JPH04179469A - Particle manipulative system - Google Patents

Abstract

PURPOSE:To facilitate the immobilization of particles by such a method that the particles reached the intersection of cross-type flow channels are immobilized through suction at the opening and manipulated with a probe set at a second opening facing to the first opening and then returned into the flow channels. CONSTITUTION:A sample liquid 31 containing cells 8 is allowed to flow into cross-shaped flow channels using syringes 33,43 controlled by a control unit 6, and the cells 8 are brought close to an opening 14 while observing the state in the flow channels with a microscope 51. The diameter of this opening 14 is smaller than that of the cells; therefore, the cells are immobilized at the opening and manipulated with a probe 21 for a second opening 13. After manipulation, the cells are forcedly made to flow using the syringes 43,33 toward a recovering section 7. A microscope 51 is equipped with an image input unit 52, and data are transferred via a processing section 53 to a control section 6, and a sample liquid feed section 3, immobilizing section 4, micromoving section 22 and suction-delivery section 23 are controlled at the control section 6, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to devices for retaining and manipulating particles, such as cells, in fluids.

[Conventional technology]

The conventional device uses a particle manipulation probe such as a pipette mounted on a fine movement table, as described in Japanese Patent Application Laid-Open No. 61-267723. In order to perform operations such as injecting drugs containing genes into cells existing in a liquid in a petri dish or on a glass slide under a microscope, and removing the nucleus into 9 sections, the cells are placed in a liquid in a glass tube with an inner diameter slightly smaller than the size of the cells. Floating cells were sucked up and fixed, and other small pipettes were inserted into the cells to perform operations such as injecting drugs and removing nuclei.

[Problem to be solved by the invention]

In the above-mentioned conventional technology, when a pipette is brought close to suck up microscopic cells floating under a microscope, the resulting flow causes the cells to escape, and it takes a long time to fix them. In addition, in order to forcefully suction cells that are far away, the suction pressure was increased too much, which could damage the cells.

Furthermore, the relative positioning of the pipette that holds the particles and the instrument that manipulates the particles is not easy, and the operation takes time and the accuracy of the operation is poor.

An object of the present invention is to facilitate the fixation of particles using a pipette or the like and to shorten the operation time.

Another object of the present invention is to facilitate the relative positioning of a pipette for holding particles and a device for manipulating particles, thereby making it possible to shorten the time for manipulating particles and improve the precision of manipulation.

[Means to solve the problem]

In order to achieve the above object, the present invention includes a first flow path partially or entirely made of a transparent member, and a first fluid drive that drives a fluid containing particles flowing through the first flow path. an opening in the first flow path that faces the particle manipulation device, which includes a source, a first control unit that controls the first fluid driving source, and an optical device such as a microscope that observes the state within the flow path; , the diameter of the first opening is smaller than the diameter of the particles in the fluid,
Furthermore, a second flow path connected to the first opening is provided, and the second flow path has a second drive source that moves the fluid in the second flow path, and a second drive source that controls the second flow path. A particle manipulation probe having an outer diameter smaller than the diameter of the second opening is provided in the second opening.

[Effect]

A fluid containing particles is caused to flow toward the opening by a first driving source. The vicinity of the opening is observed using a microscope, and the fluid is stopped when the particles to be manipulated reach just in front of the opening. next,
The fluid in the first flow path is sucked through the second opening by the second driving source. With this suction, particles within the fluid approach the first opening. Since the diameter of the first opening is smaller than the diameter of the particles, the particles cannot pass through the first opening and are fixed to the first opening. Next, the particles are manipulated using a needle or tubular particle manipulation probe placed in a second opening opposite the first opening.

Next, the fluid in the second flow path is pushed out into the first flow path by the second driving source, and the particles fixed in the first opening are returned into the first flow path. Further, the first active source causes the manipulated particles to flow downstream of the opening for collection.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG.

The configuration of the device is shown at the bottom of FIG.

Functionally, there are an inlet 11 and an outlet 12 for a sample solution 31 containing cells, and an insertion inlet 1 for inserting a probe 21 for particle manipulation.
3, a particle manipulation jig 1 (a transparent one made by processing a glass tube, etc.) having a cross-shaped flow path ending with a fixing port 14 for particle fixation, and an injection port 11 of the particle manipulation jig 1. a sample liquid supply unit 3 that supplies a sample liquid 31 from the sample liquid, a collection unit 7 that collects treated cells coming out from the discharge port 12, and a fixing port 1.
4, a fixing part 4 that fixes and separates cells, a fine movement part 22 that slightly moves the probe 21 for cell manipulation, and a suction part 4 that is connected to the insertion port and that sucks and discharges liquid from the tip of the probe 21. A discharge section 23, an image processing section 5 that detects and processes images in the particle manipulation jig 1, and a sample liquid supply section 3, a fixing section 4, a fine movement section 22, and a suction/discharge section based on the processing results of the image processing section 5. The system controller 6 is classified as a system control unit 6 that coordinately controls the 23.

Insertion port 13 of particle manipulation jig 1. The structure near the fixed port 14 is as shown in the upper part of FIG.

The insertion port 13 extends slightly toward the probe 21 side, and there is a liquid seal portion 9 (for example, a magnetic fluid seal) between the probe 21 and the insertion port to allow the probe 21 to move. Note that if the insertion port 13 is sufficiently thin, liquid will not leak due to surface tension and there is no need for sealing.

The sample liquid supply section 3, fixing section 4, and suction/discharge section 23 have similar configurations. That is, sample tubes 37, 47, 237 containing various liquids, valves 32, 42, 232, syringes 33, 43, 233, and pipes 35, 45, . ．． 235 and pipes 36, 46, 236
and pipes 38, 4 that connect the particle manipulation jig 1 and each part.
8, 238, and an interface 34, 44, 234 that controls the opening and closing of the valves 32, 42, 232, activates the syringe, and interfaces with the system control unit 6.

The recovery unit 7 includes a test tube 771 containing physiological saline 71, a test tube 772 for cell recovery, a valve 72, pipes 751, 752 connecting them, a particle manipulation jig 1, and the valve 72.
It consists of a pipe 78 that connects the

The fine movement unit 22 includes a probe 21 and a fine movement stage 221 that carries the probe 21 and operates in the XYZ directions with an accuracy of less than a micrometer, and an interface 22 that interfaces the fine movement stage 221 and the system control unit 6.
Consists of 2.

The image processing unit is composed of an image input device 52 such as a television camera attached to the microscope J151, and an information processing unit 53 that processes the image, identifies the position and type of cells, and transfers the data to the system control unit 6. .

The operation of the apparatus having the above configuration is shown in FIGS. 2 to 7. The upper half of the figure is an enlarged view of the vicinity of the insertion port and fixing port, and the lower half is a diagram showing the overall operation.

First, the preparation operation is performed as follows.

(1) The valve 32.42 connects the syringe 33.43 and the injection port 11 of the particle manipulation jig 1. Fixed port 14 to pipe 35.45
and are connected via pipes 38 and 48. In this state, the syringe 33.43 is operated to draw the physiological saline 71 in the test tube 771 from the pipe 78 into the outlet 12, and through the particle manipulation jig 1 to pipes 38.48 and 35°45. . At the same time, a liquid 231 such as a drug in a test tube 237 is sucked into a syringe 233 through a pipe 236 and a pipe 235 by a valve 232 (FIG. 2).

(2) Close the valve 72 and open the pipe 35 by the valves 32 and 42.
45. After operating the syringe 33.43 to discharge the air in the syringe 33.43 through the pipes 36 and 46,
The sample liquid and physiological saline 41 in the test tube 37.47 are aspirated into the syringe 33.43. At the same time, the pipe 235 and the pipe 238 are connected by the valve 232, and the syringe 233
The liquid 231 in the syringe 233 is moved to the probe 2.
1 (Figure 3).

Next, a particle movement operation is performed.

(3) Pipe 78 by valve 72. The outlet 12 and the test tube 772 are connected via a pipe 752 . At the same time, valve 32
connects the pipe 35 and the pipe 38, drives the syringe 32, and transfers the sample liquid 31 in the syringe 33 to the particle manipulation jig 1.
Send it inside.

The image processing unit 5 monitors the image inside the particle manipulation jig 1 and identifies the cells 8 to be manipulated in the sample liquid based on their size, morphological characteristics, etc., and when the cells 8 reach the vicinity of the fixing port 14, The drive of the syringe 33 is stopped and the cells 8 are stopped near the fixed port 14.

Furthermore, cells are fixed and manipulated (Figure 4).

(4) Connect pipe 38 and pipe 36 by valve 32,
The syringe 43 is driven and sucked into the fixed port 14 of the cell 8. At the same time, the cell 8 is monitored by the image processing unit 5.
After confirming that the syringe 43 is fixed, stop driving the syringe 43 (Fig. 5).

(5) Drive the fine movement stage 221 to move the probe 21 according to the data indicating the shape and position of the cell from the image processing unit 5.
The operation is performed on the cell 8 by. At this time, if necessary, the syringe 233 is driven to inject the drug into the cells 8 and remove the nucleus (FIG. 6).

Finally, - Collect the treated cells 8.

(6) Drive the syringe 43 to push out the physiological saline 41 inside the syringe 43 and separate the cells 8 from the fixed port 14. Next, the syringe 33 is driven to send the sample liquid 31 to the particle manipulation jig 1, and the operation returns to (4) suction operation to the fixed port 14. The treated cells 8 are collected into the test tube 772 of the collection unit 7 via the outlet 12 along the flow of the sample liquid 31 (
(Figure 7).

According to the present invention, the cells are sent to the vicinity of the fixation port and fixed by suction, making it easy to fix the cells and prevent damage to the cells.

In addition, since the cell detection process and the syringe operation are performed automatically in coordination, there is a possibility that a large number of cells can be manipulated in a short period of time, instead of the long and skilled manual work. Also,
Since the position for fixing the cells is fixed and there is no need to focus the microscope during cell manipulation, highly accurate processing is possible.

Furthermore, if the outer shape of the particle manipulation jig 1 is rectangular and the flow path is also rectangular, refraction of light passing through the particle manipulation jig 1 can be prevented and an accurate cell image can be captured.

Next, another embodiment of the present invention is shown in FIG.

FIG. 8 is a diagram showing the vicinity of the fixed port 14. Fixed port 14
A suction tube 49 for suctioning and fixing cells is inserted into the tube, and is connected to the fixing part 4 by a vibrator 48 as in the embodiment shown in FIG.

The operation is similar to the embodiment shown in FIG.

According to this embodiment, the inhalation tube can be replaced depending on the size of the cells, making it possible to manipulate many types of cells. In addition, while the cells are fixed in the tube, the tube is removed and the cells are removed.
You can also take out pieces.

Next, another embodiment of the present invention is shown in FIG.

The configuration of the apparatus shown in FIG. 9 is as follows.

The basic configuration is the same as that of the apparatus shown in FIG. 1, but a laser light source 54 is newly installed at a position facing the microscope 51 with the particle manipulation jig 1 in between. The laser beam 541 passes through the channel in the particle manipulation jig 1 and reaches the objective lens of the microscope 21 . At this time, a wire 512 that blocks the laser beam is placed in front of the objective lens so that the laser beam does not directly enter the objective lens. Further, a highly sensitive photodetector 513 such as a photomultiplier tube is attached to the microscope 51, and an optical filter 514 that transmits light of a specific wavelength is inserted in the optical path from the particle manipulation jig 1 to the photodetector 513. .

The output signal from the photodetector 513 is sent to a signal processor 315, where it is processed and sent to an information processing section 53.

The operation is almost the same as that of the apparatus shown in FIG. 1, but the signal from the laser optical system described above is used when detecting and classifying cells. Generally, cells are classified by dyeing the cells and classifying them based on their morphology, or by adding a fluorescent substance, binding it to specific molecules within the cell, and irradiating it with monochromatic light obtained from a laser, etc., and measuring the amount of fluorescence generated. There are ways to perform classification. This embodiment adds the latter classification based on fluorescence.

According to this embodiment, classification based on fluorescence can be performed, which increases the number of types of cells that can be handled. Furthermore, since the number of classification items increases, the classification of cells becomes more accurate.

Additionally, in another embodiment of the invention, a microscope.

Use a confocal optical microscope (scanning laser microscope).

Confocal optical microscopy can also provide a three-dimensional view of intracellular microstructures with higher resolution (resolving power approximately 30% higher) than conventional microscopes. Therefore, by controlling micro-movement parts based on intracellular 3D information from a confocal optical microscope, operations on chromosomes and organelles that are smaller than the intracellular nucleus can be performed at a level that is almost impossible to perform manually. ) becomes possible. Furthermore, since the deformation state of cells near the fixed port can be captured three-dimensionally, rheological information such as cell stiffness can be obtained.

〔Effect of the invention〕

According to the present invention, particles can pass through a certain flow path and be stopped near the fixing opening, and since there is no movement of the probe that disturbs the flow, particles can be easily fixed by suction operation from the opening, and the operation is easy. This has the effect of shortening time and reducing damage to particles.

Furthermore, since the fixed position of the particle is always the same, the position of the operating instrument relative to the particle can be kept constant, eliminating the need for a mechanism for moving the operating instrument, and improving the precision of operation.

[Brief explanation of the drawing]

FIG. 1 shows a cross section (a) of a main part and a system diagram (a) of an embodiment of the present invention.
b), FIGS. 2 to 7 are explanatory diagrams of the operation of one embodiment of the present invention, FIG. 8 is a partial sectional view of another embodiment of the present invention, and FIG. 9 is a partial sectional view of another embodiment of the present invention. It is a system diagram. 1... Particle manipulation jig, 3... Sample liquid supply section, 4...
・Fixing part, 5... Image processing part, 6... System control part, 7 Collection part, 8... Cell, 9... Seal part, 11...
...Inlet, 12...Drain port, 13...Insertion port, 14
...Fixed port, 21...Probe, 22...Fine movement part,
23...Aspiration/discharge Fig. 1 (E) Fig. 2 (Ku) Fig. 30 (QJ 'lA4 Fig. (Ku) 75z #2 (#+) No. gi

Claims (1)

[Claims] 1. A first channel partially or entirely made of a transparent member, and a first fluid driving source that drives a fluid containing particles flowing through the first channel. , a particle manipulation device comprising a first control unit that controls the first fluid drive source and an optical device that observes a state within the flow path, wherein an opposing opening is provided in the first flow path; The diameter of the first opening is made smaller than the diameter of the particles in the fluid, and a second flow path connected to the first opening is provided, and the second flow path is connected to the inside of the second flow path. a second driving source that drives the fluid;
A second control unit for controlling the second drive source is provided, and a particle manipulation probe having an outer diameter smaller than the diameter of the second opening is installed in the second opening. Characteristic particle manipulation device.