JP2017205089A - Pressure controller, manipulation system and pressure control method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pressure controller, manipulation system, and pressure control method, which can efficiently and suitably fix a minute object irrespective of operator's skill and technique.SOLUTION: According to the present invention, there is provided a pressure control apparatus to which fixing means for fixing a minute object is attached by internal pressure. Here, the apparatus comprises: a pressure control section for adjusting the internal pressure of the fixing means; an imaging section comprising: an imaging unit that images a minute object; and a calculation section that calculates a position of a minute object based on an image obtained from the imaging section. The pressure control section begins suction by fixing means in a state that the minute object is being separated from the fixing means, and then determined whether the minute object is fixed to the fixing means based on the information about a change in the position of the minute object received from the calculation section.SELECTED DRAWING: Figure 13

Description

  The present invention relates to a pressure control device, a manipulation system, and a pressure control method.

  In the field of biotechnology, a micromanipulation system that performs a minute operation on a minute object such as injecting a DNA solution or a cell into a cell or egg under a microscope is known. While fixing the position of the micro object with a fixing pipette for fixing the micro object, the operation pipette is inserted into the position of the operation object of the micro object to perform the injection operation. The fixing operation of the minute object is performed by recognizing the position of the minute object with a microscope and performing the suction fixation by bringing the fixing pipette close to the minute object.

  The pressure control device for cell adsorption described in Patent Document 1 below maintains the adsorption and retention of cells in a good state by controlling the suction amount of the pressure control unit based on the deformation amount of the cells adsorbed by the fixing pipette. it can.

JP-A-5-322716

  When operating the fixing pipette and the operation pipette with a joystick etc. to perform the fixing operation of the micro object and the injection operation to the micro object, a high level of skill of the operator is required, There may be variations in operation depending on the operator's senses. In order to reduce the burden on the operator's work, automation of the fixing operation of the minute object is required. In Patent Document 1, an operator performs a moving operation of a fixing pipette, and the tip of the fixing pipette is brought into contact with a desired cell to perform adsorption. For this reason, in the operation | movement of the movement of a pipette for fixation, and the operation | work of adsorption fixation, damage to a micro target object and the variation in operation may arise.

  It is an object of the present invention to provide a pressure control device, a manipulation system, and a pressure control method capable of efficiently and suitably performing a fixing operation of a minute object regardless of the skill level and skill of an operator.

  A pressure control device according to an aspect of the present invention is a pressure control device to which a fixing means for fixing a minute object by an internal pressure is mounted, and a pressure control unit that adjusts the internal pressure of the fixing means; An imaging unit that images the minute object; and a calculation unit that calculates a position of the minute object based on an image obtained from the imaging unit, and the pressure control unit includes: The suction by the fixing means is started in a state away from the fixing means, and the micro object is fixed to the fixing means based on the information on the change in the position of the micro object received from the calculation unit. Judge that.

  According to this, since the pressure control unit can start the suction by the fixing unit in a state where the micro object is separated from the fixing unit, the pressure control unit starts the suction in a state where the fixing unit is in contact with the micro object. Compared to the case, the moving operation of the fixing means can be easily performed, and damage to the minute object can be suppressed. Further, since it can be determined that the minute object is fixed to the fixing means by image recognition of the minute object, it is not necessary to perform image recognition of the fixing means, and the fixing operation can be easily performed.

  In the pressure control apparatus according to one aspect of the present invention, the calculation unit calculates a moving speed of the micro object from a change in the position of the micro object, and the pressure control unit moves the micro object. The internal pressure of the fixing means is adjusted based on the speed information. According to this, since the suction by the fixing means is performed with an appropriate internal pressure in accordance with the moving speed of the minute object, damage to the minute object can be suppressed.

  In the pressure control apparatus according to an aspect of the present invention, the pressure control unit adjusts the internal pressure of the fixing unit so that the moving speed of the minute object is within a predetermined range. According to this, since the minute object is prevented from coming into contact with the fixing means at an excessive speed, damage to the minute object can be suppressed.

  In the pressure control apparatus according to an aspect of the present invention, the pressure control unit starts the suction in a state where the fixing unit is in contact with the bottom of the sample holding member on which the minute object is placed. According to this, since the suction force of the fixing means acts on the minute object reliably, the minute object can be moved and fixed to the fixing means.

  In the pressure control device according to an aspect of the present invention, the pressure control unit may be configured to detect the micro object when a moving speed of the micro object is equal to or lower than a predetermined speed after the micro object starts moving. It is determined that the object is fixed to the fixing means, and the internal pressure of the fixing means is maintained. According to this, it is possible to determine whether or not the minute object is fixed from the moving speed of the minute object calculated based on the image recognition. Therefore, it is not necessary to perform image recognition of the fixing means, and the fixing operation can be easily performed by simplifying the calculation of the calculation unit.

  In the pressure control device according to an aspect of the present invention, the pressure control unit stops the suction when the position of the minute object does not change for a predetermined period after the suction by the fixing unit is started. According to this, since there is a possibility that the image-recognized object is not the operation target minute object, it is possible to perform operations on the other operation object minute objects.

  The calculation unit calculates the position of the center of gravity of the minute object based on the image captured by the imaging unit. According to this, the position and moving speed of the minute object can be accurately calculated by calculating the center of gravity of the minute object.

  A manipulation system according to an aspect of the present invention provides a first manipulator including any one of the above-described pressure control devices, a sample stage on which a minute object is placed, and the fixing means for fixing the minute object. A second manipulator including an operation unit for operating the minute object held by the fixing unit, and a control unit for controlling the sample stage, the fixing unit, the operation unit, and the imaging unit. . According to this, the first manipulator provided with the fixing means and the pressure control device perform the fixing operation of the minute object while suppressing the damage of the minute object, and the operation on the minute object fixed to the fixing means. Can be executed by the second manipulator.

  A pressure control method according to an aspect of the present invention is a pressure control method of a pressure control device to which a fixing means for fixing a minute object by an internal pressure is mounted, the pressure adjusting the internal pressure of the fixing means The control unit starts the suction by the fixing unit in a state in which the micro object is separated from the fixing unit, and the calculation unit is based on an image obtained from the imaging unit that images the micro object. The step of calculating the change in the position of the minute object, and the pressure control unit fixes the minute object to the fixing means based on the information on the change in the position of the minute object received from the calculation unit. Determining that it has been done.

  According to this, since the pressure control unit can start suction by the fixing means in a state where the minute object is separated from the fixing means, the fixing means can be compared with the case where the fixing means is brought into contact with the minute object. Can be easily performed, and the damage to the minute object can be suppressed. Since it can be determined that the minute object is fixed to the fixing means by the image recognition of the minute object, it is not necessary to perform image recognition of the fixing means, and the fixing operation can be easily performed.

  According to the present invention, a minute object can be fixed efficiently and suitably regardless of the skill level and skill of the operator.

Drawing 1 is a figure showing typically composition of a manipulation system provided with a pressure control device concerning an embodiment. FIG. 2 is a cross-sectional view showing an example of a fine movement mechanism. FIG. 3 is a control block diagram of the manipulation system. FIG. 4 is a flowchart showing an example of the pressure control operation of the manipulation system. FIG. 5 is a plan view schematically showing the position of the cell in the imaging region of the camera. FIG. 6 is a schematic diagram of cells detected by image processing. FIG. 7 is a cross-sectional view showing a schematic configuration of the first pipette, the medium and the cells held by the sample holding member. FIG. 8 is a graph schematically showing the relationship between the moving speed of cells and time. FIG. 9 is a graph schematically showing the relationship between the pump driving speed and time. FIG. 10 is a plan view schematically showing the relationship between the first pipette and the cells in the first steady state. FIG. 11 is a graph schematically showing the relationship between the cell position in the Xc axis direction and time in the first steady state. FIG. 12 is a graph schematically showing the relationship between the position of the cell in the Yc-axis direction and time in the first steady state. FIG. 13 is a plan view schematically showing the relationship between the first pipette and the cells in the second steady state. FIG. 14 is a graph schematically showing the relationship between the position of the cell in the Xc-axis direction and time from the first steady state to the second steady state. FIG. 15 is a graph schematically showing the relationship between the position of the cell in the Yc axis direction and time from the first steady state to the second steady state.

  DESCRIPTION OF EMBODIMENTS Embodiments (embodiments) for carrying out the present invention will be described in detail with reference to the drawings. The present invention is not limited by the contents described in the following embodiments. The constituent elements described below include those that can be easily assumed by those skilled in the art and those that are substantially the same. Furthermore, the constituent elements described below can be appropriately combined.

  FIG. 1 is a diagram schematically illustrating a configuration of a manipulation system including a pressure control device according to an embodiment. The manipulation system 10 is a system for operating a sample that is a minute object under a microscope. In FIG. 1, the manipulation system 10 includes a pressure control device 60, a first manipulator 14, a second manipulator 16, and a controller 43 that controls the manipulation system 10. A first manipulator 14 and a second manipulator 16 are separately arranged on both sides of the microscope unit 12.

  The pressure control device 60 includes a pressure control unit 45, a suction pump 29, the microscope unit 12, and a calculation unit 46C (not shown in FIG. 1) included in the controller 43. The pressure control device 60 is a device for adjusting the internal pressure of the first pipette 25 attached to the first manipulator 14 and performing an operation of fixing the sample, which is a minute object, to the first pipette 25.

  The microscope unit 12 includes a camera 18 including an image sensor, a microscope 20, and a sample stage 22. The sample stage 22 can support the sample holding member 11 such as a petri dish, and the microscope 20 is disposed immediately above the sample holding member 11. In the microscope unit 12, the microscope 20 and the camera 18 are integrated, and includes a light source (not shown) that emits light toward the sample holding member 11. The camera 18 may be provided separately from the microscope 20. In FIG. 1, the microscope 20 and the camera 18 are arranged above the sample holding member 11, but may be arranged below the sample holding member 11.

  The sample holding member 11 stores a solution containing a sample. When the sample of the sample holding member 11 is irradiated with light and the light reflected by the sample of the sample holding member 11 enters the microscope 20, an optical image related to the sample is magnified by the microscope 20 and then captured by the camera 18. The sample can be observed based on the image captured by the camera 18.

  As shown in FIG. 1, the first manipulator 14 includes a first pipette holding member 24, an XY axis table 26, a Z axis table 28, a drive device 30 that drives the XY axis table 26, and Z And a driving device 32 for driving the axis table 28. The first manipulator 14 is a manipulator having a three-axis configuration of X axis-Y axis-Z axis. In the present embodiment, one direction in the horizontal plane is the X-axis direction, and a direction that intersects the X-axis direction in the horizontal plane is a direction that intersects the Y-axis direction, the X-axis direction, and the Y-axis direction (that is, the vertical direction). Is the Z-axis direction.

  The XY axis table 26 can be moved in the X axis direction or the Y axis direction by driving of the driving device 30. The Z-axis table 28 is arranged on the XY axis table 26 so as to be movable up and down, and is movable in the Z-axis direction by driving of the drive device 32. The driving devices 30 and 32 are connected to the controller 43.

  The first pipette holding member 24 is connected to the Z-axis table 28, and a first pipette 25, which is a capillary tip, is attached to the tip. The first pipette holding member 24 moves as a moving area in the three-dimensional space according to the movement of the XY axis table 26 and the Z axis table 28, and the sample accommodated in the sample holding member 11 is moved to the end of the first pipette 25. Can be fixed to. The internal pressure of the first pipette 25 is controlled by the pressure controller 45 and the suction pump 29, and the sample is fixed by the suction of the first pipette 25. That is, the first manipulator 14 is a fixing manipulator used for fixing a minute object, and the first pipette 25 is a holding pipette used as a fixing means for the minute object.

  The second manipulator 16 shown in FIG. 1 includes a second pipette holding member 34, an XY axis table 36, a Z axis table 38, a driving device 40 that drives the XY axis table 36, and a Z axis table 38. And a driving device 42 for driving the motor. The second manipulator 16 is a manipulator having a three-axis configuration of X axis-Y axis-Z axis.

  The XY axis table 36 is movable in the X axis direction or the Y axis direction by driving of the driving device 40. The Z-axis table 38 is arranged on the XY axis table 36 so as to be movable up and down, and can be moved in the Z-axis direction by driving of the driving device 42. The driving devices 40 and 42 are connected to the controller 43.

  The second pipette holding member 34 is connected to a Z-axis table 38, and a second pipette 35 made of glass is attached to the tip. The second pipette holding member 34 can move as a moving area in the three-dimensional space according to the movement of the XY axis table 36 and the Z axis table 38, and can artificially manipulate the sample stored in the sample holding member 11. . That is, the second manipulator 16 is an operation manipulator used for manipulation of a minute object (DNA solution injection operation, perforation operation, etc.), and the second pipette 35 is an injection used as a minute object injection operation means. Pipette.

  The XY axis table 36 and the Z axis table 38 are configured as a coarse movement mechanism (three-dimensional movement table) that coarsely drives the second pipette holding member 34 to an operation position of a sample or the like accommodated in the sample holding member 11. Has been. Further, a fine movement mechanism 44 is provided as a nanopositioner at a connecting portion between the Z-axis table 38 and the second pipette holding member 34. The fine movement mechanism 44 is configured to support the second pipette holding member 34 so as to be movable in the longitudinal direction (axial direction) and to finely drive the second pipette holding member 34 along the longitudinal direction (axial direction). Is done.

  FIG. 2 is a cross-sectional view showing an example of a fine movement mechanism. As shown in FIG. 2, the fine movement mechanism 44 includes a piezoelectric actuator 44 a that drives the second pipette holding member 34. The piezoelectric actuator 44 a includes a cylindrical housing 87, rolling bearings 80 and 82 provided inside the housing 87, and a piezoelectric element 92. The second pipette holding member 34 is inserted in the axial direction of the housing 87. The rolling bearings 80 and 82 rotatably support the second pipette holding member 34. The piezoelectric element 92 expands and contracts along the longitudinal direction of the second pipette holding member 34 according to the applied voltage. A second pipette 35 (see FIG. 1) is attached and fixed to the distal end side (left side in FIG. 2) of the second pipette holding member 34.

  The second pipette holding member 34 is supported by the housing 87 via the rolling bearings 80 and 82. The rolling bearing 80 includes an inner ring 80a, an outer ring 80b, and a ball 80c provided between the inner ring 80a and the outer ring 80b. The rolling bearing 82 includes an inner ring 82a, an outer ring 82b, and a ball 82c provided between the inner ring 82a and the outer ring 82b. The outer rings 80 b and 82 b are fixed to the inner peripheral surface of the housing 87, and the inner rings 80 a and 82 a are fixed to the outer peripheral surface of the second pipette holding member 34 via the hollow member 84. Thus, the rolling bearings 80 and 82 are configured to rotatably support the second pipette holding member 34.

  A flange portion 84 a that protrudes radially outward is provided at a substantially central portion in the axial direction of the hollow member 84. The rolling bearing 80 is disposed on the distal end side in the axial direction of the second pipette holding member 34 with respect to the flange portion 84a, and the rolling bearing 82 is disposed on the rear end side with respect to the flange portion 84a. An inner ring 80a of the rolling bearing 80 and an inner ring 82a of the rolling bearing 82 are disposed across a flange portion 84a serving as an inner ring spacer. The outer peripheral surface of the second pipette holding member 34 is threaded, and lock nuts 86 and 86 are screwed into the second pipette holding member 34 from the front end side of the inner ring 80a and the rear end side of the inner ring 82a. The axial positions of the rolling bearings 80 and 82 are fixed.

  The annular spacer 90 is disposed coaxially with the rolling bearings 80 and 82 on the axial rear end side of the outer ring 82b. An annular piezoelectric element 92 is disposed substantially coaxially with the spacer 90 on the rear end side in the axial direction of the spacer 90, and a lid 88 of the housing 87 is disposed on the rear end side in the axial direction. The lid 88 is for fixing the piezoelectric element 92 in the axial direction, and has a hole through which the second pipette holding member 34 is inserted. The lid 88 may be fastened to the side surface of the housing 87 with a bolt (not shown), for example. The piezoelectric elements 92 may be arranged in a rod shape or prismatic shape so as to be substantially equidistant in the circumferential direction of the spacer 90, or may be a square tube having a hole portion through which the second pipette holding member 34 is inserted.

  The piezoelectric element 92 is in contact with the rolling bearing 82 via the spacer 90. The piezoelectric element 92 is connected to a controller 43 as a control circuit via a lead wire (not shown). The piezoelectric element 92 expands and contracts along the axial direction of the second pipette holding member 34 in response to an applied voltage from the controller 43, and finely moves the second pipette holding member 34 along the axial direction. . When the second pipette holding member 34 finely moves along the axial direction, this fine movement is transmitted to the second pipette 35 (see FIG. 1), and the position of the second pipette 35 is finely adjusted. Further, when the second pipette holding member 34 vibrates in the axial direction by the piezoelectric element 92, the second pipette 35 also vibrates in the axial direction. In this way, the fine movement mechanism 44 enables more accurate operations when manipulating a minute object (such as a DNA solution or cell injection operation or a perforation operation) and improves the perforation action by the piezoelectric element 92. it can.

  The fine movement mechanism 44 described above is provided in the second manipulator 16 for manipulating the minute object, but may be provided in the first manipulator 14 for fixing the minute object as shown in FIG. It can be omitted.

  Next, control of the manipulation system 10 by the controller 43 will be described with reference to FIG. FIG. 3 is a control block diagram of the manipulation system.

  The controller 43 includes hardware resources such as a CPU (Central Processing Unit) as a calculation unit, a hard disk as a storage unit, a RAM (Random Access Memory), and a ROM (Read Only Memory). The controller 43 performs various calculations based on a predetermined program stored in the storage unit 46B, and outputs a drive signal so that the control unit 46A performs various controls according to the calculation results.

The control unit 46A controls the focusing mechanism 81 of the microscope unit 12, the drive device 30 of the first manipulator 14, the drive device 32, the drive device 40 of the second manipulator 16, the drive device 42, the injection pump 39, and the pressure control unit 45. To do. The controller 46A outputs a drive signal to each via a driver, an amplifier, or the like provided as necessary. The controller 46A supplies a drive signal V _xy and a drive signal V _z (see FIG. 1) to the drive devices 30, 32, 40, and 42, respectively. Drive 30,32,40,42, the drive signal _{V xy,} drives the X-Y-Z-axis direction based on the driving signal _{V z.} The controller 46A may control the fine movement mechanism 44 by supplying the nanopositioner control signal V _N (see FIG. 1) to the fine movement mechanism 44.

  The controller 43 is connected with a joystick 47 and an input unit 49 such as a keyboard or a touch panel as information input means. The controller 43 is connected to a display unit 48 such as a liquid crystal panel. The display unit 48 displays a microscope image acquired by the camera 18 and various control screens. In the case where a touch panel is used as the input unit 49, the touch panel may be overlapped on the display screen of the display unit 48 so that the operator can perform an input operation while confirming the display image on the display unit 48.

  A known joystick 47 can be used. The joystick 47 includes a base and a handle portion that stands upright from the base, and the drive devices 30 and 40 can be driven in an XY manner by operating the handle portion to incline. The drive devices 32 and 42 can be Z-driven by twisting. The joystick 47 may include a button 47A (see FIG. 1) for operating each drive of the suction pump 29 and the infusion pump 39.

As shown in FIG. 3, the controller 43 further includes an image input unit 43A, an image processing unit 43B, an image output unit 43C, and a calculation unit 46C. An image signal V _PIX (see FIG. 1) captured by the camera 18 through the microscope 20 is input to the image input unit 43A. The image processing unit 43B receives the image signal from the image input unit 43A and performs image processing. The image output unit 43C outputs the image information processed by the image processing unit 43B to the display unit 48. The calculation unit 46C receives image information from the image processing unit 43B. The calculation unit 46C determines the position of a cell or the like that is a micro target imaged by the camera 18 based on the image information after the image processing, the nucleus of a cell that is an operation target for performing an injection operation with the second pipette 35, and the like. The position can be detected. For example, the calculation unit 46C can detect the presence or absence of cells or the like in the imaging region of the camera 18 based on the image information. The calculation unit 46C may detect the positions of the first pipette 25 and the second pipette 35. The image input unit 43A, the image processing unit 43B, the image output unit 43C, and the calculation unit 46C are controlled by the control unit 46A.

  For example, the image processing unit 43B performs binarization processing and filter processing on the image signal received from the image input unit 43A in order to detect the position of the cell to be fixed and the position of the cell nucleus. The image processing unit 43B converts the grayscale image into a monochrome image based on a predetermined threshold value that is converted into a grayscale image signal. Then, the image processing unit 43B performs edge extraction processing and pattern matching based on the monochrome image obtained by the binarization processing and the filter processing. Based on the processing result, the calculation unit 46C can detect the position of the cell and the position of the nucleus of the cell. As will be described later, the calculation unit 46C can detect the position of the center of gravity of the cell and calculate the moving speed of the cell from the change in the position of the center of gravity of the cell.

  The control unit 46A controls the first manipulator 14, the second manipulator 16, and the pressure control unit 45 based on the position information of the cells and the like from the calculation unit 46C and the presence / absence information of the cells and the like. In the present embodiment, the control unit 46A automatically drives the first manipulator 14 and the second manipulator 16 in a predetermined sequence. Such sequence driving is performed by the control unit 46A sequentially outputting a drive signal to each based on the calculation result of the CPU by a predetermined program stored in advance in the storage unit 46B. In the present embodiment, the pressure control unit 45 performs the cell fixing operation by controlling the suction amount of the suction pump 29 based on the cell position information received from the calculation unit 46C. Further, the pressure control unit 45 can determine whether or not the cells are fixed to the first pipette 25 based on the cell position information received from the calculation unit 46C.

  Next, a cell fixing method of the manipulation system 10 will be described. FIG. 4 is a flowchart showing an example of the pressure control operation of the manipulation system. The manipulation system 10 of the present embodiment performs a fixing operation for each cell on a plurality of cells placed on the sample holding member 11, and performs an injection operation on the fixed cells. Further, the fixing operation and the injection operation are repeatedly executed for a plurality of cells. The controller 43 may automatically perform operations on a plurality of cells.

  First, the calculation unit 46C calculates the position of the center of gravity of the cell 100 to be fixed (FIG. 4, step ST11). In the present embodiment, the cell 100 that is a micro object is a fertilized egg. The cell 100 is not limited to a fertilized egg, and is a target on which an operation such as a DNA solution injection operation or a perforation operation is performed under a microscope, and may be, for example, an egg cell or other cells.

  FIG. 5 is a plan view schematically showing the position of the cell in the imaging region of the camera. FIG. 5 shows the position of the lower right corner as (x, y) with the upper left corner of the imaging region 18A of the camera 18 as the origin (0, 0). The center position C of the imaging region 18A is (x / 2, y / 2). Here, one direction in the imaging region 18A is defined as an Xc axis direction, and a direction intersecting the Xc axis direction is defined as a Yc axis direction. The length of the imaging region 18A in the Xc-axis direction is, for example, 750 μm, and the length of the imaging region 18A in the Yc-axis direction is, for example, 500 μm.

  The control unit 46A drives the sample stage 22 so that the cell 100 is positioned in the vicinity of the center position C of the imaging region 18A. The center of gravity 100G of the cell 100 is preferably located at the center position C, but may be located, for example, in a region 18B indicated by hatching in FIG. The region 18B has a range of 3 / x or more and 2 / x or less in the Xc-axis direction, and a range of 3 / y or more and 2 / y or less in the Yc-axis direction. As described above, a plurality of cells are placed on the sample holding member 11, and as shown in FIG. 5, cells 101 other than the cells 100 to be fixed are present in the imaging region 18A. May be.

  The camera 18 images the cell 100 located in the vicinity of the center position C. FIG. 6 is a schematic diagram of cells detected by image processing. The image processing unit 43B performs binarization processing and filter processing on the image of the cell 100. As a result, the edges of the zona pellucida 100A and the cell membrane 100B of the cell 100 are extracted. When performing phase difference observation using light diffraction and interference, it is possible to extract edges by observing light due to a so-called halo effect that appears at the edge of the transparent zone 100A and the edge of the cell membrane 100B.

  The calculation unit 46C fills the region inside the edge of the zona pellucida 100A and the region inside the edge of the cell membrane 100B with respect to the image of the cell 100 received from the image processing unit 43B. Recognize as Then, the calculation unit 46C calculates the position of the center of gravity 100G of the cell 100 by a known method such as a differentiation method. The calculation unit 46C calculates the position of the center of gravity 100G of the cell 100 every predetermined time, and stores it in the storage unit 46B. The step time for calculating the position of the center of gravity 100G is preset information, and is stored in the storage unit 46B via the input unit 49. This step time can be set to 500 ms, for example. The calculation unit 46C can calculate the moving speed of the cell 100 from information on the position of the center of gravity 100G for each step time. The pressure control unit 45 receives the position information and movement speed information of the cell 100 from the calculation unit 46C, and determines whether the cell 100 is moving.

  At this time, the control unit 46 </ b> A outputs a drive signal to the drive devices 30 and 32 to move the tip of the first pipette 25 to the vicinity of the cell 100. FIG. 7 is a cross-sectional view showing a schematic configuration of the first pipette, the medium and the cells held by the sample holding member. As shown in FIG. 7, the sample holding member 11 is a dish-like member that holds the cells 100 and 101. The sample holding member 11 is made of a transparent member so that the cells 100 and 101 can be imaged. The sample holding member 11 holds a medium 52, and cells 100 and 101 are dispersed inside the medium 52. Further, paraffin oil 54 is filled on the culture medium 52. Thereby, evaporation of the culture medium 52 is suppressed while ensuring the fluidity of the culture medium 52. In FIG. 7, two cells 100 and 101 are shown, but three or more cells may be held.

  The control unit 46A fixes the tip of the first pipette 25 in the vicinity of the cell 100. For example, the tip of the first pipette 25 is separated from the central position C of the imaging region 18A shown in FIG. 5 by −250 μm or more and −0 μm or less, for example, −100 μm in the Xc axis direction, for example, +0 μm or more in the Yc axis direction, It is about +250 μm or less, for example, +100 μm apart. Further, as shown in FIG. 7, the tip of the first pipette 25 is in contact with the bottom 11 a of the sample holding member 11 and is located inside the culture medium 52.

  An oil 55 is filled in the internal space of the first pipette 25. The oil 55 is preferably a chemically inert liquid. For example, a fluorine-based inert liquid containing a fluorine-based compound is used. In a state where the first pipette 25 is fixed in the vicinity of the cell 100, the oil 55 contacts the medium 52 through the opening 25 a of the first pipette 25. That is, an air layer is not interposed between the oil 55 and the culture medium 52. Since the internal space of the first pipette 25 is filled with the oil 55 in this way, when a suction pressure (negative pressure) is applied by the suction pump 29, the minute object is easily held in the opening 25a of the first pipette 25. It becomes possible to do.

  Next, the pressure control unit 45 outputs a drive signal to the suction pump 29 and starts the suction of the first pipette 25 (FIG. 4, step ST12). When the suction is started, the tip of the first pipette 25 is fixed at a position in the vicinity of the cell 100 and away from the cell 100. In the present embodiment, since it is not necessary to bring the tip of the first pipette 25 into contact with the cell 100, the tip of the first pipette 25 falls within a predetermined range near the cell 100 without strictly controlling the position. It only has to be. Thereby, the movement operation of the 1st pipette 25 can be performed easily, and the damage of the cell 100 by the contact of the 1st pipette 25 can be suppressed.

  FIG. 8 is a graph schematically showing the relationship between the moving speed of cells and time. FIG. 9 is a graph schematically showing the relationship between the pump driving speed and time. FIG. 10 is a plan view schematically showing the relationship between the first pipette and the cells in the first steady state. FIG. 11 is a graph schematically showing the relationship between the cell position in the Xc axis direction and time in the first steady state. FIG. 12 is a graph schematically showing the relationship between the position of the cell in the Yc-axis direction and time in the first steady state.

  The pump driving speed shown in FIG. 9 is that when the suction pressure (negative pressure) is applied by the suction pump 29, the interface 55a (see FIG. 10) of the oil 55 in the internal space of the first pipette 25 is the first pipette 25. It shows the speed of movement along the axial direction. That is, when the suction pressure of the suction pump 29 is large, the driving speed Vp is large, and when the suction pressure of the suction pump 29 is small, the driving speed Vp is small.

  As shown in FIG. 9, the pressure controller 45 starts the suction of the first pipette 25 with the suction pump 29 at the driving speed Vp0. The driving speed Vp0 at the start of suction is about 50 μm / s to 200 μm / s, for example, 50 μm / s. In this example, the pressure controller 45 drives the suction pump 29 so that the interface 55a of the oil 55 moves 50 μm per second. The driving speed Vp0 is obtained in advance because it varies depending on the position of the center of gravity 100G of the cell 100 and the position of the tip of the first pipette 25. The set numerical value of the drive speed Vp0 is stored in the storage unit 46B via the input unit 49 before the suction pump 29 is driven. The driving speed Vp0 at the start of the suction of the suction pump 29 is set to a value such that the cells 100 do not reach an excessive speed due to the suction of the suction pump 29 even when the cells 100 are arranged in the vicinity of the first pipette 25. It is preferable to set.

After starting the suction, the calculating unit 46C calculates the position of the center of gravity 100G of the cell 100 for each step time described above, and calculates the moving speed Vc of the cell 100 (FIG. 4, step ST13). For example, the calculation unit 46C calculates the difference between the positions of the center of gravity 100G at time t and time t-1. The position of the center of gravity 100G at time t is (X _t , Y _t ), and the position of the center of gravity 100G at time t-1 is (X _t−1 , Y _t−1 ). The difference between the positions of the center of gravity 100G at time t and time t-1, that is, the moving distance D of the center of gravity 100G is expressed by Equation (1). The moving speed Vc of the cell is calculated by dividing the moving distance D by the step time.

D = ((X _t −X _t−1 ) ² + (Y _t −Y _t−1 ) ² ) ^1/2 (1)

The pressure control unit 45 receives information on the moving speed Vc of the cell 100 from the calculation unit 46C, and determines whether or not the cell 100 is in the first steady state (FIG. 4, step ST14). Here, the “first steady state” indicates a state where the cell 100 has not moved after the start of suction, that is, a state where the moving speed Vc is equal to or lower than a predetermined speed Vc1. As shown in FIGS. 8 to 10, in the first steady state SS1, the cell 100 is spaced from the first pipette 25 and is in a fixed position. The position of the center of gravity 100G of the cell 100 at the start of suction is defined as (x ₁ , y ₁ ). As shown in FIG. 11, from the time of suction start to time _{t 1,} the Xc axis direction (see FIG. 5), the position of the center of gravity 100G cell 100 is constant at _{x 1.} As shown in FIG. 12, the position of the center of gravity 100G of the cell 100 in the Yc axis direction (see FIG. 5) is constant at y ₁ from the start of suction to time t ₁ .

  When the moving speed Vc of the cell 100 is smaller than the predetermined speed Vc1 after the suction starts, the pressure control unit 45 determines that the cell 100 is in the first steady state SS1 (FIG. 4, step ST14, Yes). The predetermined speed Vc1 is about 20 μm / s or more and 100 μm / s or less, for example, 20 μm / s. When the cell 100 is in the first steady state SS1, the pressure control unit 45 determines whether a predetermined time has elapsed after the start of suction (FIG. 4, step ST15-1). When the predetermined time has elapsed (FIG. 4, step ST15-1, Yes), the cell 100 may not be the operation target cell, so the pressure control unit 45 stops the fixing operation on the cell 100. The controller 46A drives the sample stage 22 to execute processing for another cell 101 (FIG. 4, step ST20). Here, the predetermined time after the start of suction is about 1 second to about 5 seconds, for example, 3 seconds.

When the predetermined time has not elapsed after the start of suction (FIG. 4, step ST15-1, No), the pressure control unit 45 changes the drive speed of the suction pump 29 (FIG. 4, step ST15-2). As shown in FIGS. 8 and 9, at time t _1, when the moving speed Vc of the cell 100 is smaller than the predetermined speed Vc1, the pressure control unit 45, the driving speed Vp of the suction pump 29, suction at the start of The driving speed is set to be greater than Vp0. The suction pressure of the suction pump 29, the cell 100 begins to move toward the opening 25a of the first pipette 25, as shown in FIG. 8, after time _{t 1,} the moving velocity Vc of the cell 100 is increased. Then, the moving velocity Vc of the cell 100 becomes the predetermined speed Vc1 higher at time _{t 11.}

If the moving speed Vc of the cell 100 is equal to or higher than the predetermined speed Vc1 after starting the suction, the pressure control unit 45 determines that the cell 100 has started moving instead of the first steady state SS1 (FIG. 4, step ST14, No). 8, cells 100, after the start of suction, in the period up to time _{t 11,} a first steady state SS1, time _{t 11} and later, in the period up to time _{t 2, the} a mobile state MS.

  As shown in FIG. 7, suction by the first pipette 25 is performed in a state where the tip of the first pipette 25 is in contact with the bottom 11 a of the sample holding member 11. Since the opening 25a of the first pipette 25 faces the bottom 11a of the sample holding member 11, the suction pressure by the first pipette 25 acts in the direction along the bottom 11a. Therefore, since the suction pressure by the first pipette 25 acts on the cell 100 reliably, the cell 100 can be moved. Moreover, since the movement of the cell 100 is suppressed and moves along the bottom part 11a, the cell 100 can be reliably fixed to the first pipette 25 while the damage of the cell 100 is suppressed. it can.

  In the movement state MS (see FIG. 8) of the cell 100, the calculation unit 46C calculates the movement speed Vc of the cell 100 described above (FIG. 14, step ST16). The pressure control unit 45 determines whether or not the cell 100 is in the second steady state based on the information on the moving speed Vc of the cell 100 received from the calculation unit 46C (FIG. 14, step ST17). Here, the “second steady state” means a state in which the movement speed Vc of the cell 100 becomes smaller than a predetermined speed Vc2 after the movement of the cell 100 is started, that is, the cell 100 is opened in the first pipette 25. The state fixed to 25a is shown. The predetermined speed Vc2 is about 5 μm / s or more and 20 μm / s or less, for example, 10 μm / s. When the moving speed Vc of the cell 100 is smaller than the predetermined speed Vc2, the pressure control unit 45 determines that the cell 100 is in the second steady state SS2, and the moving speed Vc of the cell 100 is equal to or higher than the predetermined speed Vc2. In this case, it is determined that the cell 100 is in the moving state MS.

  FIG. 13 is a plan view schematically showing the relationship between the first pipette and the cells in the second steady state. FIG. 14 is a graph schematically showing the relationship between the position of the cell in the Xc-axis direction and time from the first steady state to the second steady state. FIG. 15 is a graph schematically showing the relationship between the position of the cell in the Yc axis direction and time from the first steady state to the second steady state.

As shown in FIG. 13, in the second steady state SS2, the cell 100 is fixed to the opening 25a of the first pipette 25. The position of the center of gravity 100G of the cell 100 at the start of suction is (x ₁ , y ₁ ), and the position of the center of gravity 100G of the cell 100 in the second steady state SS2 is (x ₂ , y ₂ ). As shown in FIG. 14, the position of the center of gravity 100G cells 100 in Xc axis direction (see FIG. 5) is moved from _{x 1} to _{x 2} in the period from time _{t 1} to time _{t 2,} the time _{t 2} later x ₂ certain to become. As shown in FIG. 15, the position of the center of gravity 100G cells 100 in Yc axis direction (see FIG. 5) is moved from _{y 1} to _{y 2} in the period from time _{t 1} to time _{t 2,} the time _{t 2} later y ₂ certain to become. In this example, time _{t 2} later, the moving speed of the cell 100 is smaller than the predetermined speed Vc2, the second steady state SS2.

  Next, the pressure control operation of the pressure control unit 45 in the movement state MS and the second steady state SS2 will be described in detail with reference to FIG. 4, FIG. 8, and FIG. When the moving speed Vc of the cell 100 is equal to or higher than the predetermined speed Vc2, the pressure control unit 45 determines that the cell 100 is not in the second steady state SS2 (FIG. 14, step ST17, No). And the pressure control part 45 changes the drive speed of the suction pump 29 based on the information of the moving speed Vc of the cell 100 received from the calculation part 46C (FIG. 14, step ST18).

  In the pressure control device 60 of the present embodiment, the calculation unit 46C calculates the moving speed of the cell 100 from the change in the position of the cell 100, and the pressure control unit 45 performs the first based on the information on the moving speed of the cell 100. The internal pressure of the pipette 25 is adjusted. The moving speed Vc of the cell 100 and the driving speed of the suction pump 29 are in an inversely proportional relationship. If the moving speed Vc of the cell 100 is large, the pressure control unit 45 decreases the driving speed Vp of the suction pump 29 in order to decrease the moving speed Vc. Moreover, if the moving speed Vc of the cell 100 is small, the pressure control unit 45 increases the driving speed of the suction pump 29 in order to increase the moving speed Vc.

  According to this, since the cell 100 can be moved at an appropriate speed, the fixing operation of the cell 100 can be reliably performed. Further, damage when the cell 100 comes into contact with the first pipette 25 can be suppressed.

Specifically, as shown in FIGS. 8 and 9, at time t ₁₁ , in order to increase the moving speed Vc of the cell 100, the pressure control unit 45 increases the driving speed Vp of the suction pump 29 to the upper limit speed Vpmax. Enlarge. Thus, from the time _{t 11} to time _{t 14,} the moving velocity Vc of the cell 100 is increased. Here, the upper limit speed Vpmax of the suction pump 29 is a value that can suppress the destruction of the cells 100 even when the cells 100 are sucked at the maximum driving speed Vp in a state where the cells 100 are fixed to the tip of the first pipette 25. Set to The upper limit speed Vpmax of the suction pump 29 is a preset value and is stored in the storage unit 46B via the input unit 49.

As shown in FIGS. 8 and 9, the pressure control unit 45 at time _{t 14,} in order to reduce the moving velocity Vc of the cells 100, to change the driving speed Vp to a value smaller than the upper limit speed Vpmax. The moving velocity Vc of the cell 100 slightly increases from time _{t 14} by inertia until time _{t 16.} The pressure control unit 45, the period from time _{t 14} to time _{t 2, the} to decrease the moving velocity Vc of the cells 100, to reduce the driving speed Vp of the suction pump 29 in a stepwise manner. The moving velocity Vc is time _{t 16} after the cells 100, until time _{t 18,} corresponding to the decrease of the driving speed Vp, smaller. Thus, the pressure control unit 45 can change the internal pressure of the first pipette 25 by changing the driving speed Vp of the suction pump 29 according to the moving speed Vc of the cell 100. Thereby, since the cell 100 can be moved at an appropriate speed, the cell 100 can be reliably fixed. Further, damage when the cell 100 comes into contact with the first pipette 25 can be suppressed.

In the period from time _{t 18} to time _{t 19,} since the cell 100 approaches the first pipette 25, susceptible to suction pressure of the first pipette 25, the moving velocity Vc of the cell 100 is increased. At time _{t 19,} the cell 100 is in contact with the tip of the first pipette 25, the moving velocity Vc decreases. Cells 100 at time _{t 2} becomes smaller than the predetermined speed Vc2, the second steady state SS2. That is, the cell 100 is fixed to the opening 25 a of the first pipette 25. The pressure control unit 45, the time _{t 19} after, the moving velocity Vc of the cell 100 is reduced, in order to increase the moving velocity Vc, increases the driving speed Vp at a time _{t 2} and time _{t 21.} As described above, damage to the cell 100, even when increasing the driving speed Vp at a time t ₂ and time t ₂₁ is not to occur, the upper limit speed Vpmax driving speed Vp is set.

  In the pressure control device 60 of the present embodiment, the pressure control unit 45 adjusts the internal pressure of the first pipette 25 so that the moving speed of the cell 100 is within a predetermined range. Specifically, in the movement state MS of the cell 100, the pressure control unit 45 may adjust the driving speed Vp using a control technique such as PID (Proportional-Integral-Differential) control. By so doing, the cell 100 moves while maintaining a constant movement speed Vc, so that the cell 100 can be prevented from colliding with the first pipette 25 at an excessive speed, and damage to the cell 100 can be suppressed.

  When the moving speed Vc of the cell 100 becomes smaller than the predetermined speed Vc2, the pressure control unit 45 determines that the cell 100 is in the second steady state SS2 (FIG. 14, step ST17, Yes). Then, it is determined that the fixing of the cell 100 to the opening 25a of the first pipette 25 is completed (FIG. 14, step ST19). Thus, the pressure control unit 45 can determine whether the cell 100 is fixed based on the image recognition of the cell 100 from the moving speed of the center of gravity 100G of the cell 100. Accordingly, since it is not necessary to perform image recognition of the first pipette 25, the calculation process of the calculation unit 46C can be simplified. Further, since it is not necessary to strictly control the position of the first pipette 25, the fixing operation can be easily performed.

Then, the pressure control unit 45, after the fixation of the cell 100 is completed, at time _{t 3} when 9, to 0 the driving speed Vp of the suction pump 29. That is, the position of the interface 55a (see FIG. 13) of the oil 55 filled in the internal space of the first pipette 25 is fixed, and the internal pressure of the first pipette 25 is maintained constant. Thus, since the suction pump 29 is driven so that the internal pressure of the first pipette 25 becomes constant, the fixed state of the cells 100 is maintained.

  The cell 100 fixed to the first pipette 25 is subjected to operations such as DNA solution injection operation and perforation operation by the second pipette 35 (see FIG. 1). After the operation on the cell 100 is completed, the pressure control device 60 can repeatedly perform the fixing operation from step ST11 to step ST19 in FIG. 4 on the other cells held on the sample holding member 11.

  As described above, the pressure control device 60 of the present embodiment is a pressure control device 60 to which the first pipette 25 (fixing means) for fixing the cell 100 (micro object) by internal pressure is attached. The pressure control unit 45 that adjusts the internal pressure of the first pipette 25, the camera 18 (imaging unit) that images the cell 100, and the calculation unit 46C that calculates the position of the cell 100 based on the image obtained from the camera 18 The pressure control unit 45 starts aspiration by the first pipette 25 in a state where the cell 100 is separated from the first pipette 25, and uses the information on the change in the position of the cell 100 received from the calculation unit 46C. Based on this, it is determined that the cell 100 is fixed to the first pipette 25.

  In the pressure control method of the present embodiment, the pressure control unit 45 that adjusts the internal pressure of the first pipette 25 starts the suction by the first pipette 25 in a state where the cells 100 are separated from the first pipette 25 (FIG. 14 and step ST12), and the calculation unit 46C calculates the moving speed (change in position) of the cell 100 based on the image obtained from the fixing means and the imaging unit that images the cell 100 (FIG. 14, step ST13). ) And the step of determining that the cell 100 is fixed to the first pipette 25 based on the information on the change in the position of the cell 100 received from the calculation unit 46C (FIG. 14, step ST17, Yes).

  According to this, since the pressure control unit 45 can start suction by the first pipette 25 in a state where the cell 100 is separated from the first pipette 25, the first pipette 25 is in contact with the cell 100. As compared with the case where the suction is started, the moving operation of the first pipette 25 can be easily performed, and the damage of the cell 100 can be suppressed. Further, since it can be determined that the cell 100 is fixed to the first pipette 25 by image recognition of the cell 100, it is not necessary to perform image recognition of the first pipette 25, and the fixing operation can be easily performed. it can. Therefore, the cell 100 can be fixed efficiently and suitably regardless of the skill level and skill of the operator.

  In the pressure control device 60 of the present embodiment, the pressure control unit 45 starts aspiration in a state where the first pipette 25 is in contact with the bottom 11a of the sample holding member 11 on which the cell 100 is placed. According to this, since the suction force of the first pipette 25 acts on the cell 100 reliably, the cell 100 can be moved and fixed to the first pipette 25. Moreover, since the cell 100 moves along the bottom part 11a, the vertical movement is suppressed. For this reason, the collision between the cell 100 and the first pipette 25 or the like can be suppressed, and damage to the cell 100 can be suppressed.

  In the pressure control device 60 of the present embodiment, the pressure control unit 45 fixes the cell 100 to the first pipette 25 when the moving speed of the cell 100 becomes a predetermined speed or less after the cell 100 starts moving. The internal pressure of the first pipette 25 is maintained. According to this, it can be determined whether or not the cell 100 is fixed from the moving speed of the cell 100 calculated based on the image recognition. Therefore, it is not necessary to perform image recognition of the first pipette 25, and the calculation operation of the calculation unit 46C can be simplified and the fixing operation can be easily performed.

  In the pressure control device 60 of the present embodiment, the pressure control unit 45 stops the suction when the position of the cell 100 does not change for a predetermined period after the suction by the first pipette 25 is started. According to this, since there is a possibility that the image-recognized target object is not the operation target cell 100, it is possible to perform operations on other operation target cells.

  In the pressure control device 60 of the present embodiment, the calculation unit 46C calculates the position of the center of gravity 100G of the cell 100 based on the image captured by the camera 18. According to this, the position and moving speed of the cell 100 can be accurately calculated by calculating the center of gravity 100G of the cell 100.

  The manipulation system 10 of the present embodiment includes any one of the pressure control devices 60 described above, the sample stage 22 on which the cell 100 is placed, and the first manipulator 14 including the first pipette 25 for fixing the cell 100. The second manipulator 16 including the second pipette 35 (operation means) for operating the cell 100 held by the first pipette 25, the sample stage 22, the first pipette 25, the second pipette 35, and the camera 18 are controlled. And a control unit 46A. According to this, the first manipulator 14 including the first pipette 25 and the pressure control device 60 perform the fixing operation of the cell 100 while suppressing the damage of the cell 100, and the first operation for the fixed cell 100 is performed. It can be executed by the two manipulators 16.

  The pressure control device 60, the manipulation system 10, and the pressure control method of the present embodiment may be changed as appropriate. For example, it is preferable to appropriately change the shapes and the like of the first pipette 25, the second pipette 35, and the like according to the type of the minute object and the operation on the minute object. The cell moving speed Vc shown in FIG. 8 and the driving speed Vp of the suction pump 29 shown in FIG. 9 are merely examples, and can be changed as appropriate. The calculation unit 46C calculates the position of the center of gravity 100G of the cell 100 by image processing of the cell 100, but is not limited thereto. For example, the center position of the cell 100 may be calculated. In the fixing operation of the cell 100, a part of the procedure may be omitted as appropriate, or the procedure may be replaced and executed.

DESCRIPTION OF SYMBOLS 10 Manipulation system 11 Sample holding member 12 Microscope unit 14 1st manipulator 16 2nd manipulator 18 Camera 20 Microscope 22 Sample stage 24 1st pipette holding member 25 1st pipette 26 XY axis table 28 Z axis table 30, 32 Drive device 34 Second pipette holding member 35 Second pipette 36 XY axis table 38 Z axis table 40, 42 Drive device 43 Controller 44 Fine movement mechanism 45 Pressure control unit 46A Control unit 46B Storage unit 46C Calculation unit 60 Pressure control device 80, 82 Rolling bearing 100, 101 cell 100A zona pellucida 100B cell membrane 100G center of gravity

Claims (9)

A pressure control device equipped with a fixing means for fixing a minute object by internal pressure,
A pressure controller for adjusting the internal pressure of the fixing means;
An imaging unit for imaging the minute object;
A calculation unit that calculates the position of the minute object based on an image obtained from the imaging unit;
The pressure control unit starts suction by the fixing unit in a state in which the micro object is separated from the fixing unit, and based on the information on the change in the position of the micro object received from the calculation unit, A pressure control device that determines that the minute object is fixed to a fixing means. The calculation unit calculates a moving speed of the minute object from a change in the position of the minute object,
The pressure control device according to claim 1, wherein the pressure control unit adjusts an internal pressure of the fixing unit based on information on a moving speed of the minute object.   The pressure control device according to claim 1, wherein the pressure control unit adjusts an internal pressure of the fixing unit so that a moving speed of the minute object is within a predetermined range.   4. The pressure control unit according to claim 1, wherein the pressure control unit starts the suction in a state where the fixing unit is in contact with a bottom portion of a sample holding member on which the minute object is placed. 5. Pressure control device.   The pressure control unit determines that the micro object is fixed to the fixing means when the moving speed of the micro object becomes a predetermined speed or less after the micro object starts moving, The pressure control device according to any one of claims 1 to 4, wherein an internal pressure of the fixing means is maintained.   The pressure control device according to claim 1, wherein the pressure control unit stops the suction when the position of the minute object does not change for a predetermined period after the suction by the fixing unit is started.   The pressure control device according to any one of claims 1 to 6, wherein the calculation unit calculates a position of a center of gravity of the minute object based on an image captured by the imaging unit. The pressure control device according to any one of claims 1 to 7,
A sample stage on which a minute object is placed;
A first manipulator comprising the fixing means for holding the micro object;
A second manipulator comprising operating means for operating the minute object held by the fixing means;
A manipulation system comprising the sample stage, the fixing means, the operating means, and a control unit for controlling the imaging unit. A pressure control method of a pressure control device equipped with a fixing means for fixing a minute object by internal pressure,
A pressure control unit that adjusts the internal pressure of the fixing unit starts suction by the fixing unit in a state in which the minute object is separated from the fixing unit;
A step of calculating a change in the position of the micro object based on an image obtained from an imaging unit that images the micro object;
The pressure control method includes a step of determining that the micro object is fixed to the fixing unit based on information on a change in the position of the micro object received from the calculation unit.