JP3665681B2 - Micromanipulator and cell plate used for it - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a micromanipulator for aspirating a micro sample selected from micro samples dispersed and suspended in a medium liquid under a microscope field of view, and a cell plate used therefor.
[0002]
[Prior art]
For example, Escherichia coli, which is a micro sample, is frequently used for genetic manipulation. In this case, after performing genetic manipulation by dispersing in a predetermined medium solution, the E. coli dispersed in the medium fluid is used as a medium. The whole solution is inhaled with a micropipette or the like, transplanted into a culture tank and cultured to replicate a large amount of DNA.
However, not all Escherichia coli dispersed in the medium liquid is genetically manipulated due to individual differences, and when inhaling this with a micropipette, multiple Escherichia coli are inhaled at the same time. Escherichia coli is a mixture of genetically engineered and non-genetically engineered ones, 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 manipulated, only genetically manipulated E. coli can be obtained in a yield of 100%. .
[0003]
[Problems to be solved by the invention]
However, when only one small specimen such as a microorganism or microparticle is taken out under the field of view of an optical microscope, there is no problem if the size is large to some extent. However, it was extremely difficult to take out while confirming with a microscope that there was no contamination of other minute specimens.
For example, when a microorganism having a length of 100 μm is taken out, if a lens having a resolution of 10 μm, a focal depth of 300 μm, NA = 0.034, and a field diameter of 10 mm is used, there is no contamination of other microorganisms by using a 200 μm diameter micropipette. However, microorganisms that are only about 1 μm long, such as Escherichia coli, have a resolution of 0.26 μm, NA = 1.3 (100 times oil immersion objective lens), which is the limit of optical lenses. Even if a lens is used, the depth of focus is only 0.2 μm and the field of view is only 200 μm. Therefore, when a 1 mm ³ medium liquid is sucked into a micropipette, it is confirmed that other microorganisms are not mixed with a microscope. Cannot inhale.
[0004]
In addition, even if the microscopic sample is large enough to be confirmed with a microscope, if a large number of microorganisms or microparticles are present in the 1 mm ³ medium liquid sucked into the micropipette, a large number of individuals can be sucked at the same time. May adhere to the outside of the tip of the micropipette and mix when dripping the medium liquid. Eventually, even though a large amount of the medium liquid can be sucked and a plurality of microorganisms can be taken out at the same time, 1 It is difficult to take out only the individual, and this has the trouble of having to repeat the dilution operation.
[0005]
Furthermore, even if the density of micro-analytes in the medium is small, micropipettes follow the movement of micropipets that move relatively quickly (several μm to several tens of μm / s), such as E. coli. When the pipette is operated, the medium liquid is swirled by the micropipette, and the microorganisms to be taken out are swept away by the flow, which causes a problem that they cannot be captured.
[0006]
For this reason, a sample feeding channel for flowing a medium liquid in which a minute sample is dispersed and a sample separation channel for taking out only one minute sample flowing in the sample feeding channel by flowing a medium liquid in which the minute sample is not dispersed are formed in parallel. At the same time, there has been proposed a method in which each of these channels is communicated with a connection channel and only one micro sample is taken out by intermittently connecting the connection channel with a bubble valve (USP 5100267).
The bubble valve is configured to form a bubble channel through which a liquid medium containing air bubbles flows so as to intersect the connection channel in parallel with the sample supply channel and the sample separation channel, and an intersection between the bubble channel and the connection channel. The connection channel is made conductive by filling the medium liquid and the bubble is blocked by positioning the liquid, but the medium liquid supplied by the bubble channel, the sample feeding channel, and the sample separation channel are filled. It is extremely difficult to balance the pressure with the liquid medium, for example, when air bubbles or liquid medium is sent through the bubble channel, the liquid medium flows in the other channels due to the influence of the liquid, so other channels It is extremely difficult to intermittently control only the connected channel without affecting it. Yes, it was not practical.
[0007]
Therefore, the present invention creates a state in which only one micro sample exists in the medium liquid to be inhaled at a time, and sucks the medium liquid, so that even a fast-moving microorganism such as E. coli can be confirmed without using a microscope. The technical challenge is to ensure that only individual pieces can be separated and taken out.
[0008]
In order to solve this problem, a micromanipulator according to the present invention is a micromanipulator for aspirating a microsample selected from microsamples dispersed and suspended in a medium liquid held in a cell plate with a micropipette. The cell plate has a first cell for storing a medium liquid in which a large number of minute specimens are dispersed and suspended in a plate body, and a second cell for storing a medium liquid in which the minute specimens are not suspended. A lid that is slid in the horizontal direction while being in sliding contact with the plate main body is disposed at the upper surface opening of each cell, and a micro sample is placed between the cells. free movement narrow guide path blocking is formed through a laser beam to a small analyte chosen from among micro analyte dispersed suspended in pooled liquid medium in the first cell of the cell plate A laser trapping means for trapping the minute specimen by irradiating, and in a state where the minute specimen is trapped by the laser trapping means, the cell specimen is moved or laser light is scanned to remove the minute specimen from the first cell. Sample separation means for moving to the second cell through the guide path, and sample suction means for sucking the micro sample moved to the second cell with a micropipette capable of moving back and forth in the second cell. It is characterized by that.
[0009]
In the cell plate according to the present invention, a first cell that stores a medium liquid in which a micro sample is dispersed and suspended and a second cell that stores a medium liquid in which the micro sample does not float are opened on the plate body. And a lid that is slid in the horizontal direction while being in sliding contact with the plate main body and arranged to open and close between the cells. It is characterized in that a narrow guide path that prevents free movement is formed .
[0010]
According to the present invention, after the medium liquid in which the micro sample is not suspended is injected and stored in the second cell formed on the cell plate, the medium liquid in which the micro sample is dispersed and suspended is injected into the first cell. Store. At this time, a narrow guiding path that prevents the free movement of the micro sample is formed between the first cell and the second cell so that the cells communicate with each other. Never move to the second cell.
[0011]
Next, a specific micro-analyte in a large number of micro-analytes dispersed and suspended in the medium liquid stored in the first cell is irradiated with a laser beam to trap the micro-analyte, and then in a state where this is trapped, The cell plate is moved or the laser beam is scanned to forcibly move the micro sample from the first cell through the guide path to the second cell.
At this time, only one individual trapped by the laser light is moved to the second cell, and there is only one minute specimen in the second cell. Therefore, the medium liquid in the second cell is micropipette. If it is aspirated, only one minute specimen is surely aspirated.
[0012]
In addition, a lid that slides in the horizontal direction while being in sliding contact with the plate main body is arranged at the upper surface opening of each cell, so that a medium liquid that does not float a micro sample is injected into the second cell. At the time of storage, the upper surface opening of the second cell is covered with a lid that slides in the horizontal direction, and then the medium liquid in which the micro sample is dispersed and suspended is injected into the first cell and stored. If the upper surface opening of the first cell is covered with a lid that slides in the horizontal direction, the first cell and the second cell filled with the medium liquid and the guide path are sealed and the medium liquid is less likely to evaporate. media fluid due to evaporation is prevented from flowing between the cells.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be specifically described below with reference to the drawings.
FIG. 1 is a flow sheet illustrating an example of a micromanipulator according to the present invention, FIG. 2 is a perspective view illustrating an example of a cell plate, and FIGS. 3A to 3F are explanatory diagrams illustrating an operation procedure thereof.
[0014]
In the figure, reference numeral 1 denotes a micromanipulator for taking out one microorganism 3 selected from microorganisms (microscopic specimens) dispersed and suspended in a medium liquid held on a cell plate 2, and the cell plate 2 is a stage of an inverted microscope 4. A micropipette 6 that is placed on 5 and sucks the microorganisms 3 is disposed so as to be able to advance and retreat with respect to the cell plate 2. This micromanipulator 1 includes a laser trapping means 7 for irradiating a microorganism 3 selected from microorganisms dispersed and suspended in a medium liquid under a field of view of a microscope 4 and trapping the microorganism 3 at the irradiation position, and a microorganism In a state where 3 is trapped, the cell plate 2 is moved or a laser beam is scanned to separate the microorganism 3 from other microorganisms, and the separated specific microorganism 3 is micropipette. 6 is provided with a sample aspirating means 9 for aspirating the inside.
[0015]
The cell plate 2 that holds the medium liquid includes a first cell 11A that stores a medium liquid in which a large number of microorganisms are dispersed and suspended on a 0.17 mm plate body 10 that serves as a cover glass of the inverted microscope 4, and a microorganism. The second cell 11B for storing the medium liquid not floating is formed at a predetermined interval with the upper surface opened, and a narrow guide for preventing the free movement of the minute specimen between the cells 11A and 11B. A path 12 is formed to penetrate each cell 11A and 11B .
[0016]
Accordingly, the guide path 12 is formed with an optical plane whose bottom surface serves as a cover glass of the inverted microscope 4, and a buffer cell 11 </ b> C for storing a medium liquid in which microorganisms do not float open in the middle of the guide path 12. Thus, the guiding path 12 includes a first guiding path 12a that communicates the first cell 11A and the buffer cell 11C, and a second guiding path 12b that communicates the buffer cell 11C and the second cell 11B. Formed from.
[0017]
Then, each cell 11 A- 11 C are formed in the large diameter recess 13A~13C as a surface formed medium injection opening of the plate body 10, the upper opening of each cell 11 A- 11 C, the recess Lids 14A to 14C that are opened and closed by sliding in the horizontal direction while being in sliding contact with the bottom surfaces of 13A to 13C are arranged.
In this case, the surface recess 13A～13 C and the lid 14A~14C are in contact with each other is optically polished so as to contact slidably with gap wavelength order of light. Thereby, even if the lids 14 </ b> A to 14 </ b> C are slid, each cell can be opened and closed without creating a flow in the guide path 12.
In the cell plate 2 for separating E. coli, each of the recesses 13A to 13C has a diameter of about 9 mm × depth of about 2 mm, and each of the cells 11A to 11C has a diameter of about 2 mm × depth of about 1.8 mm, between the cells 11A and 11C and between the cells 11C, 11C, The lengths of the guide paths 12a and 12b communicating between 11B are about 9 mm, and the cross sections of the guide paths 12a and 12b are about 0.1 mm in length and width.
[0018]
In the inverted microscope 4, an objective lens 16 is disposed below a stage 5 disposed so as to be movable in the X and Y directions by a stage moving device 15, and a CCD for photographing the inside of the cell plate 2 on its optical axis. The camera 17 is set, and an image photographed by the CCD camera 17 is displayed on the display device 18.
The laser trapping means 7 traps the microorganisms 3 in the first cell 11A with laser light that passes through the objective lens 16 of the inverted microscope 4 and is in the optical path of the laser light output from the laser light source 19. Includes a scanning device 21 having a scanning mirror 20x, 20y for moving the irradiation position of the laser beam in the X direction and the Y direction and a dichroic mirror 22, and the reflected laser beam reflected by the dichroic mirror 22 is reflected. The objective lens 16 is transmitted and condensed in the first cell 11A.
For example, an immersion objective lens having a magnification of 100 times and NA = 1.30 is used as the objective lens 16, and a semiconductor laser is used as the laser light source 19, for example.
[0019]
Further, in the specimen separating means 8, the cell plate 2 is moved by the stage moving device 15 or the laser light is scanned by the scanning device 21 while the specific microorganism 3 is trapped by the laser trapping means 7. The microorganism 3 is moved from the first cell 11A through the guide path 12 to the second cell 11B to be separated from other microorganisms in the first cell 11A.
[0020]
The sample aspirating means 9 of the micropipette 6 performs aspiration / discharge by changing a volume in the pipette 6 by displacing a diaphragm or the like formed of an elastic membrane, and controls the temperature in the pipette 6 to control the temperature. Arbitrary means such as suction / ejection using the volume change of air based on the change can be adopted.
In addition, the micropipette 6 is attached to the tip of a robot arm (not shown), and is configured to reciprocate between, for example, a microplate (not shown) on which a medium is formed. Is discharged onto a predetermined microplate.
[0021]
Reference numeral 30 denotes a control device for controlling the micromanipulator 1. The CCD camera 17, the keyboard 31, and the mouse 32 are connected to the input side, and the display device 18 and the laser light source 19 are connected to the output side. The drive device 33, the driver 34 of the laser scanning device 21, and the stage moving device 15 are connected.
[0022]
The above is an example of the device of the present invention. Next, its operation will be described with reference to FIGS.
First, the cell plate 2 is set on the stage 5 and, as shown in FIG. 3A, a clean medium liquid free of microorganisms is poured into the recess 13B where the second cell 11B is formed, and the lid 14B is used as the medium. When the lid 14B is immersed in the liquid, the upper surface opening of the second cell 11B is closed by sliding the lid 14B. At this time, the medium liquid in the second cell 11B flows through the guiding path 12b by capillary action and flows out into the buffer cell 11C.
[0023]
Next, as shown in FIG. 3B, a clean medium liquid without microorganisms is poured into the recess 13C in which the buffer cell 11C is formed, and the lid body 14C is removed when the lid body 14C is immersed in the medium liquid. The upper surface opening of the buffer cell 11C is closed by sliding. At this time, the medium liquid in the buffer cell 11C flows through the guide path 12a by the capillary phenomenon and flows out into the first cell 11A.
[0024]
Then, as shown in FIG. 3C, a medium liquid in which a large number of microorganisms are dispersed and suspended is poured into the recess 13A in which the first cell 11A is formed, and the lid body 14A is immersed in the medium liquid when the lid body 14A is immersed in the medium liquid. 14A is slid to close the upper surface opening of the first cell 11A.
At this time, the sliding contact surface between the lid 14A and the concave portion 13 A is because it is optically polished, by sliding the lid 14A, the liquid medium is guideway 12a in the first cell 11A, through 12b, It does not flow into the buffer cell 11C or the second cell 11B.
Further, since the upper surface openings of the cells 11A to 11C are all closed by the lids 14A to 14C and the lids 14A to 14C are immersed in the medium liquid, The medium liquid stored in the recesses 13A to 13C outside the cells 11A to 11C blocked by the bodies 14A to 14C will evaporate, and the influence will not be exerted in the cells 11A to 11C. Therefore, the medium liquid in the cells 11 </ b> A to 11 </ b> C is maintained in a stopped state, and no flow is formed in the guide path 12.
[0025]
In this state, while observing the inside of the first cell 11A with the display device 14 and irradiating the arbitrary microorganism 3 with laser light by the scanning device 22, the microorganism 3 is located at that position as shown in FIG. Is trapped.
In this state, as shown in FIG. 3 (e), by moving the stage 5 by the stage moving device 15, the specific microorganism 3 trapped by the laser beam passes from the first cell 11A through the guide path 12a to the buffer cell 11C. Furthermore, it moves to the 2nd cell 11B through the guidance path 12b.
[0026]
At this time, since the guide path 12 that communicates the first cell 11A and the second cell 11B is formed narrow enough to prevent free movement of microorganisms, other microorganisms in the first cell 11A guide The probability of swimming on the road 12 and reaching the second cell 11B is extremely low, and therefore, only the special microorganism 3 trapped by the laser light exists in the second cell 11B.
[0027]
In this example, since the buffer cell 11C is formed in the middle of the guide path 12, even if the microorganisms swim and pass from the first cell 11A through the guide path 12a and reach the buffer cell 11C, the buffer cell 11C is buffered again. It is almost impossible to reach the second cell 11B from the cell 11C through the guide path 12b. Therefore, only the specific microorganism 3 trapped by the laser beam exists in the second cell 11B, and other microorganisms exist. do not do.
[0028]
Finally, as shown in FIG. 3 (f), the lid 14B is slid to open the upper surface opening of the second cell 11B, and all the medium liquid in the second cell 11B is sucked with the micropipette 6. Since there is only the microorganism 3 trapped by the laser beam in the medium liquid, one microorganism can be used without using a particularly thin one as the micropipette 6 and without confirming the inside of the second cell 11B with the microscope 4. Can be reliably sucked.
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 together with the medium solution. Thus, for example, genetically engineered Escherichia coli can be purely cultured from one bacterium at a yield of 100%.
[0029]
Note that the buffer cell 11C is not limited to being formed in the middle of the guide path 12, but can be formed in an arbitrary number as necessary, and may not be provided in some cases.
Further, the micro sample is not limited to a microorganism such as Escherichia coli but may be a fine particle.
Furthermore, in this example, the case where the first cell 11A and the second cell 11B are formed one by one on the cell plate has been described. However, the present invention is not limited to this, and a plurality of the second cells 11B are formed. You may make it connect with 11 A of 1st cells via the guidance path 12. FIG. In this case, after injecting a liquid medium in which microorganisms are not suspended into all the second cells 11B and closing the upper surface openings with the lids 14B, finally, the microorganisms are dispersed and suspended. The medium liquid may be injected into the first cell 11A, and the upper surface opening may be closed with the lid 14A.
[0030]
【The invention's effect】
As described above, according to the present invention, a specific micro-analyte in a large number of micro-analytes dispersed and suspended in the medium liquid stored in the first cell is irradiated with laser light to trap the micro-analyte, The micro sample is forcibly moved from the first cell to the second cell through the guide path by moving the cell plate or scanning with laser light while keeping this trapped. Therefore, when only one individual trapped by the laser beam is moved to the second cell, there is only one minute specimen in the second cell, so the medium liquid in this second cell is aspirated with a micropipette. For example, even a fast-moving microorganism such as Escherichia coli has an excellent effect that it can be reliably separated and taken out without checking with a microscope.
[Brief description of the drawings]
FIG. 1 is a micromanipulator according to the present invention.
FIG. 2 is a cell plate used for this.
FIGS. 3A to 3F are explanatory diagrams showing an operation procedure. FIG.
[Explanation of symbols]
1 ... Micromanipulator 2 ... Cell plate 3 ... Specific microorganism (micro sample)
6. Micropipette 7 ... Laser trapping means 8 ... Separation means 9 ...・ Suction means 10... Plate body 11A... First cell 11B... Second cell 11C. ··· Buffer cells 12, 12a, 12b ··· Guidance paths 13A to 13C ··· depressions 14A to 14C ··· lid

Claims (5)

A micromanipulator for aspirating a microsample selected from microsamples dispersed and suspended in a medium liquid held in a cell plate with a micropipette,
The cell plate has a first cell for storing a medium liquid in which a large number of minute specimens are dispersed and suspended in a plate body, and a second cell for storing a medium liquid in which the minute specimens are not suspended. A lid that is formed at a predetermined interval and that opens and closes by sliding in the horizontal direction while being in sliding contact with the plate body is disposed at the upper surface opening of each cell. A narrow guiding path that prevents movement is formed through,
Laser trapping means for irradiating a laser beam to a micro sample selected from micro samples dispersed and suspended in a medium liquid stored in the first cell of the cell plate, and trapping the micro sample;
A sample that moves the cell plate or scans the laser beam while the micro sample is trapped by the laser trapping means, and moves the micro sample from the first cell to the second cell through the guide path. Separating means;
A micromanipulator comprising: a sample aspirating means for aspirating a micro sample that has moved to the second cell with a micropipette that can advance and retreat into the second cell. The plate body, a first cell in which the micro sample to storing distributed floating the liquid medium, the second cell the micro specimen storing liquid medium which is not suspended are respectively the upper face is opened are formed apart a predetermined distance, each A lid that is slid in the horizontal direction while being in sliding contact with the plate body is disposed at the upper surface opening of the cell, and a narrow guiding path that prevents free movement of a minute specimen between the cells. A cell plate for a micromanipulator, characterized in that is formed through .   The cell plate for a micromanipulator according to claim 2, wherein one or more buffer cells for storing a medium liquid in which a micro sample is not suspended are formed in the middle of the guide path with an upper surface opened.   4. The cell plate for a micromanipulator according to claim 2, wherein the guide path is formed by an optical plane whose bottom surface serves as a cover glass for an inverted microscope. The cell plate for micromanipulators according to claim 2 , wherein a recess is formed on the surface of the plate main body so as to immerse each lid body in which the upper surface opening of each cell is closed in a medium liquid stored in each cell. .

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