferprozesses in der Größe des Pipettendurchmessers (hier 6 μm) ist es wichtig, daß die Pipette senkrecht auf das zu pickende Objekt trifft Dabei kann eine Abweichung von maximal ± 20 ° toleriert werden Damit bildet sich kein Wasserfϊlm zwischen Kapillarenwand und Agarsubstrat, der umliegende Objekte beim Transferprozeß in Mitleidenschaft ziehen konnte Zur Vorbereitung des Pickens wird über einen Unterdruck aus einem Vorratsgefaß eine Pufferlosung in die Kapillare 1 eingesogen Hierbei reicht es aus, daß nur der verjungte Teil der Kapillare mit der Pufferlosung gefüllt ist Nun wird das zu pickende Objekt bzw. die von dem Substrat 9 zu separierende Bakterie 10 durch eine koordinatengesteuerte Bewegung des Mikroskopverschiebetisches unterhalb der Kapillare 1 positioniert. Hierbei wird der Kapillaren- innendruck auf -300 mbar gegenüber Umgebungsdruck eingestellt. Unter gleich- zeitger Mikroskopbeobachtung wird die Kapillare 1 direkt über der zu pickendenIn this way, the observation of the transfer process can take place undisturbed. For the high spatial resolution of the transfer process in the size of the pipette diameter (here 6 μm), it is important that the pipette hits the object to be picked perpendicularly.A deviation of up to ± 20 ° can be tolerated.Therefore, no water film forms between the capillary wall and Agar substrate, which could affect surrounding objects during the transfer process. To prepare for picking, a buffer solution is sucked into the capillary 1 from a storage container using an underpressure. It is sufficient that only the tapered part of the capillary is filled with the buffer solution The object to be picked or the bacterium 10 to be separated from the substrate 9 is now positioned below the capillary 1 by means of a coordinate-controlled movement of the microscope displacement table. The internal capillary pressure is set to -300 mbar compared to the ambient pressure. With simultaneous microscope observation, the capillary 1 is placed directly over the one to be picked
Bakterie 10 auf das Agarosesubstrat 9 aufgesetzt. Anschließend wird die Mikrokapillare 1 über die Höhenverstellung am Mikromanipulator 3 angehoben und als Resultat bleibt im Mikroskopbild die leere Substratfläche zurück. Zum Ausspülen der Bakterie 10 wird entweder die Mikrokapillare 1 oder der Verschiebetisch zu einer entsprechenden Stelle auf dem Zielsubstrat gefahren.Bacteria 10 placed on the agarose substrate 9. Subsequently, the microcapillary 1 is raised via the height adjustment on the micromanipulator 3 and as a result the empty substrate area remains in the microscope image. To rinse out the bacterium 10, either the microcapillary 1 or the displacement table is moved to a corresponding location on the target substrate.
Hierbei wird der Innendruck auf + 100 mbar erhöht. Während die Kapillare 1 auf das Zielsubstrat (hier die Randzone auf dem Substrat 9) aufgesetzt wird, findet der Ausspülprozeß statt. Nachdem die Kapillare 1 wiederum von dem Zielsubstrat abgehoben wurde, wird die ausgespülte Bakterie im Phasenkontrastbild des Mikroskops erneut sichtbar. Auf diese Weise wurden in vier Beispielen jeweils 10 Bakterien von einem Ausgangssubstrat auf ein Zielsubstrat, das Nährstoff enthielt, übertragen. Nach einer Anwachszeit von einigen Tagen bildeten sich in 50 - 60 % der Fälle aus den einzelnen Bakterien Kolonien. In einem Test der Vitalitätsrate der Ausgangspopulation wurde ein Wert von 60 % bestimmt, so daß hiermit der Transferprozeß als sehr schonend angesehen werden kann. Alternativ können die Bakterien können in eine Flüssigkeit z.B. PBS-Puffer ausgebracht werden. Diese Flüssigkeit kann sich in den Vertiefungen (Wells) von handelsüblichen 96- oder 384-Mikrotiterplatten befinden.The internal pressure is increased to + 100 mbar. While the capillary 1 is placed on the target substrate (here the edge zone on the substrate 9), the rinsing process takes place. After the capillary 1 has been lifted off the target substrate again, the rinsed-out bacterium becomes visible again in the phase contrast image of the microscope. In this way, in four examples, 10 bacteria in each case were transferred from a starting substrate to a target substrate which contained nutrient. After a growth period of a few days, colonies developed from the individual bacteria in 50 - 60% of the cases. A value of 60% was determined in a test of the vitality rate of the initial population, so that the transfer process can be regarded as very gentle. Alternatively, the bacteria can be placed in a liquid e.g. PBS buffer are applied. This liquid can be found in the wells of commercially available 96 or 384 microtiter plates.
Neben dem unmittelbar nacheinander stattfindenden Pick- und Ausspülprozeß wurden auch mehrere Bakterien hintereinander in einem Arbeitsgang gepickt und entsprechend nacheinander am Zielsubstrat wieder ausgespült. Zu diesem Zweck werden der Mikromanipulator 3 und die Spritze 5 so gesteuert, daß bei einer gleichbleibenden Unterdruckeinstellung einzelne Bakterien nacheinander aufgepickt werden. Hierbei werden die Bakterien aufgesogen, indem die Kapillare 1 nacheinander auf die Substratstellen abgesenkt wird, die die zu pickenden Bakterie tragen. Anschließend werden bei einer gleichbleibenden Uberdruckeinstellung die Bakterien ausgespült, indem die Kapillare nacheinander auf verschieden Stellen des Substrates abgesenkt wirdIn addition to the picking and rinsing process, which took place immediately one after the other, several bacteria were picked one after the other in a single operation and rinsed out one after the other on the target substrate. For this purpose, the micromanipulator 3 and the syringe 5 are controlled so that individual bacteria are picked one after the other with a constant negative pressure setting. Here, the bacteria are sucked up by successively lowering the capillary 1 onto the substrate locations that carry the bacteria to be picked. The bacteria are then rinsed out with a constant overpressure setting by successively lowering the capillary to different locations on the substrate
Diese Prozedur ist vergleichsweise zeitsparend, da die Wege zwischen den zu sortierenden Objekten optimiert werden können und auch die Druckeinstellung in der Kapillare jeweils nur einmal vorgenommen werden muß Der Vorteil in der Verwendung von Bakterien besteht in der einfachen Amplifizierung durch reguläres Anwachsen. Darüber hinaus können mit Hilfe der Polymerase Chain Reac- tion (PCR) können aus einzeln sortierten Bakterien die Gensequenzen amplifiziert werden, die für die spezifische Eigenschaft der Bakterien verantwortlich sind This procedure is comparatively time-saving, since the paths between the objects to be sorted can be optimized and the pressure setting in the capillary only has to be carried out once. The advantage of using bacteria is the simple amplification by regular growth. In addition, the polymerase chain reaction (PCR) can be used to amplify the gene sequences that are responsible for the specific property of the bacteria from individually sorted bacteria

Claims

Patentansprücheclaims
1 Vorrichtung zum Transfer von Mikroobjekten, insbesondere von biologischen Objekten, von einem aktuellen Substrat (9) zu einem Zielsubstrat, mit einem verfahrbaren Pipettensystem, das mit einer Druckerzeugungs- Vorrichtung zum Ansaugen und Ausspulen der biologischen Objekte verbunden ist, dadurch gekennzeichnet, daß das Pipettensystem in einem Mikromanipulator (3) angeordnet ist und eine im wesentlichen vertikal angeordnete Mikrokapillare (1) mit einer lichten Weite von 1 μ bis 50 μm, vorzugsweise 5 μm bis 20 μm aufweist1 device for the transfer of micro-objects, in particular of biological objects, from a current substrate (9) to a target substrate, with a movable pipette system, which is connected to a pressure generating device for sucking in and spooling out the biological objects, characterized in that the pipette system is arranged in a micromanipulator (3) and has an essentially vertically arranged microcapillary (1) with a clear width of 1 μm to 50 μm, preferably 5 μm to 20 μm
2 Vorrichtung nach Anspruch 1, dadurch gekennzeichnet, daß die Mikrokapillare (1) aus einem zylindrischen Glasrohr besteht2 Device according to claim 1, characterized in that the microcapillary (1) consists of a cylindrical glass tube
3 Vorrichtung nach Anspruch 1 bis 2, dadurch gekennzeichnet, daß die Mikrokapillare (1) annähernd rechtwinklig abgebogen ist.3 Device according to claim 1 to 2, characterized in that the microcapillary (1) is bent approximately at right angles.
4 Vorrichtung nach Anspruch 1 bis 3, dadurch gekennzeichnet, daß die Mikrokapillare ( 1) aus Borosilikatglas, Aluminiumsilikatgias, oder4 Device according to claim 1 to 3, characterized in that the microcapillary (1) made of borosilicate glass, aluminum silicate glass, or
Hamatokritglas besteht.Hamatocrit glass exists.
Vorrichtung nach Anspruch 1 bis 4, dadurch gekennzeichnet, daß der Mikromanipulator (3) und die Druckerzeugungsvorrichtung so gesteuert sind, daß bei einer Unterdruckeinstellung in einem Arbeitsgang mehrere biologische Objekte nacheinander von der Mikrokapillare (1) angesaugt und anschließend im nächsten Arbeitsgang mit einer Uberdruckeinstellung nacheinander wieder ausgepult werdenApparatus according to claims 1 to 4, characterized in that the micromanipulator (3) and the pressure generating device are controlled in such a way that, when the vacuum is adjusted in one operation, several biological objects are sucked in succession by the microcapillary (1) and then in the next operation with an overpressure adjustment in succession be rewound again
Vorrichtung nach Anspruch 1 bis 5, dadurch gekennzeichnet, daß das aktuelle Substrat (9) und das Zielsubstrat aus einem mit Agar oder Agarose beschichteten Trager bestehtDevice according to Claims 1 to 5, characterized in that the current substrate (9) and the target substrate consist of a carrier coated with agar or agarose
Vorrichtung nach Anspruch 1 bis 5, dadurch gekennzeichnet, daß das Zielsubstrat aus einer Mikrotiterplatte besteht Device according to Claims 1 to 5, characterized in that the target substrate consists of a microtiter plate
EP97931772A 1996-07-19 1997-07-04 Device for separating micro objects Withdrawn EP0912720A1 (en)

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