JPH09271379A - Micromanipulator and cell used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a micromanipulator capable of surely separating a microorganism or particle from many microorganisms or particles contained in a medium, even when the microorganisms or particles are fast moving as Escherichia coli. SOLUTION: A specific microorganism 3 or particle in a medium is irradiated with laser beam, and an electric field is subsequently formed between electrodes 8A, 8B disposed in the medium in the state that the microorganism 3 or particle is trapped. Thus, microorganisms or particles except the specific microorganism 3 or particle trapped with the laser beam are moved toward the electrodes 8A, 8B and adsorbed to the electrodes 8A, 8B. Thereby, the specific microorganism 3 or particle is separated from the other microorganism group or particle group. Subsequently, the laser beam is scanned or a cell is moved to transfer the specific microorganism 3 or particle to a position for the suction of a micropipette, or the micropipette 6 is moved to the trap position. Thus, only the specific microorganism 3 or microorganism can surely be separated without using an especially fine micropipette.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a micromanipulator for selecting only one specific fine particle or microorganism while monitoring the fine particle group or microorganism group dispersed and suspended in a medium with a microscope and aspirating into a micropipette. Regarding

[0002]

2. Description of the Related Art For example, Escherichia coli is frequently used for gene manipulation. In this case, after the gene manipulation is carried out by dispersing it in a prescribed drug solution, the E. coli dispersed in the drug solution together with the drug solution is treated. It is inhaled with a micropipette or the like, transplanted into a culture tank and cultured to replicate a large amount of DNA. However, all of the E. coli dispersed in the drug solution are not genetically engineered due to individual differences, and when they are inhaled with a micropipette, multiple E. coli are inhaled at the same time, so they were cultured. Escherichia coli is in a mixed state of genetically engineered and non-genetically engineered, and the yield of genetically engineered is low. Therefore, if it can be cultured from one E. coli, it can be purely cultured. Therefore, if the E. coli is genetically modified, only the genetically modified E.
It becomes possible to obtain it at 00%.

[0003]

However, in order to aspirate only one micropipette, the tip of the micropipette must be thin so that surrounding solids are not sucked in at the same time, and the tip is easily broken. There was a problem. For microorganisms or microparticles that do not move or do not move like cells, by reducing the number of solids in the medium extremely, it is possible to suck one solid with a relatively thick micropipette. As for E. coli, etc., which have relatively fast movement (several μm to several tens of μm / s), it is difficult to operate following the movement,
It was difficult to aspirate only one.

[0006] Even if the micropipette can be moved in a follow-up manner by automatic control or the like, when the micropipette is moved in the liquid medicine, the liquid medicine is agitated to cause a flow, and the microorganisms to be sucked move themselves. Therefore, in the end, although it is possible to suck a large amount of the drug solution and simultaneously capture a plurality of microorganisms, it is difficult to aspirate only one microorganism. Therefore, a technical object of the present invention is to make it possible to reliably aspirate only one fast-moving microorganism or fine particle such as Escherichia coli.

[0005]

In order to solve this problem, the present invention provides a microscope view of specific microorganisms or fine particles from a group of microorganisms or fine particles dispersed and suspended in a medium held on a cell. A micromanipulator for sucking into a micropipette below, which is a laser trapping means for irradiating a specific microorganism or fine particle in a medium with a laser beam to trap the microorganism or the fine particle, and a microorganism other than the microorganism or the fine particle trapped by the laser beam. The microorganism group or fine particle group of (1) is trapped by the laser trapping means, and an electric adsorption means for adsorbing to one or both electrodes by forming an electric field between at least a pair of electrodes arranged in the medium. And a suction means for sucking specific microorganisms or fine particles into the micropipette that has advanced into the medium. Characterized in that was.

According to this, when a specific microorganism or fine particle in the medium is irradiated with laser light, the microorganism or fine particle is trapped at the focal position, and the movement of fast moving bacteria such as Escherichia coli is stopped. When an equal electric field is formed in the medium in this state, an electrophoretic phenomenon occurs in which the charged fine particles present in the electric field migrate toward the electrodes of opposite polarity. In addition, when an unequal electric field is formed, electrically neutral microbes and fine particles present in the electric field are polarized, and electric charges of equal positive and negative polarities are induced at both ends. Imbalance occurs, causing dielectrophoresis phenomenon in which microorganisms and particles migrate toward the higher electric field density.

Then, by this electrophoresis and dielectrophoresis,
Microorganism groups or fine particle groups existing between the electrodes move toward the electrode except for the specific microorganisms or fine particles trapped by the laser beam, and are adsorbed by the electrode, so that the specific microorganisms or fine particles are separated from other microorganism groups. Alternatively, it is separated from the fine particle group. Then, by scanning the laser light or moving the cell to move the specific microorganisms or fine particles to the micropipette suction position, or if the micropipette is advanced to the trap position, use a special thin micropipette. Only one specific microorganism or fine particle can be reliably sucked.

Dielectrophoresis, unlike electrophoresis,
The difference is noticeable in the AC electric field. That is, in electrophoresis, regardless of whether the electric field is equal or unequal, the Coulomb force acts on the charged particles, and the charged particles are attracted to the electrode of the opposite polarity,
In an alternating electric field, a charged particle reverses its direction as the electric field alternates and oscillates around a single point, but no translational motion occurs.On the other hand, in dielectrophoresis, the direction of a dipole polarized with an alternating electric field. Microorganisms or fine particles are always carried to the one having a stronger electric field (the one having a weaker dielectric constant) while reversing. In particular, when dealing with microorganisms and fine particles, since there are microorganisms and fine particles in the immediate vicinity of the electrode, the use of electrophoresis with a DC electric field in an aqueous solution that is a medium liquid may cause problems such as the effect of electrode reactions such as electrolysis. On the other hand, if the dielectrophoresis phenomenon caused by an alternating electric field is used, the electrode reaction is unlikely to occur.

Further, this unequal electric field is formed not by making the surfaces of the two plate-like electrodes face each other, but by
For example, one or both electrodes may be formed in a needle shape or a knife edge shape (linear shape) to form a three-dimensionally unequal electric field. For example, when knife-edge electrodes are opposed to each other in a medium, the electric lines of force of the electric field generated between the electrodes spread radially from one knife edge tip when viewed from the cross-sectional direction, and the other knife edge tip. Since the electric field near the tip of the electrode is strong and the electric field in the middle part of the electrode is weak, an unequal electric field is formed.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below based on the embodiments shown in the drawings. 1 is a schematic configuration diagram showing an example of a micromanipulator according to the present invention, FIG. 2 is a perspective view showing a cell used therein, FIG. 3 is a schematic diagram of an unequal electric field formed between electrodes, and FIG. 4 is an operating procedure. FIG.

The case of capturing electrically neutral microorganisms will be described as an example. In FIG. 1, 1 is a solid solid selected from a group of microorganisms such as Escherichia coli and captured from a medium solution held in a cell 2. A micromanipulator, in which the cell 2 is placed on a stage 5 of an inverted microscope 4, a micropipette 6 for sucking the microorganisms 3 is arranged so as to be able to move forward and backward in the cell 2, and at an arbitrary irradiation position. The specific microorganisms 3 trapped by the laser light by irradiating the laser light and trapping the specific microorganisms 3 at the irradiation position, by scanning the laser light or moving the stage 5 on which the cell 2 is placed. Laser trapping means 7 for separating the microorganisms from the microorganism group and transferring them to the tip position (suction position) of the micropipette 6 advanced into the medium, and the cell 2.
At least a pair of electrodes 8 disposed in the medium stored above
A group of microorganisms other than the microorganisms 3 trapped by the laser light by forming an unequal electric field between A and 8B is applied to the electrode 8
The electric adsorption means 9 to be adsorbed on A and 8B and the suction means 10 for sucking the specific microorganisms 3 when they are transferred to the tip position (suction position) of the micropipette 6 by the laser trapping means 7. It has and.

The inverted microscope 4 includes a stage moving device 11
The objective lens 12 is arranged below the stage 5 which is arranged so as to be movable in the XY directions, and the CCD camera 13 for photographing the inside of the cell 2 is set on the optical axis thereof.
The 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 in the optical path of the laser light output from the laser light source 15. The 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 the dichroic mirror 18 are interposed, and the reflected laser light reflected by the dichroic mirror 18 is an objective lens. The light is transmitted through 12 and is condensed in the cell 2. The objective lens 1
For example, an immersion objective lens having a magnification of 100 times and NA = 1.30 is used as 2, and a semiconductor laser is used as the laser light source 15.

As shown in FIG. 2, the cell 2 for holding the medium liquid is formed of, for example, an extremely thin glass plate, and its plate surface 20.
Above, a first area A ₁ for trapping specific microorganisms in the medium by irradiating laser light and a second area A ₂ for sucking the specific microorganisms trapped by the laser light with a micropipette. Are formed so as to communicate with each other via the passage 21. The partition wall 22 that partitions each of the areas A ₁ and A ₂ and the peripheral wall 23 that surrounds the periphery of the plate surface 20 are formed by adhering resin onto the plate surface 20 by, for example, a photoresist method or a printing method. The areas A ₁ and A ₂ may be formed by providing the plate surface 20 with recesses.

In the first area A ₁ , electrodes 8A and 8B that form an unequal electric field in the medium are arranged. In the electrodes 8A and 8B, a plurality of linear conductors 25 ... Are embedded in the surfaces 24 facing each other at a predetermined interval, and by applying an AC / DC voltage between the electrodes 8A and 8B, it is possible to obtain the structure shown in FIG. An unequal electric field is formed as shown in.
This is because the lines of electric force are the linear conductors 25 of one electrode 8A.
From each of the linear conductors 2 of the other electrode 8B.
5, the electric field lines have a high density in the vicinity 26 of the electrodes 8A and 8B and thus the electric field is strong, and the electric field lines in the middle portion 27 of the electrodes 8A and 8B have a low electric field line density and the electric field is weak. It is formed. The shape of the electrodes 8A and 8B is not limited to this. For example, one of the electrodes may be a point or linear electrode and the other may be a planar electrode. In short, electrically neutral microorganisms and fine particles When it is adsorbed by dielectrophoresis, it may be any as long as an unequal electric field is formed.

Further, a vibrator 28 such as a piezoelectric ceramics, a piezoelectric element, a giant magnetostrictive element or the like is attached to the back surface side of the cell 2, and the cell 2 is ultrasonically vibrated to be attracted to the plate surface 20 of the cell 2. It is designed to shake off the microorganisms and fine particles that have formed.

Reference numeral 30 denotes a control device for controlling the micromanipulator 1. The input side thereof is connected to the CCD camera 13, the keyboard 31, and the mouse 32, and the output side thereof is connected to the display device 14 and the laser light source 15. Drive device 33, a high-frequency oscillator 34 that forms a high-frequency AC electric field of about 1 MHz between electrodes 8A and 8B, a vibration generator 35 that drives oscillator 28, a laser scanning device driver 36, and stage drive device 11 are connected. I'm going.

The above is one example of the device of the present invention.
The operation will be described with reference to FIGS. First, set the cell 2 on the stage 5, and
As shown in (a), a medium liquid containing a large number of microorganisms (for example, Escherichia coli) is dropped in the first region A ₁ , and a medium liquid free of microorganisms is dropped in the second region A ₂ . If left in this state, microorganisms in the first area A ₁ will pass through the passage 2
Since it swims from 1 to the second area A ₂ , when an AC voltage is immediately applied to the electrodes 8A and 8B by the high-frequency oscillator 34 to form an unequal electric field, the microorganisms cause dielectrophoresis to cause each electrode 8A. , 8B. In this way, as shown in FIG.
Adsorbed to A and 8B and confined in the first region A ₁ . At this time, between the electrodes 8A and 8B, for example, when the resistivity of the medium liquid is 10 kΩ · cm or more, 400 V /
An alternating voltage of about 1 cm and 1 MHz is applied.

Next, when the voltage applied to the electrodes 8A and 8B is temporarily cut off, the unequal electric field disappears, and
As shown in (c), since the microorganisms adsorbed on the electrodes 8A and 8B start swimming away from the electrodes, the specific microorganisms 3 at the intermediate position between the electrodes 8A and 8B can be seen while observing this on the display device 14. On the other hand, when the scanning device 17 irradiates a laser beam, the microorganisms 3 are trapped at that position.

When an alternating voltage is applied to the electrodes 8A and 8B again, an unequal electric field is formed, as shown in FIG. 4 (d), so that the microorganisms other than the specific microorganism 3 trapped by the laser light are regenerated. Adsorbed to the electrodes 8A and 8B.
Here, by driving the scanning device 17 to scan the laser light or moving the stage 5 by the stage driving device 11, as shown in FIG. 3 is moved from the first area A ₁ through the passage 21 into the second area and positioned at the tip position (suction position) of the micropipette 6 that has been advanced into the medium liquid in advance.

When the suction means 10 is operated to suck the microorganisms 3 into the micropipette 6 while weakening the output of the laser light to the extent that the microorganisms 3 do not move to the outside of the laser spot, the micropipette 6 does not move. However, only one microorganism can be reliably captured. Also, other microorganisms electrodes 8A, without out swim to the second region A ₂ because it is attracted to 8B, the second region A ₂
Since there is only a specific microorganism trapped by the laser beam, even if the tip of the micropipette 6 is somewhat thick, only one can be reliably sucked into the micropipette 6.

Next, the micropipette 6 is moved to a microplate (not shown) on which a medium is formed by a robot arm (not shown) or the like, and the microorganisms 3 are discharged onto the microplate. Thus, for example, genetically engineered Escherichia coli can be purely cultivated from one bacterium at a yield of 100%.

The electrodes 8A and 8B are not limited to those shown in FIG. 2, but the linear conductors 25 are arranged along the longitudinal direction on the surfaces 24 facing each other as shown in FIG. 5A. Good. In this case, as shown in FIG. 5B, an unequal electric field is formed in the thickness direction. Further, when adsorbing charged particles or the like by electrophoresis, it suffices to form an electric field regardless of equality or inequality. Therefore, the electrodes 8A and 8B are made of plate-shaped conductors.
May be formed, and the surfaces thereof may be arranged to face each other.

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 formed by being superposed on a substrate 64.
The concave portion formed by the circular hole 61 and the substrate 64 becomes the first region A ₁ for trapping the specific microorganisms 3 or fine particles in the medium by irradiating the laser beam, and the circular hole 62
The concave portion formed by the substrate 64 serves as a _second region A ₂ for sucking the specific microorganisms 3 or fine particles trapped by the laser light with the micropipette. And the cell plate 63
A groove is formed on a bottom surface of the cell plate 63 to serve as a tunnel type passage 21 that connects the regions A ₁ and A ₂ when the cell plate 63 is stacked on the substrate 64. Also, on the substrate,
At the position where the circular holes 61 that will be the first area A ₁ overlap,
Thin film electrodes 8A and 8B formed by depositing aluminum are formed.

According to this, meet the medium a material that microorganisms in the first region A ₁ are mixed, it meets the medium with no microorganism in the second region A _2, the first region A ₁
Irradiate with laser light to capture any microorganisms 3,
After forming an electric field between the electrodes 8A and 8B to adsorb other microorganisms 3 to the electrodes 8A and 8B, the microorganisms 3 are transferred to the second region A ₂ through the tunnel type passage 21. Then, if the microbes 3 transferred to the second area A ₂ are sucked by the micropipette 6, only the specific microbes 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 slightly thick, only one can be surely sucked into the micropipette 6.

FIG. 7 is a perspective view showing yet another embodiment of the cell 2. In this example, a micropipette is used in a state where specific microbes 3 or fine particles in a medium are trapped by irradiating a laser beam. A region A for inhalation is formed in the electrode 6, and at least a pair of electrodes 8A, 8B forming an electric field in the medium are formed so as to sandwich the region A.

According to this, the area A is filled with a medium serving as a material in which microorganisms are mixed, and the area A is irradiated with laser light to trap any microorganisms 3 and the electrode 8
An electric field is formed between A and 8B to allow other microorganisms 3 to pass through the electrodes 8A,
Adsorb to 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 sucked, there are only specific microorganisms trapped by the laser light in the vicinity thereof. Even if the tip is slightly thicker, only one can be reliably sucked into the micropipette 6.

[0027]

As described above, according to the present invention, a specific microbe or fine particle is trapped by a laser beam from a group of microbes or a group of fine particles by a laser trapping means to stop its movement, and the trapped specific microbe or fine particle is trapped. Microorganisms or microparticles other than microbes or microparticles can be adsorbed to the electrode by an electric adsorption means, and if specific microbes or microparticles trapped by laser light are inhaled with a micropipette, the vicinity of the tip of the micropipette Since only one microbe or fine particle is positioned in the and no other microbe or fine particle is present, even if a micropipette with a relatively thick tip is used, only one microbe or fine particle can be reliably sucked. Has excellent effect.

[Brief description of 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 therein.

FIG. 3 is a schematic diagram of an unequal electric field formed between electrodes.

4 (a) to 4 (d) are explanatory views each showing an operation procedure.

5A and 5B are a perspective view and an electric field schematic view showing another electrode, respectively.

FIG. 6 is a perspective view showing another example of a cell.

FIG. 7 is a perspective view showing still another example of the cell.

[Explanation of symbols]

1 ... Micromanipulator 2 ... Cell 3 ... Microorganism 4 ... Inverted microscope 6 ... Micropipette 7 ... Laser trapping means 8A, 8B ... Electrode 9 ... Electrical adsorption means 10・ ・ ・ Suction means 20 ・ ・ ・ Plate surface A ₁・ ・ ・ First area A ₂・ ・ ・ Second area 21 ・ ・ ・ Passage

Front page continuation (72) Inventor Hiroshi Horio Koji Kawasaki, Kanagawa Prefecture 3-6-3 Chigasaki Higashi, Kanagawa, Japan Moritex Co., Ltd. Kohoku Technology Center (72) Inventor Osamu Kano Tsuzuki-ku, Yokohama, Kanagawa Prefecture Chigasaki Higashi 3-chome 32-3 Moritex Kohoku Technology Center (72) Inventor Chie Nishioka 3-32 Chigasaki Higashi 3-chome, Tsuzuki-ku, Kanagawa Prefecture Moritex Kohoku Technology Center Co., Ltd.

Claims (3)

[Claims] 1. A micropipette (6) under a visual field of a microscope (4) for a specific microorganism (3) or fine particles from a microorganism group or fine particle group dispersed and suspended in a medium held on a cell (2). A micromanipulator for sucking inside, a laser trapping means (7) for irradiating a specific microorganism (3) or fine particle in a medium with a laser beam to trap the microorganism (3) or the fine particle, and a laser trap One or both electrodes (8A, 8B) by forming an electric field between at least a pair of electrodes (8A, 8B) arranged in the medium, for the microorganisms (3) or the microorganisms or particles other than the fine particles. And an electric adsorption means (9) for adsorbing the specific microorganisms (3) or fine particles trapped by the laser trapping means (7) into the medium. Micromanipulator, characterized in that a suction means (10) for sucking in Tsu bets (6) within. 2. A cell for holding a medium liquid in which microorganisms or fine particles are dispersed and suspended when inhaling the microorganisms (3) or fine particles into a micropipette (6) under the view of a microscope. A _first area (A ₁ ) for trapping specific microorganisms (3) or fine particles in the medium by irradiation,
Specific microorganisms trapped by the laser light (3)
Alternatively, a second region (A ₂ ) for sucking fine particles with a micropipette is formed in communication with each other through a passage (21), and the first region has at least a pair of electrodes for forming an electric field in a medium. A cell having (8A, 8B) disposed therein. 3. A cell for holding a medium liquid in which microorganisms or fine particles are dispersed and suspended when inhaling the microorganisms (3) or fine particles into a micropipette (6) under a microscope field of view. Irradiation forms a region (A) for inhalation into the micropipette (6) while trapping specific microorganisms (3) or fine particles in the medium, and at least a pair of electric fields that form an electric field in the medium. Electrode (8A, 8
B) is formed so as to sandwich the region (A).