JP3547899B2 - Micromanipulator and cell used therein - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a micromanipulator that selects only specific particles or microorganisms while monitoring a group of particles or microorganisms dispersed and suspended in a medium with a microscope, and aspirates them into a micropipette.
[0002]
[Prior art]
For example, Escherichia coli is frequently used for genetic manipulation.In this case, after dispersing in a predetermined chemical solution and performing genetic manipulation, the Escherichia coli dispersed in the drug solution is inhaled with a micropipette together with the chemical solution. Then, this is transplanted into a culture tank and cultured to replicate a large amount of DNA.
However, Escherichia coli dispersed in the drug solution is not all genetically manipulated due to individual differences, and when inhaling this with a micropipette, multiple Escherichia coli are inhaled at the same time, so it was cultured. Escherichia coli is a mixture of those that have been genetically engineered and those that have not, and the yield of genetically engineered ones is low.
Therefore, if it can be cultured from one Escherichia coli, it can be purely cultured. If the Escherichia coli is genetically modified, only the genetically modified Escherichia coli can be obtained at a yield of 100%. .
[0003]
[Problems to be solved by the invention]
However, in order to aspirate only one micropipette with a micropipette, the tip of the micropipette must be thin so as not to suck in surrounding solids at the same time, and there is a problem that the tip is easily broken.
For microorganisms and microparticles that do not move or move slowly like cells, by making the number of solids in the medium extremely small, even a relatively thick micropipette can aspirate one solid, It is difficult to operate following a relatively fast movement (several μm to several tens μm / s) such as Escherichia coli or the like, and it is difficult to suck only one of them.
[0004]
Further, even if the micropipette can be moved following the automatic control or the like, when the micropipette is moved in the drug solution, the drug solution is agitated and a flow occurs, and the microorganisms to be sucked are moved. Although it is possible to simultaneously capture a plurality of microorganisms by sucking a large amount of a drug solution, it is difficult to aspirate only one microorganism.
Therefore, it is a technical object of the present invention to make it possible to reliably aspirate only one fast moving microorganism or microparticle such as Escherichia coli.
[0005]
[Means for Solving the Problems]
In order to solve this problem, the present invention provides a micropipette that aspirates a specific microorganism or microparticle from a group of microbes or microparticles dispersed and suspended in a medium held on a cell into a micropipette under a visual field of a microscope. A manipulator, a laser trapping means for irradiating a specific microorganism or fine particles in a medium with laser light to trap the microorganism or fine particles, and a group of microorganisms or fine particles other than the microorganism or fine particles trapped by the laser light, An electric adsorption means for adsorbing to one or both electrodes by forming an electric field between at least a pair of electrodes provided in the medium; and a specific microorganism or fine particles trapped by the laser trapping means in the medium. And a suction means for sucking into the micropipette advanced into the micropipette.
[0006]
According to this, when a specific microorganism or fine particle in a medium is irradiated with laser light, the microorganism or fine particle is trapped at the focal position, and the movement of even fast moving bacteria such as Escherichia coli is stopped.
In this state, when a uniform electric field is formed in the medium, an electrophoresis phenomenon occurs in which charged fine particles existing in the electric field migrate toward an electrode having an opposite polarity. In addition, when an uneven electric field is formed, electrically neutral microorganisms and fine particles existing in the electric field are polarized, and electric charges of the same amount are induced at both ends. This causes an imbalance, which causes a dielectrophoresis phenomenon in which microorganisms and fine particles migrate toward the higher electric field density.
[0007]
Then, due to the electrophoresis and dielectrophoresis, a group of microorganisms or particles present between the electrodes moves toward the electrode except for specific microorganisms or particles trapped by the laser light, and is adsorbed to the electrode. Certain microorganisms or microparticles are separated from other microbes or microparticles.
Then, by scanning the laser light, or by moving the cell to move the specific microorganisms or microparticles to the micropipette suction position, or to advance the micropipette to the trap position, use a special thin micropipette In addition, only one specific microorganism or fine particle can be reliably sucked.
[0008]
Note that dielectrophoresis is different from electrophoresis, and the difference is noticeable in an alternating electric field. That is, in electrophoresis, regardless of whether the electric field is equal or unequal, Coulomb force acts on charged particles, and the charged particles are attracted to an electrode of the opposite polarity to the polarity, In an alternating electric field, the charged particle reverses its direction with the alternation of the electric field and oscillates only around one point without causing translational motion, whereas in dielectrophoresis, the orientation of the dipole polarized with the alternation of the electric field Microorganisms or microparticles are always transported to the one with a stronger electric field (the one with a weaker dielectric constant) while reversing.
In particular, when dealing with microorganisms and microparticles, there are microbes and microparticles in the immediate vicinity of the electrode, so if electrophoresis using a DC electric field is used in an aqueous solution that is a medium solution, the effects of electrode reactions such as electrolysis may become a problem. On the other hand, if a dielectrophoretic phenomenon caused by an AC electric field is used, an electrode reaction is unlikely to occur.
[0009]
In addition, this non-uniform electric field is not formed by making the surfaces of the two plate-shaped electrodes face each other, but, for example, by forming one or both electrodes in a needle shape or a knife edge shape (linear shape). Thus, a three-dimensional uneven electric field may be formed.
For example, when a knife-edge-shaped electrode is opposed to a medium, the lines of electric force of the electric field generated between the electrodes spread radially from one knife-edge tip and see the other knife-edge tip when viewed from the cross-sectional direction. Therefore, an unequal electric field is formed in which the electric field near the electrode tip is strong and the electric field in the middle part of the electrode is weak.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be specifically described based on embodiments shown in the drawings.
FIG. 1 is a schematic diagram showing an example of a micromanipulator according to the present invention, FIG. 2 is a perspective view showing a cell used for the same, FIG. 3 is a schematic diagram of an uneven electric field formed between electrodes, and FIG. FIG.
[0011]
The case where an electrically neutral microorganism is captured will be described as an example. In the figure, reference numeral 1 denotes a micromanipulator that selects and captures one solid from a group of microorganisms such as Escherichia coli from a medium solution held in a cell 2. The cell 2 is placed on a stage 5 of an inverted microscope 4, a micropipette 6 for sucking the microorganisms 3 is provided so as to be able to advance and retreat into the cell 2, and a laser beam is emitted to an arbitrary irradiation position. The specific microorganisms 3 trapped with the laser light by scanning the laser light or moving the stage 5 on which the cell 2 is placed in a state where the specific microorganisms 3 at the irradiation position are trapped by the irradiation are replaced with the microorganisms. A laser trapping means 7 for transferring the micropipette 6 separated from the group to a tip position (suction position) of the micropipette 6 advanced into the medium; An electric adsorption means 9 for adsorbing a group of microorganisms other than the microorganisms 3 trapped by laser light to the electrodes 8A, 8B by forming an uneven electric field between at least the pair of electrodes 8A, 8B; When the specific microorganisms 3 are transferred to the tip position (suction position) of the micropipette 6 by the means 7, a suction means 10 for sucking the specific microorganisms 3 is provided.
[0012]
The inverted microscope 4 has an objective lens 12 disposed below a stage 5 which is disposed so as to be movable in the X and Y directions by a stage moving device 11, and a CCD for photographing the inside of the cell 2 on the optical axis thereof. The camera 13 is set, and an image captured by the CCD camera 13 is displayed on the display device 14.
The laser trapping means 7 is adapted to trap the microorganisms 3 in the cell 2 by the laser light transmitted through the objective lens 12 of the inverted microscope 4, and the laser trapping means 7 is provided in the optical path of the laser light output from the laser light source 15. A scanning device 17 having scanning mirrors 16x and 16y for moving the irradiation position of the laser light in the X direction and the Y direction and a dichroic mirror 18 are interposed, and the laser light reflected by the dichroic mirror 18 is used as an objective lens. 12 and is condensed in the cell 2.
As the objective lens 12, for example, a liquid immersion objective lens with a magnification of 100 and NA = 1.30 is used, and as the laser light source 15, for example, a semiconductor laser is used.
[0013]
As shown in FIG. 2, the cell 2 holding the medium liquid is formed of, for example, an extremely thin glass plate, and a specific microorganism in the medium is trapped on the plate surface 20 by irradiating a laser beam. A _first region A ₁ and a second region A ₂ for sucking a specific microorganism trapped by the laser light with a micropipette are formed so as to communicate with each other via a passage 21.
The partition wall 22 for partitioning the respective areas A ₁ and A ₂ and the peripheral wall 23 surrounding the periphery of the plate surface 20 are formed by attaching a resin to the plate surface 20 by, for example, a photoresist method or a printing method. Note that the regions A ₁ and A ₂ may be formed by providing recesses on the plate surface 20.
[0014]
Then, wherein the first region A _1, the electrode 8A to form a uniform electric field in the medium, 8B are provided.
The electrodes 8A and 8B have a plurality of linear conductors 25 embedded at predetermined intervals on surfaces 24 facing each other, and by applying an AC / DC voltage between the electrodes 8A and 8B, An uneven electric field as shown in FIG.
This is because the lines of electric force spread radially from the respective linear conductors 25 of one electrode 8A and converge on the respective linear conductors 25 of the other electrode 8B, so that the density of the lines of electric force near the electrodes 8A and 8B 26 Therefore, an uneven electric field is formed at the intermediate portion 27 between the electrodes 8A and 8B because the density of the lines of electric force is low and the electric field is weak.
The shape of the electrodes 8A and 8B is not limited to this. For example, one of the electrodes may be a point or a linear electrode and the other may be a planar electrode. When adsorbing by dielectrophoresis, it is sufficient that an uneven electric field is formed.
[0015]
A vibrator 28 such as a piezoelectric ceramic, a piezoelectric element, or a giant magnetostrictive element is attached to the back surface of the cell 2. It is designed to shake off fine particles.
[0016]
Reference numeral 30 denotes a control device for controlling the micromanipulator 1. The CCD camera 13, the keyboard 31, and the mouse 32 are connected to the input side, and the display device 14 and the driving device for the laser light source 15 are connected to the output side. 33, a high-frequency oscillator 34 for forming a high-frequency AC electric field of about 1 MHz between the electrodes 8A and 8B, a vibration generator 35 for driving the vibrator 28, a driver 36 of a laser scanning device, and the stage driving device 11 are connected.
[0017]
The above is an example of the device of the present invention. Next, the operation thereof will be described with reference to FIGS.
First, it sets the cell 2 on the stage 5, as shown in FIG. 4 (a), while dropping a liquid medium containing a large number of microorganisms in the first region A ₁ (e.g. E. coli), the second region dripping the liquid medium with no microorganism in a _2.
If left in this state, since the microorganisms in the first area A ₁ will be out swim from the passage 21 to the second region A _2, electrodes 8A immediately by the high-frequency oscillator 34, by applying an AC voltage to 8B non When an equal electric field is formed, the microorganisms cause dielectrophoresis and move toward the electrodes 8A and 8B.
In this way, each electrode 8A microorganisms as shown in FIG. 4 (b), adsorbed onto 8B, confining the first area A _1.
At this time, an AC voltage of about 400 V / cm and 1 MHz is applied between the electrodes 8A and 8B, for example, when the resistivity of the medium liquid is 10 kΩ · cm or more.
[0018]
Next, when the voltage applied to the electrodes 8A and 8B is temporarily cut off, the unequal electric field disappears, and the microorganisms adsorbed on the electrodes 8A and 8B separate from the electrodes as shown in FIG. Since the swimmer begins to swim, a specific microorganism 3 located at an intermediate position between the electrodes 8A and 8B is irradiated with a laser beam by the scanning device 17 while watching it on the display device 14, and the microorganism 3 is trapped at that position.
[0019]
When an AC voltage is applied again to the electrodes 8A and 8B, an uneven electric field is formed as shown in FIG. 4D, so that microorganisms other than the specific microorganism 3 trapped by the laser beam are again applied to the electrodes 8A and 8B. 8B.
Here, by scanning the laser beam by driving the scanning device 17 or by moving the stage 5 by the stage driving device 11, as shown in FIG. 3 is a movement from the first region a ₁ through the passage 21 into the second region, is positioned at the end position of the micropipette 6 which had been advanced to advance the liquid medium in (inhalation position).
[0020]
Then, while the output of the laser beam is weakened to the extent that the microorganisms 3 do not move out of the laser spot, the suction means 10 is operated to suck the microorganisms 3 into the micropipette 6, and the microorganisms are moved without moving the micropipette 6. Only one can be reliably captured.
Also, other microorganisms electrodes 8A, without out swim to the second region A ₂ because they are attracted to 8B, since the second region A ₂ not only specific microorganisms trapped by the laser light, micro Even if the tip of the pipette 6 is somewhat thick, only one pipette can be reliably sucked into the micropipette 6.
[0021]
Next, the micropipette 6 is moved to a microplate (not shown) on which a culture medium is formed by a robot arm (not shown) or the like, and the microorganisms 3 are discharged onto the microplate.
This allows, for example, pure cultivation of genetically engineered Escherichia coli from one bacterium at a yield of 100%.
[0022]
Note that the electrodes 8A and 8B are not limited to the case shown in FIG. 2, and a linear conductor 25 may be provided along the longitudinal direction on the surfaces 24 facing each other as shown in FIG. In this case, as shown in FIG. 5B, an uneven electric field is formed in the thickness direction.
Further, when charged particles are adsorbed by electrophoresis, it suffices to form an electric field irrespective of equality or inequality. May be.
[0023]
FIG. 6 is a perspective view showing another embodiment of the cell 2. In this example, a cell plate 63 in which two circular holes 61 and 62 are formed is superposed on a substrate 64, and the circular hole is formed. The concave portion formed by the circular hole 62 and the substrate 64 becomes a _first region A ₁ for trapping a specific microorganism 3 or fine particles in the medium by irradiating the laser beam. , the second region a ₂ where a particular microorganism 3 or particulates trapped by the laser light is sucked by the micropipette.
The bottom of the cell plate 63 is formed with a groove serving as a tunnel-type passage 21 communicating the regions A ₁ and A ₂ when the cell plate 63 is overlaid on the substrate 64.
Further, on the substrate, at a position where the first region A ₁ and comprising a circular hole 61 overlaps the thin film electrode 8A made by depositing aluminum, 8B are formed.
[0024]
According to this, the first region A ₁ is filled with a medium serving as a material containing microorganisms, the second region A ₂ is filled with a medium free of microorganisms, and the first region A ₁ is irradiated with laser light. Irradiation captures any microorganisms 3 and forms an electric field between the electrodes 8A and 8B to adsorb other microorganisms 3 to the electrodes 8A and 8B. to transfer to the region a _2.
Then, when the microorganisms 3 transferred to the second area A ₂ are aspirated by the micropipette 6, only the specific microorganisms trapped by the laser light are present in the second area A ₂ , so that the tip of the micropipette 6 is Even if it is somewhat thick, only one can be reliably sucked into the micropipette 6.
[0025]
FIG. 7 is a perspective view showing still another embodiment of the cell 2. In this example, a specific microorganism 3 or fine particles in a medium are trapped by irradiating a laser beam, and the cell 2 is placed in a micropipette 6. A region A for suction is formed, and at least a pair of electrodes 8A and 8B for forming an electric field in the medium are formed with the region A interposed therebetween.
[0026]
According to this, the region A is filled with a medium serving as a material in which microorganisms are mixed, and the region A is irradiated with a laser beam to capture any microorganisms 3 and an electric field is formed between the electrodes 8A and 8B. The other microorganisms 3 are adsorbed on the electrodes 8A and 8B.
Then, in this state, if the micropipette 6 is advanced to the irradiation position of the laser light and the trapped microorganisms 3 are aspirated, only the specific microorganisms trapped by the laser light are present in the vicinity thereof. Even if the tip is slightly thicker, only one can be reliably sucked into the micropipette 6.
[0027]
【The invention's effect】
As described above, according to the present invention, specific microorganisms or microparticles are trapped by a laser beam from a group of microbes or microparticles with a laser beam to stop their movement, and other than the specific microbes or microparticles trapped. Other microorganisms or microparticles can be adsorbed on the electrode by an electric adsorption means, and if specific microbes or microparticles trapped by laser light are inhaled by a micropipette, the microbes or microparticles will be located near the tip of the micropipette. Since only one is positioned and no other microorganisms or microparticles are present, it has a very excellent effect that only one microbe or microparticle can be reliably aspirated even if a micropipette with a relatively thick tip is used. .
[Brief description of the drawings]
FIG. 1 is a schematic explanatory view showing an example of a micromanipulator according to the present invention.
FIG. 2 is a perspective view showing a cell used for it.
FIG. 3 is a schematic view of an uneven electric field formed between electrodes.
FIGS. 4A to 4D are explanatory diagrams each showing an operation procedure.
FIGS. 5A and 5B are a perspective view and a schematic view of an electric field, respectively, showing another electrode.
FIG. 6 is a perspective view showing another example of the cell.
FIG. 7 is a perspective view showing still another example of the cell.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Micromanipulator 2 ... Cell 3 ... Microorganism 4 ... Inverted microscope 6 ... Micropipette 7 ... Laser trapping means 8A, 8B ... Electrode 9 ... Electric adsorption means 10 ... suction means 20 ... plate surface a ₁ ... first region a ₂ ... second area 21 ... passage

Claims (3)

A micro that sucks a specific microorganism (3) or a fine particle from a group of microorganisms or fine particles dispersed and suspended in a medium held on a cell (2) under a visual field of a microscope (4) into a micropipette (6). A manipulator,
A laser trapping means (7) for irradiating a specific microorganism (3) or fine particles in a medium with laser light to trap the microorganism (3) or fine particles;
A microorganism group or particles other than the microorganisms (3) or the particles trapped by the laser beam is formed by forming an electric field between at least a pair of electrodes (8A, 8B) arranged in the medium, thereby forming one or both electrodes ( 8A, 8B), an electric adsorption means (9) for adsorbing the
And a suction means (10) for sucking a specific microorganism (3) or fine particles trapped by said laser trapping means (7) into a micropipette (6) advanced into a medium. manipulator. A cell for holding a medium liquid in which microorganisms or fine particles are dispersed and suspended when the microorganisms (3) or fine particles are sucked into the micropipette (6) under the field of view of a microscope;
A _first region (A ₁ ) for trapping a specific microorganism (3) or fine particles in a medium by irradiating laser light, and a specific microorganism (3) or fine particles trapped by the laser light are micropipetted. A _second area (A ₂ ) for suction is formed in communication with the passage (21),
A cell, wherein at least a pair of electrodes (8A, 8B) for forming an electric field in a medium are arranged in the first region. A cell for holding a medium liquid in which microorganisms or fine particles are dispersed and suspended when the microorganisms (3) or fine particles are sucked into the micropipette (6) under the field of view of a microscope;
A region (A) to be sucked into the micropipette (6) is formed in a state where the specific microorganisms (3) or the fine particles in the medium are trapped by irradiating the laser beam,
A cell characterized in that at least a pair of electrodes (8A, 8B) for forming an electric field in a medium are formed sandwiching the region (A).

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