Organism manipulation method and organism manipulation device

Abstract

A method for manipulating microscopic organisms such as cells using a gas-liquid interface is provided. [Solution] There is provided a method for manipulating organisms, comprising: a bubble formation step of forming bubbles in a liquid in which the organisms are immersed; an organism attachment step of attaching the organisms to the bubbles; and an airflow control step of generating an air current in the bubbles and manipulating the position of the organisms with the air current. The bubble formation step may be performed by immersing an end of a flow path in the liquid and introducing gas into the liquid from the end, and the airflow control step may include generating an air current in the bubbles by moving the relative positions of the flow path and the liquid.

Description

[Technical Field]

[0001] The present invention relates to a method and an apparatus for manipulating an organism. [Background technology]

[0002] In cell biology research and the like, it is common to aspirate specific cells from among many cells in a culture vessel. Patent Document 1 discloses a system that assists in the aspirating of characteristic cells from among a large number of cells. [Patent Document 1] JP 2019-030263 A

[0003] [General Disclosure] In a first aspect of the present invention, there is provided a method for manipulating an organism. The method for manipulating an organism may include a bubble formation step of forming bubbles in a liquid in which the organism is immersed. The method for manipulating an organism may include an organism attachment step of attaching the organism to the bubbles. The method for manipulating an organism may include an air flow control step of generating an air flow in the bubbles and manipulating the position of the organism using the air flow.

[0004] In a second aspect of the present invention, the bubble formation step may be performed by immersing an end of a flow path in a liquid and introducing gas into the liquid from the end, and the air flow control step may include generating an air flow in the bubble by moving the relative positions of the flow path and the liquid.

[0005] In a third aspect of the present invention, the bubble forming step may include forming and maintaining a bubble at an end of the flow channel.

[0006] In a fourth aspect of the present invention, the airflow control step may include generating an airflow in the bubbles by moving the relative position between the flow path and the surface of the container storing the liquid that is in contact with the organism in a direction within a range of ±20° from the horizontal direction.

[0007] In a fifth aspect of the present invention, the airflow control step may include generating an airflow in the bubbles by moving the relative position between the flow path and the surface of the container storing the liquid with which the organism is in contact in a direction within a range of ±20° from the vertical direction.

[0008] In a sixth aspect of the present invention, the liquid and the organism may be contained in a container, and the airflow control step may include moving the relative position of the flow path and the liquid while the air bubble is in contact with a surface of the container that the organism is in contact with.

[0009] In a seventh aspect of the present invention, the airflow control step may include generating an airflow in the air bubble by changing the volume of the air bubble.

[0010] In an eighth aspect of the present invention, the airflow control step may include generating an airflow in the air bubble by increasing the volume of the air bubble.

[0011] In a ninth aspect of the present invention, the airflow control step may include generating an airflow in the air bubble by reducing the volume of the air bubble.

[0012] In a tenth aspect of the present invention, the bubble formation step may be performed by immersing an end of a flow path in liquid and introducing gas into the liquid from the end, and the flow path may include a gas supply flow path for supplying the gas and a gas recovery flow path for recovering the gas. The gas flow control step may include generating an air flow in which the gas supplied from the end of the gas supply flow path passes through the bubbles and flows toward the end of the gas recovery flow path.

[0013] In an eleventh aspect of the present invention, the flow path may have a double-pipe structure, the gas supply flow path may be one of the inner or outer flow paths in the double-pipe structure, and the gas recovery flow path may be the other inner or outer flow path in the double-pipe structure.

[0014] In a twelfth aspect of the present invention, the method may further comprise a recovery step of recovering the organisms from the liquid after the airflow control step.

[0015] A thirteenth aspect of the present invention provides an organism manipulation device for manipulating organisms. The organism manipulation device may include a flow path whose end is immersed in a liquid in which the organisms are immersed and which can introduce a gas into the liquid. The organism manipulation device may also include a bubble control unit that generates an airflow in bubbles formed by introducing the gas from the end into the liquid, and manipulates the position of the organisms using the airflow.

[0016] In the fourteenth aspect of the present invention, the bubble control unit may perform control so as to maintain the bubble formed at the end portion.

[0017] In a fifteenth aspect of the present invention, the bubble control unit may have a flow path position control unit that controls an actuator that moves through the flow path, thereby controlling the air flow in the bubble.

[0018] In a sixteenth aspect of the present invention, the bubble control unit may include a stage position control unit that controls an actuator that moves a stage that carries a container that houses the organism, thereby controlling the air flow within the bubble.

[0019] In a seventeenth aspect of the present invention, the bubble control unit may include a volume control unit that controls a pump connected to the flow path, thereby controlling the volume of the bubbles in the liquid.

[0020] An eighteenth aspect of the present invention provides an organism manipulation device. The organism manipulation device may include a flow path having an end located in a liquid containing an organism, which is stored in a container. The organism manipulation device may include a pump that introduces gas into the flow path to form bubbles. The organism manipulation device may include a position control unit that can change the position of the container or the flow path. The pump or position control unit may attach the organism to the gas-liquid interface of the bubbles. The position control unit may move the flow path in the opposite direction to the organism, relative to the central axis of the flow path, as viewed in the vertical direction.

[0021] The above summary of the invention does not list all of the necessary features of the present invention, and subcombinations of these features may also be inventions. [Brief explanation of the drawings]

[0022] [Figure 1A] An example of the device configuration of a living organism manipulation device 100 in this embodiment is shown. [Figure 1B] An example of the device configuration of a living organism manipulation device 100 in this embodiment is shown. [Figure 2A] 1 shows an example of a schematic diagram illustrating the structure of a nozzle 49 in this embodiment. [Figure 2B] 1 shows an example of a schematic diagram illustrating the structure of a nozzle 49 in this embodiment. [Figure 3A] 1 shows an example of a schematic diagram illustrating the structure of a nozzle 49 in this embodiment. [Figure 3B] 1 shows an example of a schematic diagram illustrating the structure of a nozzle 49 in this embodiment. [Figure 4] FIG. 1 shows an example of a schematic diagram illustrating an example of a method for recovering organisms in this embodiment. [Figure 5] An example of a specific configuration of the information processing device 170 in this embodiment will be described. [Figure 6] 1 shows an example of the flow of a method for manipulating an organism in this embodiment. [Figure 7A]1 shows an example of a GUI image displayed on the output unit 160 in this embodiment. [Figure 7B] 1 shows an example of a GUI image displayed on the output unit 160 in this embodiment. [Figure 7C] 1 shows an example of a GUI image displayed on the output unit 160 in this embodiment. [Figure 7D] 1 shows an example of a GUI image displayed on the output unit 160 in this embodiment. [Figure 7E] 1 shows an example of a GUI image displayed on the output unit 160 in this embodiment. [Figure 8] An example of the flow of the liquid replacement or addition process in S600 in this embodiment will be described. [Figure 9A] An example of a flow for moving the relative position between the nozzle 49 and the cell in S200 in this embodiment will be shown. [Figure 9B] An example of a flow for moving the relative position between the nozzle 49 and the cell in S200 in this embodiment will be shown. [Figure 9C] An example of a flow for moving the relative position between the nozzle 49 and the cell in S200 in this embodiment will be shown. [Figure 10A] An example of a flow for forming bubbles in S300 in this embodiment will be described. [Figure 10B] An example of a flow for forming bubbles in S300 in this embodiment will be described. [Figure 11A] 10 shows an example of a flow for executing the operation of S400 in this embodiment. [Figure 11B] An example of recovering cytoplasm and cell membrane from cells in this embodiment will be described. [Figure 11C] An example of the present embodiment in which cells are attached to air bubbles and then detached will be described. [Figure 11D] FIG. 1 shows an example of a schematic diagram illustrating a method for recovering cells in this embodiment. [Figure 11E] 1 shows an example of passaged cells in this embodiment. [Figure 11F]1 shows an example of passaged cells in this embodiment. [Figure 11G] An example of analysis of collected cells in this embodiment is shown below. [Figure 11H] FIG. 1 shows an example of a schematic diagram illustrating retained cells in this embodiment. [Figure 11I] 1 shows an example of compressed cells in this embodiment. [Figure 12A] 10 is a diagram showing an example of a flow for removing air bubbles in S500 in this embodiment. [Figure 12B] 10 is a diagram showing an example of a flow for removing air bubbles in S500 in this embodiment. [Figure 13] An example of a flow for controlling the air current in the bubble 256 to which the manipulation target 35 is attached in this embodiment will be described. [Figure 14] An example of a method for generating an air current in the bubble 256 and manipulating the position of the manipulation target 35 by the air current will be described below. [Figure 15] An example of a schematic diagram illustrating a method 914 for moving the nozzle 49 in the horizontal direction in this embodiment is shown. [Figure 16] 10 shows an example of a schematic diagram illustrating the movement direction of the operation target 35 when the nozzle 49 is moved in the horizontal direction in this embodiment. [Figure 17] An example of a schematic diagram illustrating a method 915 for moving the nozzle 49 in the vertical direction in this embodiment is shown. [Figure 18] 10 shows an example of a schematic diagram illustrating the movement direction of the operation target 35 when the nozzle 49 is moved in the vertical direction in this embodiment. [Figure 19] An example of a schematic diagram illustrating a method 917 for increasing the volume of the air bubble 256 and a method 918 for decompressing the air bubble 256 in this embodiment is shown. [Figure 20] 10 shows an example of a schematic diagram illustrating the movement direction of the operation target 35 when the volume of the bubble 256 is changed in this embodiment. [Figure 21]9 shows an example of a schematic diagram illustrating a method 913 for generating an airflow using the double-tube nozzle 49a in this embodiment. [Figure 22] 10 shows an example of a schematic diagram illustrating the movement direction of the operation target 35 when a double-tube nozzle 49a is used in this embodiment. [Figure 23] 10 is an experimental image showing the movement of the operation target 35 caused by the movement of the bubble 256 in this embodiment. [Figure 24] 10 is an experimental image showing the position control of the operation target 35 by enlarging the bubble 256 in this embodiment. [Figure 25] 1 shows an example of a hardware configuration of a computer. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described below through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. Furthermore, not all of the combinations of features described in the embodiments are necessarily essential to the solution of the invention. In the drawings, the same reference numerals are used to designate the same or similar parts, and redundant explanations may be omitted.

[0024] FIG. 1A shows an example of the device configuration of an organism manipulation apparatus 100 according to the present embodiment. The organism manipulation apparatus 100 according to the present invention manipulates microscopic organisms such as cells using an interface between gas and liquid. For example, the organism manipulation apparatus 100 can perform various operations on organisms, such as attaching an organism to an interface and then detaching the organism adhered to a solid phase. The organism manipulation apparatus 100 comprises a microscope unit 50, a camera 60, a camera 70, an air-liquid interface manipulation unit 101, an output unit 160, an information processing device 170, and an input unit 180.

[0025] The microscope unit 50 is a device for observing or displaying the manipulation target 35 by magnifying it using a microscope. The manipulation target 35 is a living organism. The living organism may be an organic life form. For example, the living organism may be a cell. As an example, the cell may be an animal cell or a plant cell. As an example, the cell may be a living cell or a dead cell. Furthermore, for example, the living organism may be a microorganism other than a cell. As an example, the microorganism may be a microorganism, a fungus, an algae, a living tissue, a spheroid, or the like. Furthermore, the living organism may include organelles within the cell.

[0026] The microscope section 50 includes a light source 1 for observing a fluorescent image, a dichroic mirror 2, an optical deflector 3, a relay lens 4, a dichroic mirror 5, an objective lens 6, a condenser lens 7, a focusing lens 8, a bandpass filter 9, a light source 10 for observing a transmitted image, a barrier filter 11, a projection lens 12, a barrier filter 13, a projection lens 14, a pinhole 15, a light source 16, and a light source 17.

[0027] The light source for fluorescence image observation 1 is a light source used when performing fluorescence image observation of the manipulation target 35. The manipulation target 35 may be labeled with one or more types of fluorescent substances, or may not be fluorescently labeled. The light source for fluorescence image observation 1 irradiates the manipulation target 35 with light that excites or reflects the manipulation target 35.

[0028] The light source 10 for transmission image observation is a light source used when performing transmission image observation of the operation target 35. The light source 10 for transmission image observation irradiates light that is transmitted through the operation target 35. The light that is transmitted through the operation target 35 may pass through the outside or inside of the nozzle.

[0029] Configurations of the microscope unit 50 other than those described above will be described later. The microscope unit 50 is not limited to the examples described above, and may have any known configuration. For example, the microscope unit 50 may have the configuration described in Japanese Patent Laid-Open No. 7-13083 or Japanese Patent No. 3814869.

[0030] The camera 60 captures a fluorescent image of the operation target 35 and generates an image. The image data generated by the camera 60 may be recorded inside the information processing device 170 (for example, in the recording unit 190 described below) and / or output to the output unit 160. For example, the camera 60 may be a camera that captures a fluorescent image, but is not limited to this. In the following description, the camera 60 is assumed to be a camera that captures a fluorescent image.

[0031] The camera 70 captures a transmission image of the operation target 35 and generates an image. Data of the image generated by the camera 70 may be recorded inside the information processing device 170 (for example, in the recording unit 190 described later) and / or output to the output unit 160. For example, the camera 70 may be a camera that captures a transmission image, but is not limited to this. In the following description, the camera 70 is assumed to be a camera that captures a transmission image.

[0032] The cameras 60 and 70 each have an imaging sensor (not shown). The cameras 60 and 70 may be cooled cameras. A cooled camera is a camera that can suppress noise caused by heat by cooling the imaging sensor. The imaging sensor may be a CMOS image sensor (Complementary Metal Oxide Semiconductor) or a CCD image sensor (Charge Coupled Device). The cameras 60 and 70 may be housed in a housing separate from the microscope unit 50.

[0033] The gas-liquid interface manipulation unit 101 manipulates the manipulation target 35 using the gas-liquid interface between the gas and the liquid. For example, the gas-liquid interface manipulation unit 101 forms gas bubbles in the liquid to manipulate living organisms (e.g., cells) in the liquid. The gas-liquid interface manipulation unit 101 has all or at least a portion of a nozzle actuator 40, a sample actuator 41, a channel imaging camera 42, a light source 45, a light source 46, a pressure generating unit 47, a sensor unit 48, a nozzle 49, a channel 51, a channel exchanging unit 53, a liquid storage unit 54, a sample lid 58, and a sample lid storage unit 59.

[0034] The nozzle actuator 40 mounts a nozzle 49 via a pressure generating unit 47 and moves the nozzle 49. As described below, a flow path 51 is formed inside the nozzle 49, and a gas-liquid interface 255, such as a bubble, is formed at the tip of the flow path 51. The nozzle actuator 40 may be movable in any of the vertical, horizontal, and up-down directions. The nozzle actuator 40 may be movable only in the up-down direction. In this case, the vertical and horizontal movements of the nozzle actuator 40 may be controlled by a stage of the microscope unit 50. The nozzle actuator 40 may be movable only in the vertical and horizontal directions. In this case, the up-down movements of the nozzle actuator 40 may be controlled by a stage of the microscope unit 50. The nozzle actuator 40 may be fixed and not move. In this case, the vertical, horizontal, and up-down movements of the nozzle actuator 40 may be controlled by a stage of the microscope unit 50. The operation of the nozzle actuator 40 is controlled by a nozzle position control unit (not shown) of the bubble generating unit in the information processing device 170.

[0035] The sample actuator 41 moves a stage (not shown) on which the container 25 is mounted. The sample actuator 41 may be operable in any of the vertical, horizontal, and up-down directions. The stage may mount a transparent container 25 that houses the manipulation target 35. The container 25 may be a culture vessel filled with liquid. The sample actuator 41 may mount one or more containers and / or tubes, but is not limited to these. The operation of the sample actuator 41 is controlled by a stage position control unit (not shown) of the bubble generation unit in the information processing device 170. The stage may be provided in the gas-liquid interface manipulation unit 101 or in the microscope unit 50.

[0036] The flow channel imaging camera 42 captures an image of the tip of the nozzle 49. The flow channel imaging camera 42 may capture an image of a bubble formed at the tip of the nozzle 49. The captured image may be sent to an image processing unit in the information processing device 170. Based on the captured image, the bubble generating unit 200 may instruct the nozzle actuator 40 and / or the sample actuator 41 to move the relative position between the nozzle 49 and the operation target 35. Note that instead of the flow channel imaging camera 42, a camera 60 or a camera 70 may capture an image of the tip of the nozzle 49. In the following description, the flow channel imaging camera 42 may be a microscope-attached camera provided in the microscope unit 50. The camera provided in the microscope unit 50 may use the fluorescent image observation light source 1, the transmitted image observation light source 10, the light source 16, the light source 17, the light source 45, and the light source 46 as illumination. The light source 16 and the light source 17 may be, but are not limited to, ring illumination.

[0037] The light source 45 and the light source 46 illuminate the nozzle 49 and / or the operation target 35. The light source 45 and the light source 46 may be, but are not limited to, ring illumination.

[0038] The pressure generating unit 47 generates a pressure to be applied to the flow path 51. The pressure generating unit 47 is connected to one end of the flow path 51 that does not contact the liquid, and supplies a preset gas to that end. For example, the pressure generating unit 47 may include a syringe pump and an actuator that reciprocates the plunger of the syringe pump. The actuator may push the plunger of the syringe pump toward the flow path 51 to supply gas to the flow path 51, and the actuator may pull the plunger of the syringe pump from the flow path 51 to suck gas from the flow path 51. The pressure generating unit 47 is controlled by a bubble generating unit in the information processing device 170.

[0039] The liquid in which the manipulation target 35 is immersed may be, but is not limited to, a complete medium, a basal medium, or a buffer solution. A complete medium is a medium containing maintenance and growth factors necessary for the maintenance and growth of cells. A basal medium is a medium containing only a small amount of proteins, amino acids, or salts. A buffer solution is a liquid that maintains a pH and osmotic pressure suitable for cell survival. Known liquids, complete mediums, basal mediums, and buffer solutions can be used.

[0040] The gas may be air. The gas may contain moisture.

[0041] The sensor unit 48 has one or more sensors and detects the state of the nozzle 49 and the liquid and gas within the nozzle 49. For example, the sensor unit 48 may detect the position, speed, and acceleration of the nozzle 49. The sensor unit 48 may detect the position of the nozzle actuator 40, the pressure generated in the pressure generating unit 47, and the position of the plunger of the syringe pump in the pressure generating unit 47. The sensor unit 48 may detect the ambient temperature and the temperature of the liquid within the container 25. The sensor unit 48 may detect the ambient humidity. The sensor unit 48 may also detect the pH of the liquid within the container 25. The sensor unit 48 may detect the temperature and humidity of the gas within the nozzle 49. The sensor unit 48 sends this information to the information processing device 170 (for example, the bubble generating unit 200 described below). Known sensors may be used for the sensor unit 48. The sensor unit 48 may be housed in a housing separate from the pressure generating unit 47, or may be housed within the pressure generating unit 47.

[0042] The nozzle 49 is a device that includes a flow path 51, which will be described later. The nozzle 49 may be rod-shaped or flat.

[0043] Liquid and gas to be sucked (suction) or discharged (supply) pass through flow path 51. Flow path 51 is provided inside nozzle 49 so as to penetrate the nozzle 49 in the longitudinal direction. The other end of flow path 51 is connected to pressure generating unit 47.

[0044] The flow path exchanging unit 53 is a device that stores and discards the nozzle 49. When replacing the nozzle 49, the flow path exchanging unit 53 may remove the nozzle 49 attached to the nozzle actuator 40 and discard it in a nozzle discarding unit (not shown) of the flow path exchanging unit 53, and may attach the nozzle 49 stored in a nozzle storage unit (not shown) of the flow path exchanging unit 53 to the nozzle actuator 40 instead. The flow path exchanging unit 53 may be omitted, in which case the nozzle 49 may be replaced by an operator.

[0045] Liquid storage unit 54 is a device that stores the liquid to be supplied to container 25 and recovers and discards the liquid from container 25. When replacing the liquid, liquid storage unit 54 may recover the liquid contained in container 25 from container 25 and discard it in a liquid disposal unit (not shown) of liquid storage unit 54, and may replenish container 25 with the liquid stored in a liquid storage unit (not shown) of liquid storage unit 54. Liquids may be replaced between liquids of the same type. Liquids may be replaced between liquids of different types. Liquid storage unit 54 may be omitted, in which case the liquid may be replaced manually by an operator.

[0046] The sample lid 58 is a lid that is attached to the container 25. The sample lid 58 may be attached to the container 25 or may be stored in a sample lid storage unit 59. The sample lid 58 may be removed from the sample lid storage unit 59 and attached to the container 25, or removed from the container 25 and stored in the sample lid storage unit 59, by a sample lid actuator (not shown). In this case, the operation of the sample lid actuator may be controlled by a sample lid control unit (not shown) of the bubble generation unit 200 in the information processing device 170. The sample lid 58 and the sample lid storage unit 59 may be omitted, in which case the sample lid 58 may be attached to and removed from the container 25 by hand by an operator.

[0047] The output unit 160 outputs the processing result of the information processing device 170. For example, the output unit 160 outputs an image that has been subjected to image processing inside the information processing device 170 (for example, an image processing unit 300, which will be described later). For example, the output unit 160 is a monitor connected to the information processing device 170.

[0048] The information processing device 170 exchanges commands and data with the microscope unit 50, the camera 60, the camera 70, the gas-liquid interface operation unit 101, the output unit 160, and the input unit 180. For example, the information processing device 170 is connected to the microscope unit 50 and the gas-liquid interface operation unit 101, and controls the microscope unit 50 and the gas-liquid interface operation unit 101.

[0049] Specifically, the information processing device 170 switches the combination of the type of objective lens 6 and / or the type of filter cube of the fluorescence filter placed in the optical path of the microscope unit 50. For example, transmission image observation and fluorescence image observation differ in both the type of filter cube placed in the optical path and the type of objective lens 6. Furthermore, the two types of fluorescence image observation differ only in the type of filter cube placed in the optical path. Furthermore, transmission image observation and fluorescence image observation also differ in the light sources used (light source 10 for transmission image observation and light source 1 for fluorescence image observation, respectively). Therefore, the information processing device 170 (for example, the imaging control unit 171, described later) may switch one or more of the filter blocks, objective lens 6, and light source depending on whether transmission image observation and / or one or more types of fluorescence image observation are to be performed.

[0050] When performing fluorescence image observation, the information processing device 170 turns on the light source for fluorescence image observation 1 and turns off the light source for transmitted image observation 10 in order to enable the light path of the light source for fluorescence image observation 1. When performing fluorescence image observation, the light emitted from the light source for fluorescence image observation 1 illuminates the operation target 35 via the dichroic mirror 2, the optical deflector 3, the relay lens 4, the dichroic mirror 5, and the objective lens 6.

[0051] If the manipulation target 35 is fluorescently labeled, the fluorescent substance in the manipulation target 35 is excited and emits fluorescence. The fluorescence emitted from the manipulation target 35 reaches the light receiving surface of the camera 60 via the objective lens 6, dichroic mirror 5, relay lens 4, optical deflector 3, dichroic mirror 2, barrier filter 13, projection lens 14, and pinhole 15 (if the microscope unit 50 is a confocal microscope). At this time, a fluorescent image of the manipulation target 35 is formed on the camera 60. Note that even if the manipulation target 35 is not fluorescently labeled, the manipulation target 35 can be observed by using the light emitted from the fluorescent image observation light source 1 that hits the manipulation target 35 and is reflected from the manipulation target 35.

[0052] The information processing device 170 turns on the light source 10 for transmission image observation and turns off the light source 1 for fluorescence image observation to enable the light path of the light source 10. When transmission image observation is performed, light emitted from the light source 10 for transmission image observation illuminates the operation target 35 via the bandpass filter 9, the condenser lens 8, and the condenser lens 7. The light that has passed through the operation target 35 reaches the light receiving surface of the camera 70 via the objective lens 6, the dichroic mirror 5, the barrier filter 11, and the projection lens 12. At this time, a transmission image of the operation target 35 is formed on the camera 70. Note that if the end of the nozzle 49 is difficult to see during fluorescence observation, transmission image observation may also be performed.

[0053] Furthermore, the information processing device 170 controls the relative positions of the nozzle 49 and stage of the gas-liquid interface manipulation unit 101. In addition to controlling the microscope unit 50 and the gas-liquid interface manipulation unit 101, the information processing device 170 may also receive images of the manipulation target 35 captured by the camera 60 and / or the camera 70 and / or images captured by the channel imaging camera 42 of the gas-liquid interface manipulation unit 101, and perform image processing such as generating a single composite image from multiple images. The information processing device 170 may also control other operations of the organism manipulation device 100 and perform data processing, etc., as necessary. The configuration of the information processing device 170 will be described later.

[0054] The input unit 180 inputs instructions and data from the operator to the information processing device 170. For example, the input unit 180 inputs instructions from the operator regarding the selection of an operation application for the operation target 35. The input unit 180 also inputs the amount of operation of the nozzle actuator 40 and / or the sample actuator 41 from the operator to the information processing device 170. For example, the input unit 180 is a keyboard or mouse connected to the organism manipulation device 100.

[0055] 1B shows another example of the device configuration of organism manipulation apparatus 100 in this embodiment. FIG. 1B shows organism manipulation apparatus 100 in which microscope unit 50 is a phase-contrast microscope or a differential interference microscope. In the case where microscope unit 50 is a phase-contrast microscope, microscope unit 50 may include objective lens 6, condenser lens 7, focusing lens 8, bandpass filter 9, transmitted image observation light source 10, barrier filter 11, projection lens 12, light source 16, light source 17, and ring diaphragm 39. When the microscope unit 50 is a differential interference microscope, the microscope unit 50 may include an objective lens 6, a condenser lens 7, a focusing lens 8, a bandpass filter 9, a light source for transmission image observation 10, a barrier filter 11, a projection lens 12, a light source 16, a light source 17, a Normarski prism 31, an analyzer (polarizing plate) 32, a polarizer (polarizing plate) 37, and a Normarski prism 38. Furthermore, the microscope unit 50 is not limited to these, and may include configurations other than those listed above. The description of FIG. 1A may be applied to the configuration of the organism manipulation apparatus 100 other than the microscope unit 50.

[0056] 2A and 2B are exemplary schematic diagrams illustrating the structure of the nozzle 49 according to this embodiment. In FIG. 2A, the nozzle 49 includes a tubular portion 253 having a flow path 51. The tubular portion 253 may be hollow and cylindrical. In this case, the cross section of the tubular portion 253 perpendicular to the axial direction has a circular shape. One end of the flow path 51 may be connected to a pump 251 (e.g., a syringe pump of the pressure generating unit 47). The pump 251 adjusts the amount of gas supplied to or drawn from the flow path 51 in response to an instruction from the information processing device 170 (e.g., the bubble generating unit 200, described later), thereby adjusting the pressure and / or volume of the bubbles.

[0057] In FIG. 2B , when end 254 of cylindrical portion 253, which is not connected to a pump (not shown: for example, a syringe pump of pressure generating unit 47), is placed in liquid 261, the pump can supply gas to flow path 51, thereby forming a bubble at end 254. In this case, a gas-liquid interface 255 is formed at the boundary between the gas in the bubble and liquid 261. Note that the shape of the bubble is not limited to a spherical shape and may be deformed depending on the shape of end 254. Here, when gas is held at end 254 of flow path 51, gas-liquid interface 255 is formed at end 254 of flow path 51. However, when both gas and liquid exist inside flow path 51, gas-liquid interface 255 may be formed at the interface between the two inside flow path 51.

[0058] When the gas-liquid interface 255 comes into contact with an organism adhered to a solid phase, such as the inner bottom surface of the container 25 in the liquid 261, the gas-liquid interface 255 can be moved to apply a force to the organism, detaching it from the solid phase and attaching it to the gas-liquid interface 255. The gas-liquid interface 255 can be moved by moving the nozzle 49 in which the bubbles are formed using the nozzle actuator 40, by moving the liquid, or by changing the volume of the bubbles. Here, the solid phase may be a surface on which adherent cells can be attached and cultured. For example, the solid phase may be glass; a resin such as polystyrene; a metal; a surface coated with one or more extracellular matrix components selected from collagen, fibronectin, laminin, polylysine, etc.; or a surface coated with various polymers (e.g., polymers whose hydrophilicity and cell adsorption properties can be controlled). Examples of the solid phase include, but are not limited to, glass, resins such as polystyrene, metal, and the like. In this embodiment, the gas-liquid interface 255 is formed by the interface between the gas and the liquid, but is not limited to this and may be changed depending on the phase or substance in contact with the interface. The method for detaching the organism from the solid phase will be described in detail later.

[0059] The opening area of ​​the channel 51 at the end 254 is not particularly limited as long as it is large enough to manipulate the organism. For example, the opening area may be larger than the adhesion area per organism. The shape of the end 254 is not particularly limited. The inner diameter of the channel 51 may be the same along the entire length of the tubular portion 253.

[0060] Furthermore, flow path 51 may be configured such that pump 251 draws in gas in bubbles to which the organisms are attached, thereby taking gas-liquid interface 225 into flow path 51 and further recovering the organisms. Alternatively, nozzle 49 may further include another flow path, separate from flow path 51, for recovering the organisms.

[0061] In the embodiment of Figures 2A and 2B, only one flow path 51 is formed in the nozzle 49, and only one pump 251 is connected to the flow path 51, which is a very simple and minimal configuration, and can reduce the maintenance and costs of the living body manipulation device 100.

[0062] 3A and 3B are schematic diagrams illustrating an example of the structure of nozzle 49 in another embodiment. In the example of Figures 2A and 2B, the flow path for forming bubbles and the flow path for recovering organisms are the same, but in the example of Figures 3A and 3B, the flow path for forming bubbles and the flow path for recovering organisms are different.

[0063] 3A, the cylindrical portion 253 of the nozzle 49 has a double structure of an outer cylinder 253a and an inner cylinder 253b. The space between the outer cylinder 253a and the inner cylinder 253b is a first flow path 51a through which gas flows, and the interior of the inner cylinder 253b is a second flow path 51b. For example, the first flow path 51a may be a gas supply flow path, and the second flow path 51b may be a gas recovery flow path.

[0064] Furthermore, the first flow path 51a and the second flow path 51b of the nozzle 49 may be connected at one end to a first pump 251a and a second pump 251b, respectively. For example, the pressure generating unit 47 may have the first pump 251a and the second pump 251b as syringe pumps, each controlled by a separate actuator. The first pump 251a and the second pump 251b adjust the pressure and / or volume of the bubbles by adjusting the amount of gas supplied or drawn into the first flow path 51a and the second flow path 51b by the actuator receiving an instruction from the bubble generating unit 200. The cross section of the cylindrical portion 253 perpendicular to the axial direction has a donut shape in the first flow path 51a and a circle in the second flow path 51b.

[0065] 3B, when end 254 of cylindrical portion 253, which is not connected to first pump 251a and second pump 251b, is placed in liquid 261, first pump 251a supplies gas to first flow path 51a, thereby forming bubbles at end 254. In this case, gas-liquid interface 255 is formed at the boundary between the gas in the bubbles and liquid 261.

[0066] When the gas-liquid interface 255 comes into contact with organisms adhered to the solid phase in the liquid 261, the gas-liquid interface 255 can be moved to detach the organisms from the solid phase and attach the organisms to the gas-liquid interface 255. The second pump 251b may take the gas-liquid interface 225 into the flow path 51 by sucking in the air bubbles to which the organisms are attached through the second flow path 51b, and recover the organisms.

[0067] In the above embodiment, the first pump 251a supplies gas to the first flow path 51a to form bubbles, and the second pump 251b draws the gas into the second flow path 51b to collect the organisms. However, the second pump 251b may supply gas to the second flow path 51b to form bubbles, and the first pump 251a may draw the gas into the first flow path 51a to collect the organisms. Furthermore, the first pump 251a and the second pump 251b may be the same syringe pump provided in the pressure generating unit 47. Either the first pump 251a or the second pump 251b may be omitted.

[0068] 3A and 3B, bubbles can be formed in one flow path to cause organisms to adhere to the gas-liquid interface 255, and the adhered organisms can be collected in the other flow path simultaneously, which has the effect of shortening the time required to collect cells. Note that, in FIGS. 3A and 3B, an embodiment is shown in which the cross-sectional shape of the cylindrical portion 253 perpendicular to the axial direction is doughnut-shaped in the first flow path 51a and circular in the second flow path 51b, but the cross-sectional shape is not limited to doughnut-shaped or circular, and as long as there are two flow paths, organisms can be attached and collected simultaneously.

[0069] 4 is a schematic diagram showing an example of a method for recovering the manipulation target 35 in this embodiment. In 290a, the manipulation target 35 is cultured on a solid phase on the inner bottom surface of the container 25. The manipulation target 35 may be cultured in a liquid 261. For example, the manipulation target 35 is an adherent cell. For example, the liquid may be a complete medium.

[0070] In 290a, pump 251 supplies gas to flow path 51 of nozzle 49, forming bubbles 256 at end 254 of nozzle 49. By bringing bubble 256 into contact with manipulation target 35, gas-liquid interface 255 between the gas and liquid 261 comes into contact with manipulation target 35. In this case, pump 251 adjusts the supply and suction of gas to maintain formed bubbles 256. This makes it possible to more easily manipulate manipulation target 35 using bubbles 256.

[0071] Next, in step 290b, the nozzle actuator 40 moves the nozzle 49 along the surface of the solid phase while keeping the bubble 256 in contact with the manipulation target 35. While FIG. 4 illustrates the nozzle actuator 40 moving the nozzle 49 from left to right, the direction of nozzle 49 movement is not limited as long as it is parallel to the surface of the solid phase. By moving the nozzle 49, the nozzle actuator 40 can detach the manipulation target 35 from the solid phase. At this time, the detached manipulation target 35 adheres to the gas-liquid interface 255 of the bubble 256. The bubble generator 200 controls the pump 251 to adjust the pressure and / or volume of the gas being supplied or drawn in, thereby changing the size of the bubble 256, thereby enabling the manipulation target 35 within a desired range to be detached. Note that instead of moving the nozzle 49 along the surface of the solid phase, the stage may be moved.

[0072] Next, in 290c, the pump 251 may suck in the gas in the flow path 51, thereby recovering the manipulation target 35 adhering to the gas-liquid interface 255. In this way, the manipulation target 35 can be selectively detached from the solid phase and recovered using the gas bubbles 256 formed in the nozzle 49.

[0073] In addition, when the manipulation object 35 is an adherent cell that is strongly adhered to a solid phase, the adhesion of the adherent cell may be relaxed in advance before carrying out the method of Fig. 4. The relaxation of the adhesion of the adherent cell can be carried out using a known method, as described below.

[0074] FIG. 4 illustrates an example in which the gas-liquid interface 255 is moved by moving the nozzle 49, thereby detaching and recovering cells. However, the movement of the gas-liquid interface 255 is not limited to the above example. For example, the volume of the bubble 256 may be increased after the bubble 256 is brought into contact with the manipulation target 35. In this case, the contact surface between the bubble 256 and the solid phase is enlarged, allowing the manipulation target to be selectively detached from the solid phase and recovered. For example, after the bubble 256 is brought into contact with the manipulation target 35, the nozzle actuator 40 may move the nozzle 49 so as to approach the solid phase. In this case, the bubble 256 is pressed against the solid phase, thereby enlarging the contact surface between the bubble 256 and the solid phase, allowing the manipulation target to be selectively detached from the solid phase and recovered.

[0075] 5 shows an example of a specific configuration of the information processing device 170 in this embodiment. The information processing device 170 has an imaging control unit 171, a recording unit 190, a bubble forming unit 200, a flow path control unit 250, a liquid control unit 260, and an image processing unit 300.

[0076] The imaging control unit 171 controls the fluorescence image observation light source 1, the objective lens 6, the fluorescence filter, the transmission image observation light source 10, the flow channel imaging camera 42, the light source 45, the light source 46, the camera 60, and the camera 70, all of which are described in FIGS. 1A and 1B. For example, when imaging conditions for the operation target 35 are input to the input unit 180, the imaging control unit 171 makes necessary adjustments for each imaging operation, such as switching the camera, switching the type of objective lens 6 in the microscope unit 50, switching the light source, switching the type of fluorescent filter, the position of the stage, and the height of the objective lens 6, in accordance with the input imaging conditions. After the imaging control unit 171 makes the necessary adjustments, one or more of the flow channel imaging camera 42, the camera 60, and the camera 70 capture images of the operation target 35 or the nozzle 49 and generate images of the operation target 35 or the nozzle 49. The one or more cameras send data of the generated images to the image processing unit 300. Furthermore, data of the generated image may be recorded in the recording unit 190 and / or output to the output unit 160.

[0077] The recording unit 190 may be, but is not limited to, a memory, an internal hard disk drive, or an external recording medium. The information processing device 170 has a central processing unit (CPU), and the CPU executes a computer program recorded in the recording unit 190 to realize the information processing device 170.

[0078] The bubble formation unit 200 controls the pressure and volume of bubbles formed in the flow path 51, the supply and suction of gas, the movement of the nozzle 49, and the movement of the stage. The bubble formation unit 200 may include all or some of a nozzle position control unit, a stage position control unit, a volume control unit, a supply control unit, and a suction control unit.

[0079] The nozzle position control unit controls the nozzle actuator 40, and controls the operation of the nozzle 49, the airflow in the bubble that accompanies the operation of the nozzle 49, and the movement of the air-liquid interface 255 that accompanies the operation of the nozzle 49. The nozzle position control unit also receives position information of the nozzle 49 from the sensor unit 48 or the nozzle actuator 40. Note that the airflow may refer to the flow or dynamics of the fluid in the bubble.

[0080] The stage position control unit controls the sample actuator 41, and controls the operation of the stage on which the container 25 containing the manipulation target 35 is mounted, the airflow in the bubbles accompanying the stage operation, and the movement of the gas-liquid interface 255 accompanying the stage operation. The stage position control unit also receives position information of the stage and the manipulation target 35 from the sensor unit 48 or the sample actuator 41.

[0081] The volume control unit controls the actuator of the pressure generating unit 47 and controls the pressure and / or volume of bubbles formed in the flow path 51 by supplying gas from the syringe pump or sucking gas into the syringe pump. The volume control unit also receives information on the pressure and / or volume of the bubbles from the nozzle actuator 40, the pressure generating unit 47, or the sensor unit 48.

[0082] Furthermore, when the nozzle 49 includes a gas supply flow path that supplies (supplies) gas and a gas recovery flow path that recovers (intakes) gas, the volume control unit or the gas supply control unit controls the first pump 251a connected to the gas supply flow path, thereby controlling the volume of gas supplied to the gas supply flow path. The volume control unit or the gas supply control unit receives information about the amount of gas supplied to the gas supply flow path from the nozzle actuator 40, the pressure generating unit 47, or the sensor unit 48.

[0083] Furthermore, if the nozzle 49 includes a gas supply flow path that supplies (intakes) gas and a gas return flow path that returns (intakes) gas, the volume control unit or the air intake control unit controls the second pump 251b connected to the gas return flow path, thereby controlling the amount (volume) of gas drawn from the gas return flow path. The volume control unit or the air intake control unit receives information about the amount of gas drawn from the gas return flow path from the nozzle actuator 40, the pressure generating unit 47, or the sensor unit 48.

[0084] The flow path control unit 250 controls the storage, attachment, and disposal of the nozzle 49. The flow path control unit 250 receives operational instructions from the operator regarding the attachment and disposal of the nozzle 49 from the input unit 180. In accordance with the received instructions, the flow path control unit 250 sends an instruction to the flow path exchanging unit 53 to remove the nozzle 49 from the flow path storage unit of the flow path exchanging unit 53 and attach the nozzle 49 to the nozzle actuator 40, or to remove the nozzle 49 attached to the nozzle actuator 40 and discard it in the flow path discard unit of the flow path exchanging unit 53.

[0085] The liquid control unit 260 controls the storage, replenishment, and disposal of liquid. The liquid control unit 260 receives operational instructions related to the replenishment and disposal of liquid from the operator via the input unit 180. In accordance with the received instructions, the liquid control unit 260 sends instructions to the liquid storage unit 54 to replenish the liquid stored in the liquid storage unit of the liquid storage unit 54 to the container 25, or to recover the liquid contained in the container 25 from the container 25 and discard it in the liquid disposal unit of the liquid storage unit 54.

[0086] The image processing unit 300 receives images captured by the flow channel imaging camera 42, the camera 60, and the camera 70 from these cameras. The image processing unit 300 may use multiple of the received images to combine them into a single composite image. For example, the image processing unit 300 may generate a composite image by combining a fluorescent image captured by the camera 60 and a transmission image captured by the camera 70. The image processing unit 300 may record the images received from these cameras and / or the composite image in the recording unit 190 and / or output the images to the output unit 160.

[0087] The energy control unit controls the difference (E1-E2) between the interfacial free energy E1 at the interface between the gas and the organism and the interfacial free energy E2 at the interface between the gas and the liquid. The value of the difference (E1-E2) may be positive, zero, or negative. The smaller the value of the difference (E1-E2), the more the organism adheres to the bubbles. Here, because the value of the interfacial free energy E1 is constant, there is little room for control by the energy control unit. Therefore, by using the energy control unit to control the interfacial free energy E2 at the interface between the gas and the liquid, the value of this difference (E1-E2) can be controlled to a preset value. Details will be described later.

[0088] Fig. 6 shows an example of the flow of a method for manipulating an organism in this embodiment. An organism that is an operation target 35 in this embodiment can be manipulated by performing the processes of S100 to S680 in Fig. 6. For convenience of explanation, the processes of S100 to S680 will be explained in order, but at least some of these processes may be performed in parallel, or the steps may be interchanged within the scope of the present invention.

[0089] First, in S100, the sample actuator 41 receives a living organism that will be the operation target 35. For example, in S100, the sample actuator 41 mounts a container 25 that contains the operation target 35 together with a liquid on a stage. In order to operate the operation target 35, the lid of the container 25 may be removed. The lid may be replaced by a lid replacement actuator or may be replaced manually by an operator. After the sample actuator 41 receives the living organism that will be the operation target 35, the information processing device 170 advances the process to S120.

[0090] Next, in S120, the camera 60 or 70 captures an image of a wide observation field including the operation target 35 to generate an image. The imaging control unit 171 sets the observation method to low-magnification transmission image imaging and instructs the camera 70 to capture an image of the observation field. The imaging control unit 171 may also set the observation method to fluorescence image imaging and instruct the camera 60 to capture an image of the observation field. The imaging control unit 171 may receive input of imaging conditions from the operator via the input unit 180. The camera 60 or 70 captures an image of the observation field. The image processing unit 300 may record the captured image in the recording unit 190 and / or output it to the output unit 160. After the camera 60 or 70 captures an image of the observation field, the imaging control unit 171 advances the process to S140.

[0091] Next, in S140, the information processing device 170 receives input regarding the manipulation object 35 and the type of manipulation from the operator via the input unit 180. The manipulation object 35 may be, but is not limited to, a single cell, a cell population (colony), the cytoplasm and / or cell membrane of a cell, or a spheroid. The type of manipulation may be, but is not limited to, retrieving the manipulation object 35, removing the manipulation object 35, holding the manipulation object 35, or compressing the manipulation object 35.

[0092] FIG. 7A is an example of a GUI (Graphical User Interface) image displayed on the output unit 160, in which the camera 60 or the camera 70 captures an image of the observation field. In FIG. 7A, cells aaa, bbb, and ccc to be manipulated are designated as the manipulation target 35 via the input unit 180. As shown in FIG. 7A, the organism to be manipulated 35 is arbitrarily designated via the input unit 180. As shown in FIG. 7A, a removal region and / or a protection region may be provided in the observation field so that the removal region and / or the protection region can be selected in the GUI image. By providing a removal region, the risk of recovering cells other than the cells to be recovered can be reduced. Furthermore, by providing a protection region, the risk of recovering cells being mistakenly removed when removing cells in the removal region can be reduced.

[0093] 7B is an example of a GUI image displayed on the output unit 160, in which the recovery and transfer destinations of the cells aaa, bbb, and ccc, which are the operation target 35, are designated as 12-well plates A1, A2, and A3, respectively. As shown in FIG. 7B, the transfer destinations are arbitrarily designated via the input unit 180. For example, the transfer destinations may be the same plate, different plates, petri dishes, microtest tubes, PCR tubes, or conical tubes.

[0094] 7C is an example of a GUI image displayed on the output unit 160, showing a table listing the ID number of the cell to be manipulated 35, the x and y coordinates of the manipulated object 35 on the sample actuator 41, the size of the manipulated object 35, and the destination of the manipulated object 35. While the table shown in FIG. 7C shows a case where the items are the ID number, x and y coordinates, size, and destination, the displayed items are not limited to these. In this way, the image processing unit 300 may output the table to the output unit 160 by specifying the manipulated object 35, the destination, etc. via the input unit 180.

[0095] FIG. 7D is an example of a GUI screen displayed on the output unit 160 for selecting the type of operation of the operation target 35. The input unit 180 receives instructions from the operator regarding the type of operation to be performed on the operation target 35, and inputs the instructions to the information processing device 170. For example, as shown in the display area 111, the operation on the cell may be, but is not limited to, passaging, or holding or moving the cell. For example, as shown in the display area 112, the operation on the cell may be, but is not limited to, pressing the cell to observe the depth of the cell. Although FIG. 7D shows an example in which the type of operation is selected using a radio button, the selection method is not limited to a radio button.

[0096] 7E is another example of a GUI screen displayed on output unit 160 for selecting the type of operation for operation target 35. For example, as shown in display area 113, the type of operation for a cell may be selected using a pull-down menu. For example, as shown in display areas 113, 114, and 115, the type of operation may be selected using a combination of a pull-down menu and radio buttons.

[0097] 7A to 7E, the input section 180 may send an instruction input by the operator to the information processing device 170. After the information processing device 170 receives the instruction, the imaging control section 171 advances the process to S160.

[0098] In S160, if the manipulation object 35 is an adherent cell that is strongly adhered to the solid phase, a step of alleviating the adhesion of the adherent cell in advance may be additionally performed. In this case, in the step of accepting the manipulation conditions in S140, the information processing device 170 may receive an input regarding whether or not to perform the process of alleviating adhesion.

[0099] The adhesion of adherent cells can be alleviated using known methods. For example, the adhesion of adherent cells can be alleviated by removing the liquid (e.g., medium), washing with a buffer solution, and then treating the adherent cells with an adhesion alleviation solution. For example, the adhesion alleviation solution may be a protease solution, a metal ion-free solution, or a chelating agent solution. One example of the adhesion alleviation solution is a trypsin-EDTA solution. The adhesion of adherent cells can be alleviated by the liquid control unit 260 or manually by an operator. After treating the adherent cells with the adhesion alleviation solution to weaken their adhesive force, the process can proceed to S160. Note that, regardless of whether the adhesion of adherent cells is alleviated or if the step is manually performed by an operator, the process can start again from the sample reception step of S100. Alternatively, instead of treatment with an adhesion alleviation solution, the adhesive force can be weakened using an adhesion alleviating substrate. For example, the adhesion alleviating substrate can be one that is alleviated in response to temperature or light irradiation.

[0100] Next, in S160, the information processing device 170 receives input from the operator via the input unit 180 regarding a liquid replacement or addition process. For example, when it is desired to adjust the adhesive force between the organism, which is the operation target 35, and the air bubbles, the information processing device 170 may receive input to perform a liquid replacement or addition process. When the information processing device 170 receives an instruction to perform a liquid replacement or addition process, the information processing device 170 may proceed with the process to S600. When the information processing device 170 receives an instruction not to perform a liquid replacement or addition process, the information processing device 170 may proceed with the process to S180.

[0101] In S600, the liquid control unit 260 replaces the liquid in the container 25 in which the operation target 35 is accommodated, or adds another liquid to the liquid in the container 25. In S600, the step of performing the liquid replacement or addition process includes steps S610 to S630, as shown in FIG.

[0102] First, in S610, the information processing device 170 receives an input from the operator regarding whether or not to remove the liquid via the input unit 180. If the information processing device 170 receives an instruction to remove the liquid, the process proceeds to S615. If the information processing device 170 receives an instruction not to remove the liquid, the process proceeds to S620.

[0103] In S615, the liquid control unit 260 controls the liquid storage unit 54 to remove the liquid. For example, the liquid control unit 260 sends an instruction to the liquid storage unit 54 to recover a preset amount of liquid contained in the container 25 from the container 25 and discard it in a liquid disposal unit of the liquid storage unit 54. At this time, the liquid storage unit 54 may recover and discard the entire amount of liquid. Alternatively, the liquid storage unit 54 may recover and discard a portion of the liquid (for example, half of the amount). After the liquid storage unit 54 has recovered the liquid, the liquid control unit 260 advances the process to S620.

[0104] In S620, the liquid control unit 260 sends an instruction to the liquid storage unit 54 to add an adhesion adjustment reagent to the container 25. The adhesion adjustment reagent adjusts the adhesion between the organism and the air bubbles. For example, the adhesion adjustment reagent may change the concentration of inorganic salts and / or the concentration of amphipathic substances in the liquid. As an example, the adhesion adjustment reagent may be a buffer solution containing or not containing at least one of calcium ions and magnesium ions, a basal medium, a complete medium, or a chelating agent. At this time, the liquid storage unit 54 replenishes the container 25 with the adhesion adjustment reagent stored in the liquid storage unit of the liquid storage unit 54.

[0105] In the above example, an example of refilling the adhesion adjustment reagent into the container 25 was described, but instead of adding the adhesion adjustment reagent to the container 25, the adhesion between the organisms and the bubbles may be adjusted by removing inorganic salts or amphipathic substances contained in the liquid by adhering them to a filter or the like.

[0106] In S630, the information processing device 170 receives an instruction from the operator via the input unit 180 as to whether or not to repeat the above series of operations. If the information processing device 170 receives an instruction to repeat the series of operations, the information processing device 170 proceeds to S610, and the liquid control unit 260 sends an instruction to the liquid storage unit 54 to remove the liquid from the container 25. If the information processing device 170 receives an instruction not to repeat the series of operations, the information processing device 170 proceeds to S180. Note that the steps and sub-steps of S600 may be performed manually by the operator, in which case the process may start again from the sample reception step of S100.

[0107] In S180, the nozzle actuator 40 attaches the nozzle 49. For example, the information processing device 170 receives an instruction from the operator via the input unit 180 to attach the nozzle 49 to the nozzle actuator 40. In accordance with the instruction, the flow path control unit 250 retrieves the nozzle 49 from the flow path storage unit of the flow path exchanging unit 53 and sends an instruction to the flow path exchanging unit 53 to attach the nozzle 49 to the nozzle actuator 40. At this time, an appropriate nozzle 49 may be selected depending on the size of the operation target 35, the type of operation, and the like. The selection of the nozzle 49 may be designated by the operator via the input unit 180, or may be automatically designated by the flow path control unit 250. After the nozzle 49 is attached to the nozzle actuator 40, the flow path control unit 250 advances the process to S200. In addition, if there is no need to attach the nozzle 49 to the nozzle actuator 40, such as when the nozzle 49 is already attached to the nozzle actuator 40 or when the nozzle actuator 40 and the nozzle 49 are integrally formed, step S180 may be omitted.

[0108] Next, in S200, the nozzle actuator 40 moves the relative position between the nozzle 49 and the operation target 35. For example, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move the relative position between the nozzle 49 and the operation target 35. In S200, the step of moving the relative position includes steps S210 to S225 as shown in FIG. 9A, steps S230 to S256 as shown in FIG. 9B, or steps S260 to S282 as shown in FIG. 9C.

[0109] FIG. 9A shows an example of a flow for moving the relative position between the nozzle 49 and the operation target 35 based on an image capturing the position of the end 254 of the nozzle 49.

[0110] First, in S210, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to operate the nozzle 49 to a preset position. The nozzle actuator 40 may be an actuator that controls the xyz position. Here, the z position may be a position in the vertical direction (a direction along gravity, also called the up-down direction or z direction), the x position may be a position in any x direction (also called the vertical direction) perpendicular to the z direction, and the y position may be a position in the y direction (also called the horizontal direction) perpendicular to the x and z directions.

[0111] The position of the nozzle 49 may be set by first focusing the camera 60 and / or camera 70 on the bottom surface of the vessel 25, then moving the focus of the camera 60 and / or camera 70 upward by a desired distance, and then using the nozzle actuator 40 to focus the tip of the nozzle 49 on the focus of the camera 60 and / or camera 70. For example, the desired distance may be equal to or less than the radius of a bubble formed at the end 254 of the nozzle 49. If the distance between the tip of the nozzle 49 and the bottom surface of the vessel 25 is equal to or less than the radius of the bubble, the bubble will come into contact with the bottom surface, allowing the organism located on the bottom surface to be manipulated using the bubble's interface. In this case, the xy position of the nozzle 49 can be set using the nozzle actuator 40 or the sample actuator 41 based on an image of the manipulation target 35 captured by the camera 60 and / or camera 70. Note that the position of the nozzle 49 may be set using a channel imaging camera 42 instead of or in addition to the camera 60 and / or camera 70.

[0112] The z-position of the nozzle 49 may be adjusted by using the flow path imaging camera 42 to capture an image of the tip of the nozzle 49 and the bottom surface of the container 25 from the side of the nozzle 49, and then the z-position may be set. Furthermore, the shape of the bubble or the amount of liquid in the flow path 51 may be confirmed by capturing an image of the nozzle 49 from the side using the flow path imaging camera 42. After the nozzle actuator 40 moves the nozzle 49 to a preset position, the bubble generation unit 200 advances the process to S215.

[0113] Next, in S215, the flow channel imaging camera 42 captures an image of the end 254 of the nozzle 49. The flow channel imaging camera 42 sends the captured image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble formation unit 200.

[0114] Next, in S220, the bubble formation unit 200 determines whether the position of the nozzle 49 differs from a preset position based on the captured image of the end 254 of the nozzle 49. For example, the bubble formation unit 200 calculates a positional difference between the captured image of the end 254 of the nozzle 49 and an image of the end 254 of the nozzle 49 at a preset xyz position (i.e., initial position), and if the difference is equal to or greater than a threshold, determines that the position of the nozzle 49 differs from the initial position.

[0115] If it is determined that the position of the nozzle 49 is different from the initial position, the bubble forming unit 200 advances the process to S225, otherwise, the process advances to S300.

[0116] In S225, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40 to move the nozzle 49 to a preset xyz position (i.e., the initial position), and sends an instruction to the nozzle actuator 40 to move by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S220. The nozzle actuator 40 receives the instruction and proceeds to S210. In S210 from the second time onwards, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move by an amount according to the amount of movement.

[0117] FIG. 9B shows an example of a flow for moving the relative position between the nozzle 49 and the operation target 35 based on the load sensed by the nozzle actuator 40.

[0118] First, in S230, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to operate the nozzle 49 to a preset position. The nozzle actuator 40 may be an actuator that controls the z position. In this case, the z position is controlled based on the load value, contact, or proximity information sensed by the nozzle actuator 40. The nozzle 49 may be positioned above an area on the bottom surface of the container 25 where no living organisms are present. After the nozzle actuator 40 moves the nozzle 49 to the preset position, the bubble formation unit 200 proceeds to S235.

[0119] Next, in S235, the sensor unit 48 measures the load, contact, or proximity information applied by the nozzle actuator 40 and sends the measured value to the bubble formation unit 200. As an example of load detection, when the nozzle 49 reaches the bottom of the container 25, the load sensed by the nozzle actuator 40 increases sharply. Therefore, by measuring the value of the load sensed by the nozzle actuator 40, the bubble formation unit 200 can determine whether the nozzle 49 has reached the bottom of the container 25. As another example of load detection, the sensor unit 48 may sense the load while the nozzle actuator 40 moves the nozzle 49 downward. Note that instead of the sensor unit 48, the nozzle actuator 40 may send the sensed load value to the bubble formation unit 200.

[0120] Next, in S240, the air bubble formation unit 200 determines whether the measured load value is equal to or less than the set load. If the measured load value is equal to or less than the set load, the air bubble formation unit 200 proceeds to S242; otherwise, the air bubble formation unit 200 proceeds to S245. As described above, the air bubble formation unit 200 calculates the difference between the set load and the measured load, and if the difference value is equal to or greater than the threshold, it determines that the nozzle 49 has not reached the bottom of the container 25.

[0121] In S242, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40 to move the nozzle 49 to a preset position, and sends an instruction to the nozzle actuator 40 to move by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S240. The nozzle actuator 40 receives the instruction and proceeds to S230. In S230 from the second time onwards, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move by an amount according to the amount of movement.

[0122] In S245, the bubble formation unit 200 sets the initial z position of the nozzle 49. For example, the bubble formation unit 200 may not move the z position of the nozzle 49 after the final step S230, and may set this position as the initial z position. Alternatively, the bubble formation unit 200 may move the nozzle 49 in the z direction by a predetermined arbitrary distance from the bottom surface of the container 25 and set this position as the initial z position. As a result, the initial z position of the nozzle 49 is located an arbitrary distance above the bottom surface. For example, the arbitrary distance may be equal to or less than the radius of the bubble formed at the end 254 of the nozzle 49. After the bubble formation unit 200 sets the initial z position of the nozzle 49, the bubble formation unit 200 proceeds to S250.

[0123] Next, in S250, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to operate the nozzle 49 to a preset xyz position (i.e., initial position). The nozzle 49 may move on the xy plane. In this case, the z position is controlled based on the load value sensed by the nozzle actuator 40. The movement of the nozzle 49 may include movement in the z direction as needed, in addition to movement on the xy plane. After the nozzle actuator 40 moves the nozzle 49 to the preset xyz position, the bubble formation unit 200 advances the process to S252.

[0124] Next, in S252, the flow channel imaging camera 42 captures an image of the end 254 of the nozzle 49. The flow channel imaging camera 42 sends the captured image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble formation unit 200.

[0125] Next, in S254, the bubble formation unit 200 determines whether the position of the nozzle 49 differs from a preset position based on the captured image of the end 254 of the nozzle 49. For example, the bubble formation unit 200 calculates a positional difference between the captured image of the end 254 of the nozzle 49 and an image of the end 254 of the nozzle 49 at a preset xyz position (i.e., initial position), and if the difference is equal to or greater than a threshold, the bubble formation unit 200 determines that the position of the nozzle 49 differs from the initial position.

[0126] If it is determined that the position of the nozzle 49 is different from the initial position, the bubble forming unit 200 advances the process to S256, otherwise it advances the process to S300.

[0127] In S256, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40 to move the nozzle 49 to a preset xyz position (i.e., the initial position), and sends an instruction to the nozzle actuator 40 to move by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S254. The nozzle actuator 40 receives the instruction, and the bubble formation unit 200 proceeds to the process at S250. In the second and subsequent times of S250, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move by an amount according to the amount of movement.

[0128] FIG. 9C shows an example of a flow for moving the relative position between the nozzle 49 and the operation target 35 based on the internal pressure of the air bubble formed at the end 254 of the nozzle 49.

[0129] First, in S260, the bubble formation unit 200 controls the pressure generation unit 47 to form a bubble at the end 254 of the nozzle 49. Prior to forming the bubble, the bubble formation unit 200 may send an instruction to the nozzle actuator 40 to move the end 254 of the nozzle 49 into the liquid. The step and substeps of forming the bubble in S260 may be the same as the step and substeps of S300 described below.

[0130] Next, in S262, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to operate the nozzle 49 to a preset position. The nozzle actuator 40 may be an actuator that controls the z position. In this case, the z position is controlled based on the internal pressure value measured by the sensor unit 48. The nozzle 49 may be positioned above an area on the bottom surface of the container 25 where no living organisms are present. After the nozzle actuator 40 moves the nozzle 49 to the preset position, the bubble formation unit 200 proceeds to S264.

[0131] In S262, the tip of the nozzle 49 may detect the liquid level and control the z-position based on the value of the internal pressure measured by the sensor unit 48. The bubble-forming unit 200 maintains the internal pressure of the nozzle above or below atmospheric pressure. When the tip of the nozzle 49 reaches the liquid level, the value of the internal pressure measured by the sensor unit 48 changes due to an external force caused by deformation of the gas-liquid interface due to contact with the liquid level. Therefore, the bubble-forming unit 200 can determine whether the tip of the nozzle 49 has reached the liquid level by measuring the value of the internal pressure of the bubble. As a result, even if the position to which the nozzle 49 is moved is not preset, the bubble-forming unit 200 can send a command to the nozzle actuator 40 to control the z-position using the liquid level as a reference position, and move the position of the nozzle 49.

[0132] Next, in S264, the sensor unit 48 measures the internal pressure of the formed bubble and sends the measured internal pressure value of the bubble to the bubble formation unit 200. When the bubble reaches the bottom of the container 25, its shape is deformed due to interaction with the bottom, and the internal pressure of the bubble changes rapidly. Therefore, the bubble formation unit 200 can determine whether the bubble has reached the bottom of the container 25 by measuring the internal pressure value of the bubble. As another example of internal pressure measurement, the sensor unit 48 may measure the internal pressure while the nozzle actuator 40 moves the nozzle 49 downward, or the bubble formation unit 200 may control the pressure, or the pressure generation unit 47 may operate. Such simultaneous operation can speed up bottom detection and enable the pressure change process during bubble formation to be used as a detection indicator. Note that instead of the sensor unit 48, the nozzle actuator 40 may measure the internal pressure of the bubble and send the measured internal pressure value to the bubble formation unit 200.

[0133] Next, in S266, the bubble formation unit 200 determines whether the measured internal pressure of the bubble is within a preset internal pressure range. If the measured internal pressure is outside the preset internal pressure range, the bubble formation unit 200 proceeds to S268; otherwise, the bubble formation unit 200 proceeds to S270. As described above, the bubble formation unit 200 calculates the absolute value of the difference between the internal pressure set by the bubble formation unit 200 and the measured internal pressure of the bubble. If the difference is equal to or greater than the threshold, the bubble formation unit 200 determines that the nozzle 49 has reached the bottom of the container 25.

[0134] In S268, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40 to move the nozzle 49 to a preset position, and sends an instruction to the nozzle actuator 40 to move by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S266. The nozzle actuator 40 receives the instruction and proceeds to S262. In S262 from the second time onwards, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move by an amount according to the amount of movement.

[0135] In S270, the air bubble forming unit 200 controls the pressure generating unit 47 to remove air bubbles from the end 254 of the nozzle 49. The steps and substeps of removing air bubbles in S270 may be the same as the steps and substeps of S500 described below.

[0136] Next, in S272, the air bubble formation unit 200 sets an initial z position of the nozzle 49. Step S272 may be the same as step S245. After the air bubble formation unit 200 sets the initial z position of the nozzle 49, the air bubble formation unit 200 proceeds to the process of S274.

[0137] Next, in S274, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to operate the nozzle 49 to a preset xyz position (i.e., initial position). The movement of the nozzle 49 may be on the xy plane, and may also include movement in the z direction as necessary. After the nozzle actuator 40 moves the nozzle 49 to the preset xyz position, the bubble formation unit 200 proceeds to S276.

[0138] Next, in S276, the flow channel imaging camera 42 captures an image of the end 254 of the nozzle 49. The flow channel imaging camera 42 sends the captured image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble formation unit 200.

[0139] Next, in S280, the bubble formation unit 200 determines whether the position of the nozzle 49 differs from a preset position based on the captured image of the end 254 of the nozzle 49. For example, the bubble formation unit 200 calculates a positional difference between the captured image of the end 254 of the nozzle 49 and an image of the nozzle end 254 at a preset xyz position (i.e., initial position), and if the difference is equal to or greater than a threshold, the bubble formation unit 200 may determine that the position of the nozzle 49 differs from the initial position.

[0140] If it is determined that the position of the nozzle 49 is different from the initial position, the bubble formation unit 200 advances the process to S282, otherwise it advances the process to S300.

[0141] In S282, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble formation unit 200 determines the amount of movement of the nozzle actuator 40 to move the nozzle 49 to a preset xyz position (i.e., the initial position), and sends an instruction to the nozzle actuator 40 to move by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S280. The nozzle actuator 40 receives the instruction, and the bubble formation unit 200 proceeds to the process at S274. In S274 from the second time onwards, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move by an amount according to the amount of movement.

[0142] In S300, the bubble forming unit 200 controls the pressure generating unit 47 to expand the gas-liquid interface 255. For example, expanding the gas-liquid interface 255 may include forming a gas bubble. Prior to forming the bubble, the bubble forming unit 200 may send a command to the nozzle actuator 40 to move the end 254 of the nozzle 49 into the liquid. In S300, the step of expanding the gas-liquid interface 255 includes steps S320 to S342 as shown in FIG. 10A , or includes steps S370 to S392 as shown in FIG. 10B .

[0143] FIG. 10A shows an example of a flow for enlarging the gas-liquid interface 255 based on an image captured of the position of the end 254 of the nozzle 49.

[0144] In S320, the bubble generation unit 200 sends an instruction to the pressure generation unit 47 connected to the flow path 51 to expand the gas-liquid interface 255 at the tip of the flow path 51 (to form bubbles). For example, the bubble generation unit 200 sends an instruction to the actuator of the pressure generation unit 47 to push the plunger of the syringe pump a preset distance or to push the plunger of the syringe pump until a preset pressure is reached. As a result, the gas pushed out from the syringe pump is supplied to the flow path 51, and the gas-liquid interface 255 at the tip of the flow path 51 expands (forms bubbles). After the gas-liquid interface 255 expands (forms bubbles), the bubble generation unit 200 advances the process to S330.

[0145] Next, in S330, the flow channel imaging camera 42 captures an image of the bubble formed at the end 254 of the nozzle 49. The flow channel imaging camera 42 sends the captured image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble forming unit 200.

[0146] Next, in S340, the bubble formation unit 200 determines whether the shape of the formed bubble differs from a preset bubble shape based on the captured image of the bubble. The bubble formation unit 200 predicts the shape of the bubble to be formed at the end 254 of the nozzle 49 from information such as the set internal pressure in the nozzle 49, the inner diameter of the end 254 of the nozzle 49, the wettability of the nozzle 49 (contact angle of the liquid), the type of liquid, and the type of gas. For example, the bubble formation unit 200 may determine whether the shape of the formed bubble differs from the preset bubble shape by comparing the captured image of the bubble with the bubble shape predicted from the above information.

[0147] If the shape of the formed bubble is different from the set bubble shape, the bubble forming unit 200 advances the process to S342, otherwise, advances the process to S400.

[0148] In S342, the bubble generation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47. For example, the bubble generation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47 (for example, the distance to push or pull the plunger of the syringe pump) to form a bubble at the tip of the nozzle 49 in a predetermined shape. The bubble generation unit 200 sends an instruction to the pressure generation unit 47 to operate by the determined amount of movement. For example, the bubble generation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S340.

[0149] The operation amount may be the operation amount of the actuator of the pressure generating unit 47, or may be additional pressure applied to the syringe pump. The pressure generating unit 47 receives the instruction, and the bubble forming unit 200 proceeds to the process at S320. In the second and subsequent S320, the pressure generating unit 47 performs an operation of an amount corresponding to the operation amount.

[0150] FIG. 10B shows an example of a flow in which the gas-liquid interface 255 is expanded based on the internal pressure in the nozzle 49.

[0151] Step S370 may be the same as step S320. After completing S370, the air bubble forming unit 200 advances the process to S380.

[0152] Next, in S380, the sensor unit 48 measures the internal pressure in the nozzle 49 and sends the measured value of the internal pressure in the nozzle 49 to the bubble formation unit 200. Note that instead of the sensor unit 48, the nozzle actuator 40 may measure the internal pressure in the nozzle 49 and send the measured value of the internal pressure to the bubble formation unit 200.

[0153] Next, in S390, the bubble formation unit 200 determines whether the measured value of the internal pressure in the nozzle 49 is within a preset internal pressure range. If the measured value of the internal pressure is outside the set internal pressure range, the bubble formation unit 200 proceeds to S392; otherwise, the bubble formation unit 200 proceeds to S400. For example, the bubble formation unit 200 may calculate the difference between the preset internal pressure and the measured internal pressure in the nozzle 49, and if the difference is equal to or greater than a threshold, determine that the set internal pressure has not been reached.

[0154] In S392, the bubble formation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47 (for example, the distance to push or pull the plunger of the syringe pump) in order to achieve the set internal pressure in the nozzle 49. The bubble formation unit 200 sends an instruction to the pressure generation unit 47 to operate by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S390.

[0155] The operation amount may be the operation amount of the actuator of the pressure generating unit 47, or may be additional pressure applied to the syringe pump. The pressure generating unit 47 receives the instruction, and the bubble forming unit 200 proceeds to the process at S370. In the second and subsequent S370, the pressure generating unit 47 performs an operation according to the operation amount.

[0156] In S400, the gas-liquid interface manipulation unit 101 executes a manipulation on the manipulation target 35. For example, the bubble generation unit 200 sends an instruction to the gas-liquid interface manipulation unit 101 to execute a manipulation on the manipulation target 35 based on an instruction received via the input unit 180. In S400, the step of executing the manipulation includes steps S410 to S460, as shown in FIG. 11A . For example, the manipulation may be removal of unwanted cells, recovery or movement of cell membranes and / or cell membranes, cell recovery, cell retention, or cell compression.

[0157] Fig. 11A is an example of a flow for executing an operation on an operation object 35. Fig. 11A illustrates an example in which the operation object 35 is a cell. Note that the operation object 35 is not limited to a cell, and may be another living organism.

[0158] First, in S410, if an instruction to remove unnecessary cells was received in S140, the information processing device 170 advances the process to S412. In S410, if an instruction not to remove unnecessary cells was received in S140, the information processing device 170 advances the process to S420.

[0159] In S412, the bubble forming unit 200 causes the cells to adhere to the formed bubbles and then removes them.

[0160] For example, the bubble formation unit 200 sends a command to the nozzle actuator 40 to move the nozzle 49 in the x, y, and z directions to the position where the target cell is located. After the nozzle actuator 40 moves the nozzle 49 to the target position, the bubble formation unit 200 may move the nozzle 49 and / or the stage to bring the gas-liquid interface 255 of the bubble into contact with the cell.

[0161] As an example, nozzle actuator 40 or sample actuator 41 identifies the location of the target cell from an image of the target cell captured by camera 60 or camera 70, and moves nozzle 49 to align the center of nozzle 49 with the target position. After the cell contacts gas-liquid interface 255 of the bubble, nozzle actuator 40 may collect the target cell in flow channel 51, as shown by 290a to 290c in Figure 4, and liquid storage unit 54 may discard the cell in a liquid disposal unit of liquid storage unit 54.

[0162] As another example, the bubble-forming unit 200 may form an air-liquid interface 255 of a predetermined size in the step and substep of S300, and control the expansion of the air-liquid interface 255 to cause the cells to adhere to and detach from the air-liquid interface 255. After the liquid control unit 260 removes the unnecessary cells, the bubble-forming unit 200 advances the process to S500.

[0163] In S420, if an instruction to collect cytoplasm and / or cell membrane was received in S140, the bubble-forming unit 200 advances the process to S422, otherwise, the process advances to S430.

[0164] In S422, the bubble-forming unit 200 uses the formed bubbles to separate the cytoplasm and / or cell membrane, attaching them to the bubbles and recovering them. For example, the bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles at the tip of the flow path 51 and attach the target cells to the bubbles. Next, the bubble-forming unit 200 compresses the cells with the bubbles to separate only the cytoplasm and / or cell membrane, and attaches them to the bubbles. For example, the bubble-forming unit 200 controls the pressure-generating unit 47 to increase the internal pressure of the bubbles, expand the bubbles, or control the nozzle actuator 40 to move the nozzle 49 toward the cells, thereby pressing the bubbles against the cells and compressing them. Then, the bubble-forming unit 200 moves a portion of the cells that has bulged outward due to the compression so as to separate it from the cells at the air-liquid interface, thereby separating the cytoplasm and / or cell membrane from the cells. As a result, the bubble forming unit 200 controls the nozzle actuator 40 or the pressure generating unit 47 to cut off the necessary cytoplasm and / or cell membrane of the manipulation target 35.

[0165] For example, the bubble generation unit 200 may send an instruction to the nozzle actuator 40 to move the nozzle 49 in the x, y, and z directions from its initial position to the location where the target cell is located. After the nozzle actuator 40 moves the nozzle 49 to the target position, the bubble generation unit 200 may use the nozzle actuator 40 or the sample actuator 41 to move the nozzle 49 and / or the stage to bring the gas-liquid interface 255 of the bubble into contact with the cell. As an example, the bubble generation unit 200 may identify the location of the target cell from an image of the target cell captured by the camera 60 or the camera 70, and then control the nozzle actuator 40 to move the nozzle 49 so that the center of the nozzle 49 is aligned with the target position. Here, the location of the target cell may be identified by an operator. In this case, the bubble generation unit 200 may receive input from the input unit 180 from the operator regarding the location of the target cell and identify the location.

[0166] It is known that cell membranes have portions that exhibit relatively soft physical properties and portions that exhibit relatively hard physical properties due to differences in the lipid composition of the membrane components. The bubble-generating unit 200 controls the nozzle actuator 40 or the pressure-generating unit 47 to compress cells with bubbles. Here, the compression of cells with bubbles may be achieved by the bubble-generating unit 200 forming a predetermined size of an air-liquid interface 255 in step and substep S300 and controlling the expansion of the air-liquid interface 255 to adhere the cells to the air-liquid interface 255 and compress the cells. Next, by utilizing the outward expansion of the relatively soft portion of the cell membrane, the expanded portion may be attached using the air-liquid interface 255 and moved away from the cell to detach the cell membrane. The cell membrane may then be collected in the flow channel 51 while still attached to the air-liquid interface 255. Furthermore, when the cell membrane is cut off in this manner, the cell membrane swells due to being pushed by the cytoplasm from the inside, so the cut cell membrane contains cytoplasmic components inside, which can be collected in the flow channel 51. After the nozzle actuator 40 has cut off the necessary cytoplasm and / or cell membrane portions, the bubble formation unit 200 proceeds to S434.

[0167] FIG. 11B illustrates the recovery of cytoplasm and cell membrane from cells according to this embodiment. The bubble generator 200 controls the pressure generator 47 and the nozzle actuator 40 to move the relative positions of HeLa cells (human cervical cancer cells) cultured in the solid phase of the container 25 closer to the nozzle 49 so that the cells come into contact with the gas-liquid interface 255 of the bubble (802a). Next, the bubble generator 200 controls the gas-liquid interface 255 to press against the cells, thereby compressing the cells (802b). The soft portion of the cell membrane was observed to bulge outward due to the compression (arrow 802b). Next, the gas-liquid interface 255 was moved away from the cells, detaching the cytoplasm and cell membrane from the bulging portion (arrow 802c). Finally, the detached cytoplasm and cell membrane were attached to the gas-liquid interface 255 and recovered (802d). When performing the operation of FIG. 11B, the operation may be performed by expanding the gas-liquid interface 255, by moving the nozzle 49, or by moving the stage.

[0168] Next, in S434, the bubble formation unit 200 determines whether the instruction received in S140 includes continuously collecting the cytoplasm and / or cell membrane of the operation target 35. If the determination is affirmative, the bubble formation unit 200 proceeds to S435, and if the determination is negative, the bubble formation unit 200 proceeds to S500.

[0169] In S435, the bubble forming unit 200 controls the pressure generating unit 47 to remove the bubbles formed at the end 254 of the nozzle 49. Here, when the bubbles are removed, the manipulation object 35 may be collected at the same time. For example, the manipulation object 35 may be collected in the liquid present in the flow path 51. The steps and substeps of removing the bubbles in S435 may be the same as the steps and substeps of S500, and the details thereof will be described later.

[0170] Next, in S436, the bubble formation unit 200 controls the pressure generation unit 47 and the nozzle actuator 40 to draw in gas to form a new bubble at the end 254 of the nozzle 49. The bubble formation unit 200 sends a command to the nozzle actuator 40 to remove the nozzle 49 from the liquid. After the nozzle actuator 40 removes the nozzle 49 from the liquid, the pressure generation unit 47 may pull the plunger of the syringe pump to draw in the required amount of gas.

[0171] Next, in S437, the air bubble forming unit 200 controls the pressure generating unit 47 to form a new air bubble at the end 254 of the nozzle 49. The contents of S300 already described may be applied to the step and sub-steps of S437. After the pressure generating unit 47 forms an air bubble at the end 254 of the nozzle 49, the air bubble forming unit 200 advances the process to step S420.

[0172] In S430, the bubble-forming unit 200 determines whether or not an instruction to collect cells has been received in S140. If the determination is affirmative, the bubble-forming unit 200 proceeds to S432, and if the determination is negative, the bubble-forming unit 200 proceeds to S440.

[0173] In S432, the bubble-forming unit 200 controls the pressure generating unit 47 to form bubbles and cause the cells to adhere to the bubbles. The bubble-forming unit 200 may detach the cells adhered to the bubbles from the solid phase as necessary.

[0174] For example, the bubble-forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 in the x, y, and z directions from the initial position to the position where the target cell is present. After the nozzle actuator 40 moves the nozzle 49 to the target position, the bubble-forming unit 200 controls the pressure generating unit 47 to supply gas to the flow channel 51 and form a bubble at the tip of the flow channel 51. Next, the bubble-forming unit 200 may control the nozzle actuator 40 or the sample actuator 41 to move the nozzle 49 or the stage, thereby bringing the gas-liquid interface 255 of the bubble into contact with the cell.

[0175] As an example, the nozzle actuator 40 or the sample actuator 41 identifies the location of the target cell from an image of the target cell captured by the camera 60 or the camera 70, and moves the nozzle 49 to align the center of the nozzle 49 with the target position. After the nozzle actuator 40 or the sample actuator 41 moves the nozzle 49 to the target position, the bubble generator 200 controls the pressure generator 47 to supply gas to the flow channel 51 and form a bubble at the tip of the flow channel 51. Next, the bubble generator 200 may use the nozzle actuator 40 or the sample actuator 41 to move the nozzle 49 and / or the stage to bring the gas-liquid interface 255 of the bubble into contact with the cell. For example, after the cell has been brought into contact with the gas-liquid interface 255 of the bubble, the nozzle actuator 40 may detach the cell as needed by, for example, moving the gas-liquid interface 255.

[0176] After the bubble-forming unit 200 controls the pressure generating unit 47 to form bubbles and attach the cells to the bubbles, the bubble-forming unit 200 proceeds to S434. The steps from S434 onward may be the same as those already described. In this case, in step S434, the continuous collection of material is not limited to only cytoplasm or only cells, but may be both cytoplasm and cells.

[0177] FIG. 11C shows how an established cell line is attached to a bubble and then detached according to this embodiment. The bubble-forming unit 200 controls the pressure generating unit 47 and the nozzle actuator 40 (or the sample actuator 41 instead of the nozzle actuator 40) to move the relative positions of the HeLa cells cultured in the solid phase of the container 25 closer to the nozzle 49 so that the cells come into contact with the gas-liquid interface 255 of the bubble (804a). Next, the cells come into contact with the gas-liquid interface 255 of the bubble (804b). Next, the nozzle actuator 40 moves the gas-liquid interface 255 (804c), and the cells are attached to the gas-liquid interface 255 and then detached (804d). Note that the operation shown in FIG. 11C may be performed by expanding the gas-liquid interface 255, by moving the nozzle 49, or by moving the stage.

[0178] FIG. 11D shows a schematic diagram of the process of repeating steps S430, S432, S434, S435, S436, and S437. Continuous collection of cytoplasm and / or cell membranes and continuous collection of cells may be performed as shown in FIG. 11D. At 810a, the nozzle actuator 40 immerses the nozzle 49 in the liquid, and then the bubble generator 200 controls the pressure generator 47 to supply air to a syringe pump (not shown) to form a bubble at the end 254 of the nozzle 49. Next, cells adhering to the bottom of the container 25 are detached by attaching them to the gas-liquid interface 255 of the bubble. The syringe pump then draws gas into the flow channel 51, where they are collected. At 810b, the nozzle actuator 40 lifts the nozzle 49 out of the liquid, and the syringe pump draws gas (e.g., air) into the flow channel 51. Next, in 810c, after the nozzle actuator 40 places the nozzle 49 in the liquid, the syringe pump supplies air to the end 254 of the nozzle 49 to form a new bubble, and the cells adhering to the bottom of the container 25 are collected by adhering to the gas-liquid interface 255 of the bubble. In this way, the bubble-forming unit 200 controls the pressure generating unit 47 and the nozzle actuator 40, allowing two cells (populations) to be continuously collected into the flow channel 51 via the gas without mixing. A photograph showing two cells (populations) actually collected using this method with air between them is also shown. Note that while FIG. 11D illustrates an example in which the nozzle 49 is moved, the operation of FIG. 11D may be performed by moving the stage instead of moving the nozzle 49.

[0179] Figure 11E shows the recovery and passage of established cell lines using this embodiment. By controlling the pressure generating unit 47 and the nozzle actuator 40 using the bubble generating unit 200, established cell lines HeLa cells (human cervical cancer cells, 820a), HT29 cells (human colon cancer cells, 820b), and KatoIII cells (human gastric signet ring cell cancer cells, 820c) were attached, detached, and recovered from the solid phase of the container 25 using the gas-liquid interface 255 of the gas bubbles. They were then released into culture medium in another container and cultured for 1.5 days (820a and 820b) and 2 days (820c). It was confirmed that all cells proliferated after passage. In other words, this embodiment enabled cell proliferation without damaging cell viability, even when the established cell lines were detached using the gas-liquid interface 255 of the gas bubbles. When the operation of FIG. 11E is performed, the operation may be performed by moving the nozzle 49 or by moving the stage.

[0180] FIG. 11F shows the iPS cells recovered and passaged according to this embodiment. The bubble generator 200 controls the pressure generator 47 and the nozzle actuator 40 to attach, detach, and recover a colony of cultured iPS cells from the solid phase of the container 25 using the gas-liquid interface 255 of the bubbles. The colony was then released into a liquid medium in another container and cultured for four days, after which a transmission image was observed. For comparison, iPS cells passaged using a conventional cell passage method (mechanical passage) were used. No morphological differences were observed between the iPS cells (830a) of this embodiment and the iPS cells (830b) of the comparative example. After 10 days of culture, the iPS cells of this embodiment and the comparative example were stained with alkaline phosphatase. Furthermore, the iPS cells of this embodiment and the comparative example were passaged three times and cultured for 30 days, after which the iPS cells of this embodiment and the comparative example were stained with alkaline phosphatase. As a result, no difference in staining was observed between the iPS cells of this embodiment (831a, cultured for 10 days; 832a, cultured for 30 days) and the iPS cells of the comparative example (831b, cultured for 10 days; 832b, cultured for 30 days). It is known that alkaline phosphatase is highly expressed in undifferentiated iPS cells that maintain self-renewal capacity. In other words, according to this embodiment, even when iPS cells were detached and collected using air-liquid interface 255, the maintenance of undifferentiated capacity was not affected, and the iPS cells were able to proliferate while maintaining their undifferentiated state. The operation of FIG. 11F may be performed by moving nozzle 49 or by moving the stage.

[0181] FIG. 11G shows the recovery and analysis of established cell lines using this embodiment. HeLa cells, an established cell line, were detached and recovered from the solid phase of the container 25 using the gas-liquid interface 255 of the gas bubbles. The bubble generator 200 controlled the pressure generator 47 and the nozzle actuator 40 to select one, four, and eight HeLa cells from the solid phase and detach them using the gas-liquid interface 255 of the gas bubbles. These cells were then recovered together with 7.5 nL of liquid medium (835a). The recovered HeLa cells were then released into 12.5 μL of cell lysis reagent and lysed (835b). cDNA was synthesized from β-actin mRNA in the cell lysate, and PCR was performed (835c). As a result, the amount of cDNA detected was roughly proportional to the number of cells recovered. In other words, this embodiment allows the recovery of one cell or any number of cells using the gas-liquid interface 255 for molecular biological analysis. When the operation of FIG. 11G is performed, the operation may be performed by moving the nozzle 49 or the stage.

[0182] Next, in S440, the bubble formation unit 200 determines whether or not it has received an instruction to hold and image the cell in S140. If the determination is affirmative, the bubble formation unit 200 proceeds to S442, and if the determination is negative, the bubble formation unit 200 proceeds to S450.

[0183] In S442, the bubble-forming unit 200 controls the flow so that cells adhere to the formed bubbles and retain the adhered cells. For example, the bubble-forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 in the x, y, and z directions from its initial position to the location where the target cells are located. After the nozzle actuator 40 moves the nozzle 49 to the target position, the bubble-forming unit 200 controls the pressure generating unit 47 to supply gas to the flow channel 51 and form a bubble at the tip of the flow channel 51. The bubble-forming unit 200 may use the nozzle actuator 40 or the sample actuator 41 to move the nozzle 49 and / or the stage to bring the gas-liquid interface 255 of the bubble into contact with the cells. For example, the nozzle actuator 40 or the sample actuator 41 identifies the location where the target cells are located from an image of the target cells captured by the camera 60 or the camera 70, and moves the nozzle 49 to align the center of the nozzle 49 with the target position. After the cells come into contact with the gas-liquid interface 255 of the bubble, the bubble-forming unit 200 advances the process to S444.

[0184] Here, the location of the target cell may be determined by the operator. In this case, the bubble-generating unit 200 may receive input from the input unit 180 regarding the location of the target cell from the operator and determine the location. Although the above example describes the case where the tip of the bubble is brought into contact with the cell, the bubble may also be brought into contact with the cell by bringing the side of the bubble into contact with the cell. In this case, the nozzle actuator 40 or the sample actuator 41 may move the center of the nozzle 49 to align it with the vicinity of the target cell. Furthermore, for example, after the cell is brought into contact with the gas-liquid interface 255 of the bubble, the nozzle actuator 40 may move the gas-liquid interface 255 to detach the cell as needed.

[0185] Next, in S444, the imaging control unit 171 sends an instruction to the camera 60 or the camera 70 to capture an image of the cells held in the bubbles. The camera 60 or the camera 70 captures an image and sends the image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble forming unit 200.

[0186] After the camera 60 or 70 captures an image of the held cell, the bubble forming unit 200 advances the process to S500.

[0187] FIG. 11H is a schematic diagram showing how an established cell line is held at the gas-liquid interface 255 of a bubble and observed. Suspended or loosely adhered cells move freely in the culture medium even with slight vibrations, making them difficult to observe using a microscope or other devices. In 840a, the bubble generator 200 controls the nozzle actuator 40 to place the end 254 of the nozzle 49 in the liquid medium in the container 25 where the suspended cells (manipulation target 35) are cultured. Next, the pressure generator 47 supplies gas to the flow path 51 to form a bubble at the tip of the nozzle 49, forming the gas-liquid interface 255. The bubble generator 200 controls the nozzle actuator 40 or the pressure generator 47 to attach the suspended cells (the manipulation target 35) to the formed gas-liquid interface 255. Next, in 840b, the pressure generator 47 controls the internal pressure of the bubble to temporarily hold the cells at the gas-liquid interface 255 of the bubble. Next, in 840c, the pressure generating unit 47 draws gas from the flow path 51 to shrink the bubble. The cells held in this state can be observed using a microscope or the like. Alternatively, the pressure generating unit 47 may hold the cells without shrinking the bubble, and the held cells may be observed using a microscope or the like. In this way, according to this embodiment, the cells are held at the gas-liquid interface 255 of the bubble, allowing them to be observed without moving.

[0188] Furthermore, in the case of adherent cells scattered on the solid phase of the container 25, observation using a microscope or the like requires moving the stage to obtain a wide field of view. In 842a, the bubble-forming unit 200 controls the nozzle actuator 40 to place the end 254 of the nozzle 49 in the liquid medium of the container 25 in which the adherent cells (the manipulation target 35) are cultured. Next, the pressure-generating unit 47 supplies gas to the flow path 51 to form a bubble at the tip of the nozzle 49, thereby forming a gas-liquid interface 255. Next, the bubble-forming unit 200 controls the nozzle actuator 40 or the pressure-generating unit 47 to control the gas-liquid interface 255, thereby adhering the cells to the gas-liquid interface 255 and detaching them. Next, in 842b, the pressure-generating unit 47 temporarily holds the cells at the gas-liquid interface 255 of the bubble. Next, in 842c, the pressure-generating unit 47 draws gas from the flow path 51 to shrink the bubble. The cells held in this state are present in a narrow range on the same z-plane, allowing them to be observed simultaneously using a microscope, etc. In this way, by holding the cells at the gas-liquid interface of the bubble, the field of view to be observed can be made smaller.

[0189] As an example of such an observation technique, Kato III cells, which are floating cells, are scattered on a solid phase, and some of the cells are attached to the gas-liquid interface 255 (844a). After that, the bubble generator 200 controls the internal pressure of the bubble via the pressure generator 47, thereby shrinking the bubble and retaining the cell at the gas-liquid interface 255, and when the retained cell is brought into focus, the surrounding cells move out of focus (844b). At this time, when the stage is moved, the surrounding cells move, but the cell retained at the gas-liquid interface 255 does not move, making it easy to observe the retained cell under a microscope (844c).

[0190] In S450, the bubble formation unit 200 determines whether or not an instruction to compress and image the cell has been received in S140. If the determination is affirmative, the bubble formation unit 200 proceeds to S452, and if the determination is negative, the process proceeds to S460.

[0191] In S452, the bubble-forming unit 200 controls the pressure generating unit 47 and the nozzle actuator 40 to compress the cell using a bubble. For example, the bubble-forming unit 200 controls the pressure generating unit 47 to form a bubble at the tip of the flow channel 51 and bring the bubble into contact with the cell to be manipulated 35. When performing the operation of FIG. 11H, the operation may be performed by moving the nozzle 49 or by moving the stage.

[0192] As an example, the bubble formation unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 in the x, y, and z directions from the initial position to the position where the target cell is present. As an example, the bubble formation unit 200 identifies the position where the target cell is present from an image of the target cell captured by the camera 60 or the camera 70, and controls the nozzle actuator 40 to move the nozzle 49 so that the center of the nozzle 49 is aligned with the target position.

[0193] The location of the target cell may be determined by the operator. In this case, the bubble-forming unit 200 may receive input from the input unit 180 regarding the location of the target cell from the operator and determine the location. Furthermore, the bubble may contact the cell by bringing the tip of the bubble into contact with the cell, or by bringing the side of the bubble into contact with the cell. When bringing the tip of the bubble into contact with the cell, the nozzle actuator 40 or the sample actuator 41 may move so that the center of the nozzle 49 is directly above the target cell. When bringing the side of the bubble into contact with the cell, the nozzle actuator 40 or the sample actuator 41 may move so that the center of the nozzle 49 is near the target cell. In this case, the bubble may be formed next to the cell, and the nozzle 49 may be moved to gradually compress the cell from the side.

[0194] Next, after the nozzle actuator 40 moves the nozzle 49 to the desired position, the bubble-forming unit 200 controls the pressure generating unit 47 to supply gas into the flow path 51 and form a bubble at the tip of the flow path 51 at the desired position. The bubble-forming unit 200 may move the nozzle 49 and / or the stage using the nozzle actuator 40 or the sample actuator 41 to bring the gas-liquid interface 255 of the bubble into contact with the cell. If the position of the nozzle 49 is fixed, the gas-liquid interface 255 may be brought into contact with the cell by moving the stage. The bubble-forming unit 200 may control the gas-liquid interface 255 to expand via the pressure generating unit 47, thereby bringing the cell into contact with the gas-liquid interface 255.

[0195] As an example, the bubble-forming unit 200 operates the plunger of the syringe pump of the pressure generating unit 47 at a preset operating amount to form a bubble of a preset volume, and then moves the nozzle 49 and / or the stage using the nozzle actuator 40 or the sample actuator 41 so that the nozzle 49 is positioned at a position where the bubble presses against the cell. Next, the bubble-forming unit 200 may control the pressure generating unit 47 to expand the bubble formed at the tip of the flow channel 51, or control the nozzle actuator 40 to move the nozzle 49 toward the cell and press the bubble against the cell, thereby compressing the cell.

[0196] As another example, the bubble forming unit 200 may move the nozzle 49 very close to the cell, and then control the pressure generating unit 47 to form a bubble of a predetermined volume at the tip of the flow path 51, thereby compressing the cell.

[0197] Next, in S454, the imaging control unit 171 sends an instruction to the camera 60 or the camera 70 to capture an image of the compressed cell. The camera 60 or the camera 70 captures an image and sends the image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the output unit 160.

[0198] Alternatively or additionally, sensor unit 48 or nozzle actuator 40 may measure the pressure with which the bubbles compress the cells and send the measured pressure value to bubble formation unit 200. Camera 60 or 70 may repeatedly capture images while changing the pressure with which bubble formation unit 200 compresses the cells. The pressure with which the cells are compressed can be changed by having bubble formation unit 200 control pressure generation unit 47 to change the internal pressure and / or volume of the bubbles.

[0199] The bubble generator 200 moves the nozzle 49 very close to the cell, then controls the pressure generator 47 to form a bubble at the tip of the flow channel 51. The bubble's internal pressure and / or volume can be varied to compress the entire cell or various parts of the cell while maintaining or varying the pressure applied to the cell. It is expected that various parts of the cell have different hardness depending on the composition and distribution of the cell membrane or intracellular organelles. In this way, the pressure and / or observed image obtained during bubble compression can be used as indicators to analyze the composition and distribution of the cell membrane or intracellular organelles. By compressing the cell, the cell becomes thinner, allowing for clear observation of structures deep within the cell. By expanding laterally, previously adjacent structures become separated and can be observed individually. Observing the cell while compressing it provides information on the force applied to the cell and the amount of morphological change inside and outside the cell, enabling analysis of information on cell mechanics. After the camera 60 or 70 captures an image of the compressed cell, the bubble generator 200 proceeds to S500.

[0200] Figure 11I shows the state of deep cell observation using an established cell line compressed with air bubbles according to this embodiment. The nuclei and cytoplasm of a spheroid (850a) formed from live HT29 cells were stained, and by compressing the spheroid with air bubbles, structures deep within the cell, such as the nucleus, could be observed (850b). Comparing the thick central portion in particular, the nucleus is not visible in the central portion before compression (850a), but is visible in the central portion after compression (850b). Conventionally, to observe structures deep within cells, cells have been fixed with formalin or methanol, thinly sliced, or observed using a specialized microscope specialized for deep observation. According to this embodiment, deep cell structures can be observed without a specialized microscope, even while the cells are still alive. Furthermore, in the spheroid (850a) before compression, the nuclei were closely spaced, making it difficult to identify individual nuclei. However, in the spheroid (850b) observed after compression, the spheroid expanded vertically and horizontally, creating sufficient spacing between the nuclei, allowing for individual recognition of the nuclei. Previously, microscopy systems with improved optical systems and fluorescent labeling methods have been developed to independently recognize two or more closely spaced organelles within a cell, and these systems are known as super-resolution microscopes. While these microscopy technologies increase resolution, they also narrow the field of view and require longer imaging times. According to this embodiment, two or more closely spaced organelles within a cell can be independently recognized in a live state, without the use of specialized microscopes, and within a short imaging time while maintaining the field of view. Furthermore, it is also possible to reconstruct the three-dimensional structure of the original cell from the observed image of the compressed cell and its mechanical information.

[0201] In S460, the bubble-forming unit 200 controls the air-liquid interface operating unit 101 so that necessary operations other than S410 to S450, among the instructions received in S140, are performed on the operation target 35. For example, the operations may be evaluation of cell adhesiveness or induction of cell differentiation, which will be described later. After completing S460, the bubble-forming unit 200 advances the process to S500.

[0202] In S500, the bubble forming unit 200 controls the pressure generating unit 47 to reduce the gas-liquid interface 255. Reducing the gas-liquid interface 255 may include removing the gas bubbles. Here, when reducing or removing the gas bubbles, the operation target 35 may be collected at the same time. In S500, the step of reducing the gas-liquid interface 255 includes steps S510 to S544 as shown in FIG. 12A, or includes steps S560 to S594 as shown in FIG. 12B.

[0203] FIG. 12A shows an example of a flow for reducing the gas-liquid interface 255 based on an image captured of the position of the end 254 of the nozzle 49.

[0204] In S510, the bubble-forming unit 200 controls the pressure generating unit 47 to perform a suction operation on the flow path 51, thereby drawing in the gas-liquid interface 255 from the tip of the flow path 51. At this time, the liquid is also drawn in at the same time. The drawn-in liquid may be used to separate the manipulation target 35 from the gas-liquid interface 255. The drawn-in liquid may be a liquid contained in the container 25 (for example, a culture medium), or may be another liquid stored in the liquid storage unit 54.

[0205] For example, the bubble generation unit 200 sends an instruction to an actuator of the pressure generation unit 47 to pull the plunger of the syringe pump by a preset distance or until a preset pressure is reached. Upon receiving an instruction from the volume control unit or the suction control unit in the bubble generation unit 200, the pressure generation unit 47 sucks in gas. As a result, the gas-liquid interface 255 shrinks (the bubbles are removed), and the gas-liquid interface 255 is taken into the flow path 51. After the gas-liquid interface 255 shrinks (the bubbles are removed), the bubble generation unit 200 proceeds to S520.

[0206] Next, in S520, the flow channel imaging camera 42 captures an image of the end 254 of the nozzle 49 and sends the image to the image processing unit 300. The image processing unit 300 may record the image in the recording unit 190 and / or output the image to the bubble forming unit 200.

[0207] Next, in S530, the bubble generation unit 200 determines, based on the captured image of the end 254 of the nozzle 49, whether the position of the gas-liquid interface 255 captured by the flow path 51 is different from a position previously set in the bubble generation unit 200. If the position is different, the bubble generation unit 200 proceeds to S532; otherwise, the bubble generation unit 200 proceeds to S540. For example, the bubble generation unit 200 may calculate a difference between the position of the gas-liquid interface 255 calculated based on the captured image of the end 254 of the nozzle 49 and the previously set position, and if the difference is equal to or greater than a threshold, the bubble generation unit 200 may determine that the position of the gas-liquid interface 255 captured by the flow path 51 is different from the previously set position.

[0208] In S532, the bubble generation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47. For example, the bubble generation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47 (for example, the distance to push or pull the plunger of the syringe pump) to bring the gas-liquid interface 255 up to a preset position of the gas-liquid interface 255. The bubble generation unit 200 sends an instruction to the pressure generation unit 47 to operate by the determined amount of movement. For example, the bubble generation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S530.

[0209] The operation amount may be the operation amount of the actuator of the pressure generating unit 47, or may be additional pressure applied to the syringe pump. The pressure generating unit 47 receives the instruction, and the bubble forming unit 200 proceeds to the process at S510. In the second and subsequent S510, the pressure generating unit 47 performs an operation of an amount corresponding to the operation amount.

[0210] In S540, if the instruction received in S140 includes detaching cells from the interface (e.g., cell recovery), the bubble formation unit 200 proceeds to S542; otherwise, the bubble formation unit 200 proceeds to S640.

[0211] In S542, the bubble formation unit 200 sends a command to the nozzle actuator 40 to remove the nozzle 49 from the liquid. After the nozzle actuator 40 removes the nozzle 49 from the liquid by moving the nozzle 49 upward by a preset distance, the bubble formation unit 200 proceeds to the process at S544.

[0212] Next, in S544, the bubble-generating unit 200 controls the pressure generating unit 47 to move the gas-liquid interface 255 between the gas and liquid in the flow path 51 at high speed. As a result, cells adhering to the gas-liquid interface 255 detach from the gas-liquid interface 255 and move into the liquid. For example, the bubble-generating unit 200 causes the pressure generating unit 47 to rapidly reciprocate the plunger of the syringe pump, thereby repeatedly supplying and suctioning gas into the flow path 51 and moving the gas-liquid interface 255 at high speed. In addition to or instead of this, the bubble-generating unit 200 may cause the nozzle actuator 40 to reciprocate the nozzle 49 in the vertical direction (±z directions) and / or the vertical and horizontal directions (±xy directions) at high speed, thereby moving the gas-liquid interface 255 in the flow path 51 at high speed.

[0213] The bubble-forming unit 200 may move the gas-liquid interface 255 at high speed on the spot (at the location where S542 was performed), or may move the nozzle 49 by the nozzle actuator 40 after immersing it in the liquid at the designated destination. The bubble-forming unit 200 can appropriately detach cells adhering to the gas-liquid interface 255 from the gas-liquid interface 255 by controlling the moving speed of the liquid based on information such as the internal pressure inside the nozzle 49 received from the sensor unit 48. The bubble-forming unit 200 may also vibrate the gas-liquid interface 255 by forming an electromagnetic field inside the nozzle 49.

[0214] Furthermore, the bubble-forming unit 200 may control the cells to detach from the gas-liquid interface 255 by bringing the bubbles into contact with a filter. The bubble-forming unit 200 may also control the liquid storage unit 54 to add a liquid that reduces the interfacial free energy, thereby detaching the cells from the gas-liquid interface 255. Alternatively, the bubble-forming unit 200 may control the nozzle actuator 40 and the pressure generating unit 47 to form a bubble at the tip of the nozzle 49 at a designated destination, and rub the bubble against the bottom surface of the designated destination container 25, thereby detaching the cells. The bubble-forming unit 200 may also control the pressure generating unit 47 to increase the internal pressure of the bubble, thereby pushing the cells out and detaching them. Alternatively, the destination liquid may be a liquid that reduces the interfacial free energy, thereby detaching the cells from the gas-liquid interface 255. After detaching the cells from the interface, the bubble-forming unit 200 proceeds to S640.

[0215] Furthermore, by changing the type and / or composition of the gas, the effect on the airflow can be controlled. For example, using a heavy gas or a gas with high viscosity makes the airflow less likely to move, but increases the effect on the operation target 35. On the other hand, using a light gas or a gas with low viscosity makes the airflow more likely to move, but decreases the effect on the operation target 35. The gas may be selected taking into consideration the effects of the type and / or composition of the gas.

[0216] FIG. 12B shows an example of a flow in which the gas-liquid interface 255 is reduced based on the internal pressure in the nozzle 49.

[0217] In S560, the bubble generation unit 200 controls the pressure generation unit 47 to perform a suction operation on the flow path 51, thereby drawing in the gas-liquid interface 255 from the tip of the flow path 51. Step S560 may be the same as step S510. Next, the bubble generation unit 200 advances the process to S570.

[0218] Next, in S570, the sensor unit 48 measures the internal pressure in the nozzle 49 and sends the measured value of the internal pressure in the nozzle 49 to the bubble formation unit 200. Note that instead of the sensor unit 48, the nozzle actuator 40 may measure the internal pressure in the nozzle 49 and send the measured value of the internal pressure to the bubble formation unit 200.

[0219] Next, in S580, the bubble formation unit 200 determines whether the measured value of the internal pressure in the nozzle 49 is within a preset internal pressure range. If the measured value of the internal pressure is outside the preset internal pressure range, the bubble formation unit 200 proceeds to S582; otherwise, the bubble formation unit 200 proceeds to S590. For example, the bubble formation unit 200 may calculate the difference between the preset internal pressure and the measured internal pressure in the nozzle 49, and if the difference is equal to or greater than a threshold, determine that the set internal pressure has not been reached.

[0220] In S582, the bubble formation unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generation unit 47 (for example, the distance to push or pull the plunger of the syringe pump) in order to realize the set internal pressure inside the nozzle 49. The bubble formation unit 200 sends an instruction to the pressure generation unit 47 to operate by the determined amount of movement. For example, the bubble formation unit 200 may determine the amount of movement according to the magnitude of the difference calculated in S580.

[0221] The operation amount may be the operation amount of the actuator of the pressure generating unit 47, or may be additional pressure applied to the syringe pump. The pressure generating unit 47 receives the instruction, and the bubble forming unit 200 proceeds to the process at S560. In the second and subsequent S560, the pressure generating unit 47 performs an operation of an amount corresponding to the operation amount.

[0222] In S590, if the instruction received in S140 includes detaching cells from the air-liquid interface 255 (e.g., cell recovery), the bubble-forming unit 200 proceeds to S592. Steps S590 to S594 may be the same as steps S540 to S544. After S594, the bubble-forming unit 200 proceeds to S640. If the instruction received in S140 does not include detaching cells from the air-liquid interface 255, the bubble-forming unit 200 proceeds to S640.

[0223] Next, in S640, the information processing device 170 receives an input regarding the release of the operation target 35 from the operator via the input unit 180. If the information processing device 170 has received an instruction to release the operation target 35, the information processing device 170 proceeds to S645; otherwise, the information processing device 170 proceeds to S650.

[0224] In S645, the bubble generation unit 200 may send an instruction to the nozzle actuator 40 regarding the destination of the recovered control object 35. For example, the destination of the control object 35 may be specified by the operator in the display area of ​​the GUI, as shown in FIG. 7B. The nozzle actuator 40 may immerse the nozzle 49, including the control object 35 that has adhered to or detached from the gas-liquid interface 255, in the destination liquid and release the cell into the destination liquid. After the nozzle actuator 40 releases the recovered cell into the destination liquid, the bubble generation unit 200 proceeds to S650. The cells released into the destination liquid may be observed using the microscope unit 50.

[0225] In S650, if there is another operation target 35, the bubble formation unit 200 advances the process to S660. In S650, if there is no other operation target 35, the bubble formation unit 200 advances the process to S680.

[0226] In S660, if the nozzle 49 needs to be replaced in order to operate another operation object 35, the bubble formation unit 200 advances the process to S670, and if the nozzle 49 does not need to be replaced, the process advances to S200.

[0227] In S670, the flow path control unit 250 sends an instruction to the flow path exchanging unit 53 to remove the nozzle 49 attached to the nozzle actuator 40 and discard it in a nozzle discarding unit of the flow path exchanging unit 53. Note that the nozzle may be stored with the cells taken up in the nozzle 49 without being released, and the taken-up cells may be analyzed later. In this case, the nozzle 49 may be stored in the nozzle storing unit of the flow path exchanging unit 53 without being discarded. After the flow path exchanging unit 53 discards the nozzle 49, the process proceeds to S180.

[0228] In S680, the nozzle 49 may be discarded in the same procedure as in S670. The flow path exchange unit 53 discards the nozzle 49, and the flow ends.

[0229] In the above flow, removal of unnecessary cells, recovery of cytoplasm and / or cell membrane, recovery and subculture of cells, retention of cells, and compression of cells are described as examples of manipulating the manipulation object 35. There are several other examples of manipulations in addition to those listed above.

[0230] One example of the operation is to apply a culture substrate or a drug to the solid phase on the bottom of the container 25 and evaluate the adhesiveness of these culture substrates or drugs to cells. The adhesiveness to cells can be evaluated using the internal pressure of the air bubbles when detaching the cells and the moving speed and load of the nozzle as indicators, so that the effectiveness of the culture substrate or drug on cell adhesion can be evaluated.

[0231] Another example of the operation is cell sorting. Cells are compressed with air bubbles according to the flow chart described above. It is conceivable that the cell membrane, intracellular components, or physical properties may differ depending on the type of cell. Therefore, it is conceivable that the process of cell shape change or the shape of the cell may differ after the air bubble generator 200 controls the nozzle actuator 40 and / or the pressure generator 47 to start, stop, and release the compression of the cells with air bubbles. It is also conceivable that, depending on the type of cell, some cells may burst when compressed. These can be used as indicators for cell sorting.

[0232] Another example of the operation is observing the change in shape of a cell during the process of compressing it. Following the flow described above, the cell is compressed with an air bubble. For example, during the process of compressing the cell, the pressure applied to the cell may be changed while compressing the entire cell or various parts of the cell, and the change in cell shape may be imaged.

[0233] Another example of manipulation is the rupture or cutting of cells by compressing them. By applying a large amount of pressure to cells, the cells can be ruptured or cut. By rupturing or cutting cells, it is possible to collect the cell membrane, cytoplasm, and / or organelles, and to cut connections between cells (for example, synapses, which are connections between nerve cells).

[0234] Another example of the operation is the induction of cell differentiation. It is known that differentiation of osteoblasts, muscle cells, endothelial progenitor cells, and the like can be induced by applying mechanical stimuli. Differentiation can be induced for these cells by applying pressure using bubbles with the pressure generating unit 47 according to the above-described flow.

[0235] Another example of such an operation is gene transfer into cells. Following the flow chart described above, the bubble-forming unit 200 uses bubbles to attach a vesicle-like object, such as a cell membrane, to the gas-liquid interface 255. By bringing the object into contact with the cell membrane, the contents of the vesicle are taken up by the cell through membrane fusion. At this time, by encapsulating a gene in the vesicle, the gene can be taken up into the cell. Furthermore, other polymers, not limited to genes, can also be taken up into the cell through the pores. Furthermore, when the bubble-forming unit 200 uses bubbles to compress cells at the gas-liquid interface 255 according to the flow chart described above, tiny gaps tend to form in parts of the membrane as the cells deform. At this time, by adding a gene to the cell culture medium, the gene can be taken up into the cell through the gaps. Furthermore, other polymers, not limited to genes, can also be taken up into the cell through the gaps.

[0236] Another example of the operation is cooperation with an external device such as a cell culture device such as a fermenter or a cell analysis device such as a cell sorter. According to the above-described flow chart, the bubble generator 200 may use bubbles to take cells into the flow channel 51 and release them at a specified position in the linked external device, thereby moving the cells. Alternatively, the flow channel 51 may be directly connected to the external device, so that the cells taken into the flow channel 51 can be sent to the external device and moved.

[0237] Another example of manipulation is emulsion manipulation. An emulsion is a liquid droplet in an oil liquid or an oil droplet in an aqueous solution. To stabilize the formed emulsion, an amphiphilic substance such as a surfactant may be added to the emulsion. The surfactant surrounds the liquid or oil droplets, forming a monolayer at the interface. Such monolayers are also found in some intracellular organelles, such as endosomes and lipid droplets. Following the above flow, the bubble generator 200 may use bubbles to attach the emulsion to the air-liquid interface 255 or further manipulate it.

[0238] Methods for detaching and / or recovering cells include using a special substrate that reacts to temperature or light to locally denature the substrate to detach cells, and using ultrasound to detach cells. However, these methods require a means for recovering cells. However, the method of the present invention does not require a special substrate and is equipped with both a means for detaching and a means for recovering cells. Furthermore, methods for recovering detached cells include using a liquid flow that draws in liquid, such as with an aspirator. However, this method has the potential to simultaneously recover cells and a large amount of liquid, or to entrap cells other than the target cells. However, the method of the present invention captures the air-liquid interface within a nozzle, allowing for the recovery of cells attached to the air-liquid interface. This allows for easy recovery of target cells with a very small amount of liquid, without entrapping cells other than the target cells. Furthermore, while it is known that applying a strong liquid flow when drawing in liquid can have adverse effects on cells, the method of the present invention can avoid such adverse effects.

[0239] 4, a method for detaching the manipulation object 35 from the inner bottom surface of the container 25 will be described in detail below. In this embodiment, the manipulation object 35 is moved along the air current in the air bubble 256 by controlling the air current in the air bubble 256 to which the manipulation object 35 is attached, thereby manipulating the manipulation object 35. This makes it possible to easily retrieve the manipulation object 35, for example, by detaching the manipulation object 35 from the inner bottom surface of the container 25 or by moving the detached manipulation object 35 away from the bottom surface.

[0240] Furthermore, by carrying out the method of this embodiment, it is possible not only to peel off and recover the manipulation object 35 from the inner bottom surface of the container 25, but also to perform other operations, such as, for example, pressing the manipulation object 35 from above and moving the manipulation object 35 to a predetermined location (for example, the center part) of the bubble before observing it, or by moving the manipulation object 35 laterally of the bubble, the gas-liquid interface part to which the manipulation object 35 was attached can be used to manipulate another manipulation object.

[0241] 13 shows an example of a flow for controlling the airflow in the bubble 256 to which the manipulation target 35 is attached in this embodiment. In this embodiment, the processing of S710 to S730 is performed in this order to move the manipulation target 35 along the airflow and manipulate the manipulation target 35. However, the processing of S710 to S730 does not have to be performed in this order. For example, the processing of S710 and the processing of S720 may be performed simultaneously, the processing of S720 and S730 may be performed simultaneously, the processing of S710 and S730 may be performed simultaneously, or the processing of S710 to S730 may be performed simultaneously. The control of the airflow shown in FIG. 13 may be performed during the steps of attaching and / or detaching cells, cytoplasm, or cell membranes to air bubbles 256, as shown in S422, S432, and S442 of FIG. 11A; during the step of taking the cells into nozzle 49, as shown in S435 of FIG. 11A and S500 of FIG. 6; during the step of compressing the cells, as shown in S452 of FIG. 11A; during the step of observing the cells, as shown in S444 and S454; or during other cell manipulation steps, as shown in S460 of FIG. 11A.

[0242] First, in S710, a liquid 261 and an operation target 35 are placed in a container 25, an end 254 of a nozzle 49 is placed in the liquid 261, and a gas is introduced into a flow path 51 of the nozzle 49 by a pump, thereby forming a bubble 256 in the flow path 51 of the nozzle 49 or at the end 254 of the nozzle 49. By forming the bubble 256, a gas-liquid interface 255 between the liquid 261 and the gas is formed. The step of forming the bubble 256 in S710 includes maintaining the bubble 256 formed at the end 254 of the flow path 51 of the nozzle 49. The step of forming the bubble 256 in S710 can be performed by the method shown in the description of FIG. 2B or FIG. 4 above.

[0243] Next, in S720, the manipulation target 35 is attached to the gas-liquid interface 255 of the bubble 256. In this embodiment, the bubble formation unit 200 controls the nozzle actuator 40 to move the flow path 51 of the nozzle 49, or controls the pressure generation unit 47 to change the internal pressure and / or volume of the bubble, thereby moving the gas-liquid interface 255 formed at the end 254 of the nozzle 49 and bringing the gas-liquid interface 255 into contact with the manipulation target 35 attached to the bottom of the container 25. As a result, the manipulation target 35 attached to the solid phase is attached to and / or detached from the gas-liquid interface 255 of the bubble 256. Note that the gas-liquid interface 255 of the bubble 256 to which the manipulation target 35 is attached does not have to be a perfect sphere.

[0244] Next, in S730, an airflow is generated in the bubble 256, and the position of the manipulation target 35 is manipulated by controlling the airflow. In this embodiment, the bubble generation unit 200 functions as a bubble control unit that generates an airflow in a bubble formed by introducing gas into the liquid 261 from the end 254 of the nozzle 49, and manipulates the position of the manipulation target 35 using the airflow. Note that the step of controlling the airflow in S730 may include maintaining the bubble 256 formed at the end 254 of the flow path 51 of the nozzle 49. This makes it easier to control the airflow in the bubble 256, and more reliably controls the position of the attached manipulation target 35.

[0245] 14 shows an example of a method in this embodiment for generating an air current in the bubble 256 and using the air current to manipulate the position of the manipulation target 35. As shown in Fig. 14, methods for generating an air current in the bubble 256 and using the air current to manipulate the position of the manipulation target 35 include a method 911 for generating an air current by moving the nozzle 49, a method 912 for generating an air current by changing the volume of the bubble 256, and a method 913 for generating an air current using a double-tube nozzle. Here, these methods 911 to 913 can be used in combination.

[0246] The method 911 for generating an airflow by moving the nozzle 49 is a method for generating an airflow in the bubble 256 by moving the relative position between the flow path 51 of the nozzle 49 and the liquid 261. The method 911 for generating an airflow by moving the nozzle 49 includes a method 914 for translating the nozzle 49 in a direction parallel to the bottom 25a of the container 25 (horizontal direction), a method 915 for translating the nozzle 49 in a direction perpendicular to the bottom 25a of the container 25 (vertical direction), and a method 916 for rotationally moving the nozzle 49. Here, these methods 914 to 916 can be used in combination. Note that moving the relative position between the flow path 51 and the liquid 261 may include moving the relative position between the flow path 51 and the operation target 35.

[0247] In this embodiment, the bubble forming unit 200 controls the nozzle actuator 40 to move the flow path 51 of the nozzle 49, thereby functioning as a flow path position control unit that controls the airflow in the bubble 256. Note that the bubble forming unit 200 may also function as a stage position control unit that controls an actuator that moves a stage on which a container 25 that contains an operation target 35 is mounted, thereby controlling the airflow in the bubble 256.

[0248] FIG. 15 shows an example of a schematic diagram illustrating a method 914 for moving the nozzle 49 in the horizontal direction in this embodiment. FIG. 15 shows an example in which the nozzle 49 is moved to the left (-Y direction). The relative position between the flow path 51 of the nozzle 49 and the bottom 25a of the container 25 is not limited to a completely horizontal direction (a direction perpendicular to the direction of gravity), and may be moved in a direction within a range of ±20° from the horizontal direction (±Y direction). Even if the nozzle 49 is installed at an angle, the relative position may be moved with the horizontal direction as the reference. The horizontal movement speed of the nozzle 49 is preferably 50 to 500 μm / sec.

[0249] 15(a) shows a cross-sectional view of the nozzle 49 and the bubble 256 in the length direction (Z direction) of the nozzle 49, Fig. 15(b) shows a cross-sectional view of the tip portion (portion near the bottom 25a) of the bubble 256 in a direction parallel to the bottom 25a of the container 25 (XY direction), and Fig. 15(c) shows a cross-sectional view of the middle portion of the bubble 256 in a direction parallel to the bottom 25a of the container 25 (XY direction). In Fig. 15(a) to (c), arrow F1 shown inside the bubble 256 indicates the flow of air current generated inside the bubble 256.

[0250] 15(a), when the nozzle 49 is moved leftward, a counterclockwise airflow F1 is generated inside the air bubble 256 formed at the end 254 of the nozzle 49. This airflow F1 is generated by convection caused by frictional force generated when the gas inside the air bubble 256 moves while in contact with the bottom 25a of the container 25.

[0251] As shown in (b) of Figure 15, when viewed from the +Z direction, this airflow F1 is generated in the +Y direction, which is the direction opposite to the movement direction (-Y direction) of the nozzle 49, at the tip portion (near the bottom portion 25a) of the bubble 256. Because the bubble is a closed system, as shown in (a) of Figure 15, this airflow F1 is generated in the +Z direction, which is the upward direction, behind the movement direction (-Y direction) of the bubble 256, and is generated in the -Z direction, which is the downward direction, ahead of the movement direction (-Y direction) of the bubble 256, and rotates. Similarly, as shown in (c) of Figure 15, the influence of the frictional force of the bottom surface becomes smaller at positions away from the bottom surface, so this airflow F1 describes symmetrical circles in clockwise and counterclockwise directions at the middle portion of the bubble 256.

[0252] In addition, (a) and (b) of Figure 15 show an example in which the nozzle 49 is moved horizontally while the tip of the bubble 256 is in contact with the bottom 25a of the container 25, but the nozzle 49 may also be moved horizontally while the tip of the bubble 256 is not in contact with the bottom 25a of the container 25.

[0253] Fig. 16 shows an example of a schematic diagram illustrating the movement direction of the manipulation object 35 when the nozzle 49 is moved horizontally in this embodiment. Fig. 16(a) shows a partially enlarged view of the vicinity of the bubble 256 in Fig. 15(a), and Fig. 16(b) is a view similar to Fig. 15(b). Figs. 16(a) and (b) show the manipulation object 35 attached to the gas-liquid interface 255. In Figs. 16(a) and (b), the manipulation object 35 before movement is indicated by a dashed circle 35a, and the manipulation object 35 after movement is indicated by a solid circle 35b.

[0254] As shown in (a) and (b) of Figure 16, when the nozzle 49 is moved to the left, an air flow F1 is generated at the tip of the bubble 256 (near the bottom 25a) in the +Y direction, which is opposite to the direction of movement of the nozzle 49, and the object to be operated 35 moves to position 35b in the +Y direction due to the influence of the air flow F1.

[0255] 15 and 16, an airflow F1 is generated at the tip of the bubble 256 in the direction opposite to the direction of movement of the nozzle 49, and the manipulation target 35 attached to the tip of the bubble 256 can be moved in the direction of the airflow F1. Note that when the nozzle 49 is moved to the right in Figures 15 and 16, the direction of the airflow F1 generated inside the bubble 256 and the direction of movement of the manipulation target 35 attached to the tip of the bubble 256 are opposite to the directions shown in Figures 15 and 16.

[0256] FIG. 17 shows an example of a schematic diagram illustrating a method 915 for moving the nozzle 49 in the vertical direction in this embodiment. (a) and (b) of FIG. 17 show an example of moving the nozzle 49 downward (−Z direction), and (c) and (d) of FIG. 17 show an example of moving the nozzle 49 upward (+Z direction). The relative position between the flow path 51 of the nozzle 49 and the bottom 25a of the container 25 may be moved in a direction within a range of ±20° from the vertical direction (±Z direction), rather than being limited to the vertical direction (direction along the direction of gravity). Even if the nozzle 49 is installed at an angle, the relative position may be moved based on the vertical direction. The speed of movement of the nozzle 49 in the vertical direction is preferably 10 to 100 μm / sec.

[0257] 17(a) and (c) show cross-sectional views of the nozzle 49 and the bubble 256 in the length direction (Z direction) of the nozzle 49, and FIG. 17(b) and (d) show cross-sectional views of the tip portion (portion near the bottom portion 25a) of the bubble 256 in a direction parallel to the bottom portion 25a of the container 25 (XY direction). In addition, in FIG. 17(a) to (d), arrows F2 and F3 shown inside the bubble 256 indicate the flow of air currents generated inside the bubble 256.

[0258] 17(a) to 17(d) show an example in which nozzle 49 is moved vertically while the tip of bubble 256 is in contact with bottom 25a of container 25, but nozzle 49 may be moved vertically while the tip of bubble 256 is not in contact with bottom 25a of container 25. Bottom 25a of container 25 may be the surface of the culture substrate.

[0259] As shown in (a) of FIG. 17, when the nozzle 49 is moved downward, an air current F2 is generated inside the air bubble 256 formed at the end 254 of the nozzle 49, rising along the air-liquid interface 255. As shown in (b) of FIG. 17, when viewed from the +Z direction, the air current F2 is directed radially outward from the air bubble 256. This air current F2 is generated mainly by the air bubble 256 being crushed outward. Note that even when the nozzle 49 is moved without the tip of the air bubble 256 contacting the bottom 25a of the container 25, an air current in the same direction as the air current F2 is generated due to deformation of the air bubble shape caused by resistance of the liquid and convection caused by friction with the liquid.

[0260] As shown in (c) of FIG. 17, when the nozzle 49 is moved upward, an air current F3 is generated inside the air bubble 256 formed at the end 254 of the nozzle 49, descending along the air-liquid interface 255. As shown in (d) of FIG. 17, when viewed from the +Z direction, the air current F3 is directed radially inward of the air bubble 256. This air current F3 is generated mainly by the crushed portion of the air bubble 256 attempting to return to its original shape. Note that even when the nozzle 49 is moved without the tip of the air bubble 256 contacting the bottom 25a of the container 25, an air current in the same direction as the air current F3 is generated due to deformation of the air bubble shape caused by resistance of the liquid and convection caused by friction with the liquid.

[0261] FIG. 18 shows an example of a schematic diagram illustrating the movement direction of the manipulation target 35 when the nozzle 49 is moved in the vertical direction in this embodiment. (a) and (c) of FIG. 18 show partially enlarged views of the vicinity of the bubble 256 in (a) and (c) of FIG. 17, and (b) and (d) of FIG. 18 are views similar to (b) and (d) of FIG. 17. (a) and (b) of FIG. 18 show the manipulation target 35 attached to the gas-liquid interface 255. In (a) and (b) of FIG. 18, the manipulation target 35 before movement is indicated by a dashed circle 35a, and the manipulation target 35 after movement is indicated by a solid circle 35b.

[0262] 18(a) and 18(b), when nozzle 49 is moved downward, airflow F2 is generated inside bubble 256 and flows upward along gas-liquid interface 255, causing operation target 35 to move to position 35b in the +Z direction, radially outward from bubble 256, due to the influence of airflow F2. As shown in Fig. 18(c) and 18(d), when nozzle 49 is moved upward, airflow F3 is generated inside bubble 256 and flows downward along gas-liquid interface 255, causing operation target 35 to move to position 35b in the -Z direction, radially inward from bubble 256, due to the influence of airflow F2.

[0263] As described above, by moving the nozzle 49 in the vertical direction, a radially outward airflow F2 or a radially inward airflow F3 is generated inside the bubble 256, and the object to be operated 35 attached to the gas-liquid interface 255 can be moved radially outward or radially inward.

[0264] Also, a method 916 of rotating the nozzle 49 can also generate an airflow inside the bubble 256. For example, when the nozzle 49 is rotated clockwise as viewed from the +Z direction, a counterclockwise airflow is generated in the flow path 51 of the nozzle 49 as viewed from the +Z direction, and a counterclockwise airflow is also generated inside the bubble 256. This airflow can move the manipulation target 35 attached to the gas-liquid interface 255 of the bubble 256 in the direction of the airflow.

[0265] The method 911 for generating an airflow by moving the nozzle 49 may include other forms of movement. For example, the airflow may be generated by vibrating the nozzle 49 horizontally or vertically at a specific frequency, by circularly moving the nozzle 49 around a specific center of rotation, by moving the nozzle 49 in an oblique direction relative to the bottom 25a of the container 25, by tilting the nozzle 49 at a specific angle, or by pendulum-moving the nozzle 49. Furthermore, a combination of these forms of movement may be used.

[0266] Returning to FIG. 14, the method 912 for generating an air flow by changing the volume of the air bubble 256 includes a method 917 for increasing the volume of the air bubble 256 and a method 918 for decreasing the volume of the air bubble 256 .

[0267] The volume of the bubble 256 is changed by a volume control unit, an air supply control unit, and an air intake control unit. When increasing the volume of the bubble 256, the volume control unit or the air supply control unit controls the pump of the pressure generating unit 47 connected to the gas supply flow path, thereby controlling the volume of gas supplied to the gas supply flow path. The volume control unit or the air supply control unit receives information about the amount of gas supplied to the gas supply flow path from the nozzle actuator 40, the pressure generating unit 47, or the sensor unit 48. Furthermore, it is desirable that the pressurization acceleration of the bubble 256 be 0.01 to 10 kPa / second.

[0268] When reducing the volume of the bubble 256, the volume control unit or the suction control unit controls the pump of the pressure generating unit 47 connected to the gas recovery channel, thereby controlling the volume of gas sucked from the gas recovery channel. The volume control unit or the suction control unit receives information about the amount of gas sucked from the gas recovery channel from the nozzle actuator 40, the pressure generating unit 47, or the sensor unit 48. The decompression acceleration of the bubble 256 is preferably 0.005 to 5 kPa / sec. The same pump or different pumps may be connected to the gas supply channel and the gas recovery channel.

[0269] Fig. 19 shows an example of a schematic diagram illustrating a method 917 for increasing the volume of the bubble 256 and a method 918 for reducing the volume of the bubble 256 in this embodiment. (a) and (b) of Fig. 19 show an example of increasing the volume of the bubble 256, and (c) and (d) of Fig. 19 show an example of reducing the volume of the bubble 256.

[0270] 19(a) and (c) show cross-sectional views of the nozzle 49 and the bubble 256 in the length direction (Z direction) of the nozzle 49, and FIG. 19(b) and (d) show cross-sectional views of the tip portion (portion near the bottom portion 25a) of the bubble 256 in a direction parallel to the bottom portion 25a of the container 25 (XY direction). In addition, in FIG. 19(a) to (d), arrows F4 and F5 shown inside the bubble 256 indicate the flow of air currents generated inside the bubble 256.

[0271] Although (a) to (d) of Figure 19 show an example in which the volume of the bubble 256 is changed while the tip of the bubble 256 is in contact with the bottom 25a of the container 25, the volume of the bubble 256 may also be changed while the tip of the bubble 256 is not in contact with the bottom 25a of the container 25.

[0272] 19(a), when the volume of the bubble 256 is increased, an air current F4 that rises along the gas-liquid interface 255 is generated inside the bubble 256 formed at the end 254 of the nozzle 49. As shown in FIG. 19(b), when viewed from the +Z direction, the air current F4 is directed radially outward from the bubble 256. This air current F4 is generated when the gas supplied from the pump descends through the center of the flow path 51, and the gas around the gas-liquid interface 255 is pushed back upward along the gas-liquid interface 255.

[0273] 19(c), when the volume of the bubble 256 is reduced, an airflow F5 descending along the gas-liquid interface 255 is generated inside the bubble 256 formed at the end 254 of the nozzle 49. As shown in FIG. 19(d), when viewed from the +Z direction, the airflow F5 is directed toward the radially inward direction of the bubble 256. This airflow F5 is generated when the gas sucked into the pump rises through the center of the flow path 51, causing the gas around the gas-liquid interface 255 to return downward along the gas-liquid interface 255.

[0274] Fig. 20 shows an example of a schematic diagram illustrating the movement direction of the manipulation object 35 when the volume of the bubble 256 is changed in this embodiment. (a) and (c) of Fig. 20 show enlarged partial views of the vicinity of the bubble 256 in (a) and (c) of Fig. 19, and (b) and (d) of Fig. 20 are views similar to (b) and (d) of Fig. 19. (a) and (b) of Fig. 20 show the manipulation object 35 attached to the gas-liquid interface 255. In (a) and (b) of Fig. 20, the manipulation object 35 before movement is indicated by a dashed circle 35a, and the manipulation object 35 after movement is indicated by a solid circle 35b.

[0275] 20(a) and (b), when the volume of bubble 256 is increased, an air current F4 that rises along gas-liquid interface 255 is generated inside bubble 256, and therefore, operation target 35 moves under the influence of air current F4 to position 35b in the +Z direction, radially outward of bubble 256. As shown in FIG. 20(c) and (d), when the volume of bubble 256 is reduced, an air current F5 that descends along gas-liquid interface 255 is generated inside bubble 256, and therefore, operation target 35 moves under the influence of air current F5 to position 35b in the -Z direction, radially inward of bubble 256.

[0276] As described above, by changing the volume of the bubble 256, a radially outward airflow F4 or a radially inward airflow F5 can be generated inside the bubble 256, and the object to be operated 35 attached to the gas-liquid interface 255 can be moved radially outward or radially inward.

[0277] 21 shows an example of a schematic diagram illustrating a method 913 for generating an airflow using a double-tube nozzle 49a in this embodiment. In this embodiment, the double-tube nozzle 49a has a double-tube structure including an inner tube 49b and an outer tube 49c. The double-tube nozzle 49a has a flow path 51a formed inside the inner tube 49b and a flow path 51b formed between the inner tube 49b and the outer tube 49c.

[0278] In this embodiment, flow path 51a and flow path 51b are used to control the circulation of gas within double-tube nozzle 49a. Flow path 51a and flow path 51b function as a gas supply flow path that supplies gas or a gas recovery flow path that recovers gas. Figures 21(a) and (b) show a control example in which gas is supplied from flow path 51a and recovered by flow path 51b, and Figures 21(c) and (d) show a control example in which gas is supplied from flow path 51b and recovered by flow path 51a.

[0279] The supply of gas to flow path 51a or flow path 51b is controlled by a volume control unit or an air supply control unit. The volume control unit or air supply control unit controls the pump of pressure generating unit 47 connected to flow path 51a or flow path 51b, which is a gas supply flow path, thereby controlling the volume of gas supplied to flow path 51a or flow path 51b. The recovery of gas from flow path 51a or flow path 51b is controlled by a volume control unit or an air intake control unit. The volume control unit or an air intake control unit controls the pump of pressure generating unit 47 connected to flow path 51a or flow path 51b, which is a gas recovery flow path, thereby controlling the volume of gas taken in from flow path 51a or flow path 51b.

[0280] 21(a) and (c) show cross-sectional views of the nozzle 49 and the bubble 256 in the length direction (Z direction) of the nozzle 49, and FIG. 21(b) and (d) show cross-sectional views of the tip portion (portion near the bottom portion 25a) of the bubble 256 in a direction (XY direction) parallel to the bottom portion 25a of the container 25. In addition, in FIG. 21(a) to (d), arrows F6 and F7 shown inside the bubble 256 indicate the flow of air currents generated inside the bubble 256.

[0281] Although (a) to (d) of Figure 21 show an example in which gas is supplied and recovered using flow path 51a and flow path 51b when the tip of bubble 256 is in contact with bottom 25a of container 25, gas may also be supplied and recovered using flow path 51a and flow path 51b when the tip of bubble 256 is not in contact with bottom 25a of container 25.

[0282] 21(a), when control is performed to supply gas from flow path 51a, an air current is generated in which gas supplied from the upper end of flow path 51a passes through air bubble 256 and flows toward the upper end of flow path 51b. In this case, an air current F6 is generated inside air bubble 256 formed at end 254 of nozzle 49, rising along air-liquid interface 255. As shown in FIG. 21(b), when viewed from the +Z direction, air current F6 is directed radially outward from air bubble 256.

[0283] 21(c), when control is performed to supply gas from flow path 51b, an air current is generated in which gas supplied from the upper end of flow path 51b passes through air bubble 256 and flows toward the upper end of flow path 51a. In this case, an air current F7 is generated inside air bubble 256 formed at end 254 of nozzle 49, descending along air-liquid interface 255. As shown in FIG. 21(d), when viewed from the +Z direction, air current F7 is directed toward the radially inward direction of air bubble 256.

[0284] Figure 22 shows an example of a schematic diagram illustrating the movement direction of the manipulation target 35 when using the double-tube nozzle 49a in this embodiment. Figures 22(a) and (c) show partially enlarged views of the vicinity of the bubble 256 in Figures 21(a) and (c), and Figures 22(b) and (d) are views similar to Figures 21(b) and (d). Figures 22(a) and (b) show the manipulation target 35 attached to the gas-liquid interface 255. In Figures 22(a) and (b), the manipulation target 35 before movement is indicated by a dashed circle 35a, and the manipulation target 35 after movement is indicated by a solid circle 35b.

[0285] 22(a) and 22(b), when control is performed to supply gas from flow path 51a, an air current F6 that rises along gas-liquid interface 255 is generated inside bubble 256, and therefore operation target 35 moves under the influence of air current F6 to position 35b in the +Z direction, radially outward from bubble 256. As shown in Fig. 22(c) and 22(d), when control is performed to supply gas from flow path 51b, an air current F7 that descends along gas-liquid interface 255 is generated inside bubble 256, and therefore operation target 35 moves under the influence of air current F7 to position 35b in the -Z direction, radially inward from bubble 256.

[0286] As described above, by controlling the circulation of the gas inside the double-tube nozzle 49a, a radially outward airflow F6 or a radially inward airflow F7 can be generated inside the bubble 256, and the object to be operated 35 attached to the gas-liquid interface 255 can be moved radially outward or radially inward.

[0287] As described above, by generating air currents F1 to F7 in the bubble 256, the position of the manipulation target 35 can be freely moved horizontally and / or vertically. Moving the position of the manipulation target 35 horizontally and / or vertically provides the following advantages. For example, by moving the position of the manipulation target 35 upward, the manipulation target 35 no longer comes into contact with the bottom 25a of the container 25, eliminating friction when moving the manipulation target 35. This improves the operability of, for example, the attachment and / or detachment of the manipulation target 35 in steps S422, S432, and S442 of FIG. 11A and the introduction of the manipulation target 35 into the nozzle 49 in step S435 of FIG. 11A and step S500 of FIG. 6. Furthermore, by moving the position of the manipulation target 35 upward, the manipulation target 35 is collected at the end 254 of the nozzle 49, allowing the gas-liquid interface 255 at the tip of the bubble 256 to be used as the attachment surface for the manipulation target 35. By moving the position of the manipulation object 35 upward, the manipulation object 35 is moved to the nozzle 49 end of the bubble 256, whereby the manipulation objects 35 collide with each other and can be removed from the gas-liquid interface 255. Therefore, when the manipulation object 35 is released in S645 of FIG. 6, the success rate of releasing the manipulation object 35 is improved.

[0288] By moving the position of the manipulation object 35 downward, the manipulation object 35 can be moved to the tip of the bubble 256, making it easier to compress the manipulation object 35. For example, this makes it easier to compress and / or observe the cell in S452 and S454 of FIG. 11A . By moving the position of the manipulation object 35 downward, the manipulation object 35 can be moved to the tip of the bubble 256, making it easier to observe the manipulation object 35 when observing it in S444. By moving the position of the manipulation object 35 downward, the manipulation object 35 can be moved to the tip of the bubble 256, making it possible to bring the manipulation object 35 into contact with the bottom 25a of the container 25. By moving the position of the manipulation object 35 downward, the manipulation object 35 can be moved to the tip of the bubble 256, making it less likely that the manipulation object 35 will come into contact with the wall surface of the container 25 or the inner wall of the flow channel 51 during collection. 6, the success rate of capturing the manipulation object 35 is improved. By moving the position of the manipulation object 35 downward, the manipulation object 35 is moved to the tip of the bubble 256, causing the manipulation objects 35 to collide with each other and be removed from the gas-liquid interface 255. Therefore, the success rate of releasing the manipulation object 35 is improved when releasing the manipulation object 35 in S645 of FIG. 6. By moving the position of the manipulation object 35 downward, the manipulation object 35 can be brought into contact with and / or pressed against other manipulation objects that are not attached to the bubble present in the liquid 261.

[0289] By moving the position of the manipulation target 35 in the horizontal direction, the manipulation target 35 can be moved to a certain region, and the gas-liquid interface 255 where the manipulation target 35 is not present can be used for attachment of the manipulation target 35. Therefore, when attaching the manipulation target 35 to the bubble 256 in S422, S432, and S442 of FIG. 11A, efficient attachment can shorten the operation time. By moving the position of the manipulation target 35 in the horizontal direction, the manipulation target 35 can be moved to a certain region, causing the manipulation targets 35 to collide with each other and be detached from the gas-liquid interface 255. Therefore, when releasing the manipulation target 35 in S645 of FIG. 6, the success rate of releasing the manipulation target 35 is improved. By moving the position of the manipulation target 35 in the horizontal direction, the manipulation target 35 can be brought into contact with other manipulation targets that are not attached to the bubbles present in the liquid 261.

[0290] Note that the step of attaching the control target 35 to the gas-liquid interface 255 of the bubble 256 in S720 may include control of moving the gas-liquid interface 255 so that it comes into contact with the specific control target 35. In this case, the bubble forming unit 200 acquires the horizontal positions (XY positions) of the gas-liquid interface 255 and the target control target 35 from an image captured by the camera 60 or 70, and acquires the vertical positions (Z positions) of the gas-liquid interface 255 and the target specific control target 35 from an image captured by the flow channel imaging camera 42, thereby identifying the relative positional relationship between the gas-liquid interface 255 and the specific control target 35. In addition, the sensor unit 48 may acquire the vertical position (Z position) from information on the actuator load, contact or proximity information, and internal pressure of the bubble.

[0291] Next, the bubble formation unit 200 causes the nozzle actuator 40 to move the nozzle 49 and / or the stage based on the identified relative positional relationship, thereby bringing the gas-liquid interface 255 into contact with the target operation object 35. Furthermore, the operator may move the nozzle 49 and / or the stage by inputting the amount of movement of the nozzle 49 and / or the stage based on the identified relative positional relationship, thereby bringing the gas-liquid interface 255 into contact with the specific operation object 35.

[0292] Furthermore, the step of attaching the manipulation target 35 to the gas-liquid interface 255 of the bubble 256 in S720 may be performed while detecting the pressure of the bubble 356. In this case, the sensor unit 48 detects the pressure generated in the bubble 256, and the sensor unit 48 sends this information to the bubble formation unit 200. The bubble formation unit 200 may perform feedback control of the pressure generation unit 47 based on the detected pressure.

[0293] Also, in the step of attaching the operation object 35 to the gas-liquid interface 255 of the bubble 256 in S720, the bubble forming unit 200 may acquire contact images between the gas-liquid interface 255 and the operation object 35 using the cameras 60, 70 and the flow path imaging camera 42, and grasp the contact status between the gas-liquid interface 255 and the operation object 35.

[0294] Furthermore, after the step of controlling the airflow in S730, a step of taking the manipulation object 35 from the liquid 261 into the nozzle 49 may be further included. In this case, the pump sucks in the gas in the bubbles 256 to which the manipulation object 35 is attached, thereby taking the gas-liquid interface 225 into the flow path 51 and taking the manipulation object 35 into the flow path 51, thereby taking the manipulation object 35 from the liquid 261 into the nozzle 49. This recovery step is similar to the processing performed in the step of attaching a cell, cytoplasm, or cell membrane to the bubbles 256 and taking it into the nozzle 49, shown in S435 of FIG. 11A and S500 of FIG. 6.

[0295] 23 is an experimental image showing the movement of the manipulation object 35 due to the movement of the bubble 256 in this embodiment. While the above-described embodiments in which the manipulation object 35 is moved by the air current in the bubble 256 have been described with reference to FIGS. 15 to 22, the manipulation object 35 also moves through the liquid due to the movement of the bubble 256 itself. The manipulation object 35 in FIG. 23 represents a spheroid made from HT29 cells, with the white solid line and white solid arrow indicating the movement direction and distance of the bubble from its original position, and the white dashed line and white dashed arrow indicating the movement direction and distance of the spheroid's bubble 256 from its original position on the air-liquid interface 255. For example, when the operation target 35 is attached to the gas-liquid interface 255 of the bubble 256 as shown in (a) of Fig. 23 and the bubble 256 is moved in the lower left direction as shown in (b) of Fig. 23, the operation target 35 attached to the lower left part of the bubble 256 moves along the gas-liquid interface 255 of the bubble 256 in the direction opposite to the moving direction of the bubble 256 as shown in the figure. At this time, the operation target 35 is affected by the air current in the -Z direction generated ahead of the moving direction of the bubble 256 as shown in (a) of Fig. 15 and remains near the bottom 25a, and moves under the influence of the air current in the direction opposite to the moving direction of the bubble 256 (+Y direction) as shown in (b) of Fig. 15. As shown in (c) of FIG. 23, when the bubble 256 is moved upward, the manipulation target 35 attached to the lower left portion of the bubble 256 moves along the gas-liquid interface 255 of the bubble 256 while rising away from the bottom 25a in the same direction as the movement of the bubble 256. At this time, the manipulation target 35 is influenced by an airflow in the +Z direction occurring behind the movement of the bubble 256 as shown in (a) of FIG. 15, and rises away from the bottom 25a. As shown in (c) of FIG. 15, the manipulation target 35 is influenced by an airflow occurring in the middle of the bubble 256 in the same direction as the movement of the bubble 256 (-Y direction). The movement of the manipulation target 35 away from the bottom 25a can be confirmed by the fact that the manipulation target 35 moves out of focus. Therefore, the nozzle position control unit may control the flow path 51 to move in the opposite direction from the living organism, which is the manipulation target 35, with respect to the central axis of the flow path 51 as a reference when viewed in the vertical direction.

[0296] 23(d) and (e), when the bubble 256 is moved to the right, the manipulation target 35 attached to the lower left portion of the bubble 256 moves to the right along the gas-liquid interface 255 of the bubble 256 as shown, and moves in the same direction as the movement direction of the bubble 256. At this time, since the manipulation target 35 has risen so as to move away from the bottom 25a in the previous stage, it moves under the influence of the air current generated in the middle portion of the bubble 256 in the same direction (-Y direction) as the movement direction of the bubble 256, as shown in FIG. 15(c). Such movement of the manipulation target 35 due to the movement of the bubble 256 can be combined with the movement of the manipulation target 35 due to the air current in the bubble 256.

[0297] FIG. 24 is an experimental image showing the position control of the manipulation object 35 by enlarging the bubble 256 in this embodiment. The manipulation object 35 in FIG. 24 represents a neuron derived from an iPS cell. As shown in FIG. 24(a), the nozzle 49 was adjusted so that the outer side of the inner side of the flow path 51 of the nozzle 49 was positioned above the manipulation object 35. At this time, the bubble 256 was not formed, and the gas-liquid interface 255 of the bubble 256 and the manipulation object 35 were not in contact. As shown in FIG. 24(b), the bubble 256 was enlarged, and the manipulation object 35 was attached to the gas-liquid interface 255 of the bubble 256. At this time, the manipulation object 35 was affected by the airflow in the +Z direction generated by the bubble 256, as shown in FIGS. 20(a) and 20(b), and rose away from the bottom 25a and slightly moved upward (out of focus). As shown in (c) of Figure 24, if the bubble 256 is further enlarged, the object of operation 35 moves further upward (out of focus), and the network connecting the nerve cells that make up the object of operation 35 is severed.As a result, as shown in (d) of Figure 24, the bubble 256 is taken into the flow path 51, and the object of operation 35 can be taken inside the nozzle 49.

[0298] 25 shows an example of the hardware configuration of a computer 1900 functioning as the information processing device 170. The computer 1900 according to this embodiment includes a CPU peripheral unit having a CPU 2000, a RAM 2020, a graphics controller 2075, and a display device 2080, which are interconnected by a host controller 2082, an input / output unit having a communication interface 2030, a hard disk drive 2040, and a CD-ROM drive 2060, which are connected to the host controller 2082 by an input / output controller 2084, and a legacy input / output unit having a ROM 2010, a flexible disk drive 2050, and an input / output chip 2070, which are connected to the input / output controller 2084.

[0299] The host controller 2082 connects the RAM 2020 to the CPU 2000 and graphics controller 2075, which access the RAM 2020 at a high transfer rate. The CPU 2000 operates based on programs stored in the ROM 2010 and RAM 2020 and controls each component. The graphics controller 2075 acquires image data generated by the CPU 2000 or the like in a frame buffer provided in the RAM 2020 and displays the image data on the display device 2080. Alternatively, the graphics controller 2075 may include an internal frame buffer for storing image data generated by the CPU 2000 or the like. The display device 2080 can display various information generated within the information processing device 170 (e.g., images, position information of the operation target 35, etc.).

[0300] The input / output controller 2084 connects the host controller 2082 with the communication interface 2030, hard disk drive 2040, and CD-ROM drive 2060, which are relatively high-speed input / output devices. The communication interface 2030 communicates with other devices via a network, either wired or wirelessly. The communication interface also functions as hardware for communication. The hard disk drive 2040 stores programs and data used by the CPU 2000 in the computer 1900. The CD-ROM drive 2060 reads programs or data from a CD-ROM 2095 and provides them to the hard disk drive 2040 via the RAM 2020.

[0301] The input / output controller 2084 is also connected to the ROM 2010, a flexible disk drive 2050, and a relatively slow input / output device, the input / output chip 2070. The ROM 2010 stores a boot program executed by the computer 1900 at startup and / or programs that depend on the hardware of the computer 1900. The flexible disk drive 2050 reads programs or data from a flexible disk 2090 and provides them to the hard disk drive 2040 via the RAM 2020. The input / output chip 2070 connects the flexible disk drive 2050 to the input / output controller 2084, and also connects various input / output devices to the input / output controller 2084 via, for example, a parallel port, a serial port, a keyboard port, a mouse port, etc.

[0302] The program provided to the hard disk drive 2040 via the RAM 2020 is stored in a recording medium such as a flexible disk 2090, a CD-ROM 2095, or an IC card and provided by the user. The program is read from the recording medium, installed in the hard disk drive 2040 in the computer 1900 via the RAM 2020, and executed by the CPU 2000.

[0303] The program installed in the computer 1900 and causing the computer 1900 to function as the information processing device 170 includes a bubble formation module, an energy control module, and an operation module. These programs or modules may act on the CPU 2000 or the like to cause the computer 1900 to function as the bubble formation unit 200, the liquid control unit 260, or the like.

[0304] The information processing described in these programs is read into the computer 1900 to function as the bubble generating unit 200, the liquid control unit 260, and other specific means in which the software and the various hardware resources described above work together. These specific means then perform calculations or processing of information according to the intended use of the computer 1900 in this embodiment, thereby constructing a specific information processing device 170 according to the intended use.

[0305] For example, when communication is performed between the computer 1900 and an external device, the CPU 2000 executes a communication program loaded onto the RAM 2020 and instructs the communication interface 2030 to perform communication processing based on the processing content described in the communication program. Under the control of the CPU 2000, the communication interface 2030 reads transmission data stored in a transmission buffer area or the like provided on a storage device such as the RAM 2020, the hard disk drive 2040, the flexible disk 2090, or the CD-ROM 2095 and transmits the data to the network, or writes received data received from the network to a reception buffer area or the like provided on the storage device. In this manner, the communication interface 2030 may transfer transmission and reception data to and from the storage device using a DMA (direct memory access) method. Alternatively, the CPU 2000 may transfer transmission and reception data by reading data from the source storage device or the communication interface 2030 and writing the data to the destination communication interface 2030 or the storage device.

[0306] The CPU 2000 also loads all or a necessary portion of files or databases stored in an external storage device such as the hard disk drive 2040, the CD-ROM drive 2060 (CD-ROM 2095), or the flexible disk drive 2050 (flexible disk 2090) into the RAM 2020 by DMA transfer or the like, and performs various processes on the data in the RAM 2020. The CPU 2000 then writes the processed data back to the external storage device by DMA transfer or the like. In such processes, the RAM 2020 can be considered to temporarily hold the contents of the external storage device, and therefore in this embodiment the RAM 2020 and the external storage device, etc. are collectively referred to as a memory, a recording unit, a storage device, etc.

[0307] Here, the storage device or the like stores information necessary for the information processing of the information processing device 170, such as moving image data, as needed, and supplies it to each component of the information processing device 170 as needed.

[0308] In this embodiment, various types of information, such as various programs, data, tables, and databases, are stored in such storage devices and are the subject of information processing. The CPU 2000 can also store part of the RAM 2020 in cache memory and perform read and write operations on the cache memory. Even in this configuration, the cache memory still fulfills part of the functions of the RAM 2020, and therefore, in this embodiment, the cache memory is also included in the RAM 2020, memory, and / or storage device unless otherwise specified.

[0309] Furthermore, the CPU 2000 performs various processes on the data read from the RAM 2020, including the various calculations, information processing, conditional determination, information search and replacement, and the like described in this embodiment, as specified by the instruction sequence of the program, and writes the data back to the RAM 2020. For example, when performing conditional determination, the CPU 2000 determines whether the various variables described in this embodiment satisfy a condition such as being greater than, smaller than, greater than or equal to, less than or equal to, or equal to, compared with other variables or constants, and if the condition is met (or if it is not met), branches to a different instruction sequence or calls a subroutine.

[0310] Furthermore, the CPU 2000 can search for information stored in a file or database in the storage device. For example, if a plurality of entries in which attribute values ​​of a first attribute and attribute values ​​of a second attribute are respectively associated with each other are stored in the storage device, the CPU 2000 can search for an entry in which the attribute value of the first attribute matches a specified condition from among the plurality of entries stored in the storage device, and read the attribute value of the second attribute stored in that entry, thereby obtaining the attribute value of the second attribute associated with the first attribute that satisfies a predetermined condition.

[0311] The above-described programs or modules may be stored in an external recording medium. Examples of recording media that can be used include flexible disk 2090, CD-ROM 2095, optical recording media such as DVDs or CDs, magneto-optical recording media such as MOs, tape media, and semiconductor memories such as IC cards. Alternatively, a storage device such as a hard disk or RAM provided in a server system connected to a dedicated communication network or the Internet may be used as a recording medium, and the programs may be provided to computer 1900 via the network.

[0312] In the present disclosure, the information processing device 170 has been shown to have a CPU 2000 as a processor, but the type of processor is not particularly limited. For example, a GPU, ASIA, FPGA, or the like can be used as the processor as appropriate. Furthermore, in the present disclosure, the information processing device 170 has been shown to have a hard disk drive 2040 as an auxiliary storage device, but the type of auxiliary storage device is not particularly limited. For example, instead of the hard disk drive 2040, or together with the hard disk drive 2040, another storage device such as a solid state drive may be used.

[0313] Although the present invention has been described above using embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the claims that such modifications and improvements can also be included within the technical scope of the present invention.

[0314] It should be noted that the order of execution of each process, such as operations, procedures, steps, and stages, in the devices, systems, programs, and methods shown in the claims, specifications, and drawings is not specifically stated as "before," "prior to," etc., and that the processes can be performed in any order unless the output of a previous process is used in a subsequent process. Even if the operational flow in the claims, specifications, and drawings is described using "first," "next," etc. for convenience, this does not mean that the processes must be performed in this order.

[0315] (Addendum) [Item 1] a bubble formation stage in which the organisms form bubbles in the liquid in which they are immersed; a bio-attachment step of attaching bio-organisms to the bubbles; an airflow control step of generating an airflow in the bubbles and manipulating the position of the organisms by the airflow; A method for operating an organism comprising: [Item 2] The bubble formation step is carried out by immersing the end of the flow channel in the liquid and introducing gas into the liquid from the end; The airflow control step includes generating an airflow in the bubble by moving the relative position of the flow channel and the liquid. The operation method described in item 1. [Item 3] the bubble formation step includes forming and maintaining a bubble at the end of the flow channel; The operation method described in item 2. [Item 4] The airflow control step includes generating an airflow in the bubble by moving the relative position of the flow channel and the liquid in a direction within a range of ±20° from the horizontal direction; The operation method described in item 2 or 3. [Item 5] The airflow control step includes generating an airflow in the bubbles by moving the relative position of the flow path and the liquid in a direction within a range of ±20° from the vertical direction; 5. The method of any one of items 2 to 4. [Item 6] The liquid and the organism are contained in a container; The step of controlling the air flow includes moving the relative position of the flow channel and the liquid while the air bubble is in contact with the bottom of the container; Item 5. The method of any one of items 2 to 5. [Item 7] The airflow control step includes generating an airflow in the air bubble by changing the volume of the air bubble. 7. The method according to any one of items 1 to 6. [Item 8] The airflow control step includes increasing the volume of the air bubble to generate an airflow in the air bubble. The operation method described in item 7. [Item 9] The airflow control step includes reducing the volume of the air bubble to generate an airflow in the air bubble. The operation method described in item 7. [Item 10] The bubble formation step is carried out by immersing the end of the flow channel in the liquid and introducing gas into the liquid from the end; the flow path includes a gas supply flow path that supplies gas and a gas recovery flow path that recovers the gas; the airflow control step includes generating an airflow in which gas supplied from an end of the gas supply passage passes through the air bubbles and flows toward an end of the gas recovery passage; 10. The method according to any one of items 1 to 9. [Item 11] The flow path has a double pipe structure, the gas supply flow path is one of the inner and outer flow paths in the double-pipe structure; The gas recovery flow path is the other of the inner and outer flow paths in the double pipe structure. The operating method described in item 10. [Item 12] The method further comprises recovering the organisms from the liquid after the airflow control step. 12. The method according to any one of items 1 to 11. [Item 13] An organism manipulation device for manipulating an organism, comprising: a flow channel having an end immersed in a liquid in which the organism is immersed and capable of introducing a gas into the liquid; a bubble control unit that generates an air flow in bubbles formed by introducing gas into the liquid from the end and controls the position of the organisms using the air flow; An organism manipulation device comprising: [Item 14] The bubble control unit controls the bubble formed at the end portion so as to maintain the bubble. Item 14. The organism manipulation device according to item 13. [Item 15] The bubble control unit has a flow path position control unit that controls an actuator that moves the flow path, thereby controlling the air flow in the bubble. 15. The organism manipulation device according to item 13 or 14. [Item 16] the bubble control unit has a stage position control unit that controls an actuator that moves a stage on which a container containing a living organism is mounted, thereby controlling the air flow in the bubble; 16. The organism manipulation device according to any one of items 13 to 15. [Item 17] The bubble control unit controls a pump connected to the flow path, and has a volume control unit that controls the volume of bubbles in the liquid. 17. The organism manipulation device according to any one of items 13 to 16. [Item 18] the flow path includes a gas supply flow path that supplies gas and a gas recovery flow path that recovers the gas; The bubble control unit is a gas supply control unit that controls a pump connected to the gas supply flow path, thereby controlling the amount of gas supplied to the gas supply flow path; an intake control unit that controls a pump connected to the gas recovery passage, thereby controlling the amount of gas recovered from the gas recovery passage; having 18. The organism manipulation device according to any one of items 13 to 17. [Item 19] A computer program having instructions therein, The instructions, when executed by a processor or programmable circuit, a processor or programmable circuit a bubble formation stage in which the organisms form bubbles in the liquid in which they are immersed; a bio-attachment step of attaching bio-organisms to the bubbles; an airflow control step of generating an airflow in the bubbles and manipulating the position of the organisms by the airflow; Controlling the operation, including Computer program. [Item 20] a flow channel having an end disposed in a liquid containing the organism, the flow channel being contained in a container; a pump that introduces gas into the flow path to form bubbles; a position control unit capable of changing the position of the container or the flow path, The pump or position control unit attaches the organisms to the gas-liquid interface of the bubbles, the position control unit moves the flow channel in a direction opposite to the living organisms with respect to a central axis of the flow channel as viewed in the vertical direction; Biological body manipulation device. [Explanation of symbols]

[0316] 1. Light source for fluorescence image observation 2 Dichroic mirror 3 Optical deflector 4 relay lenses 5 Dichroic mirror 6 Objective Lenses 7 Condenser Lens 8 Condenser Lens 9 Bandpass Filter 10 Light source for transmission image observation 11 Barrier Filter 12 Projection lens 13 Barrier Filter 14 Projection lens 15 Pinhole 16 light source 17 Light source 25 Container 25a bottom 35 Operation Target 40 Nozzle actuator 41 Sample actuator 42 Flow channel imaging camera 45 Light source 46 Light source 47 Pressure generating section 48 Sensor section 49 nozzles 49a Double-tube nozzle 49b Inner tube 49c outer tube 50 Microscope Section 51 Flow path 51a First flow path 51b Second flow path 53 Flow path exchange section 54 Liquid storage section 60 cameras 70 Camera 100 Biological body manipulation device 101 Operation section 111 Display area 112 Display area 113 Display area 114 Display area 115 Display area 160 Output section 170 Information processing equipment 171 Imaging control unit 180 Input section 190 Recording Department 200 Bubble forming section 250 Flow path control section 251 Pump 251a First Pump 251b Second pump 253 Cylindrical part 253a Outer cylinder 253b Inner cylinder 254 End 255 Air-liquid interface 256 Bubbles 260 Liquid control section 261 Liquid 300 Image processing unit 1900 Computer 2000 CPU 2010 ROM 2020 RAM 2030 Communication Interface 2040 hard disk drive 2050 Flexible Disk Drive 2060 CD-ROM drive 2070 I / O chip 2075 graphics controller 2080 display device 2082 Host Controller 2084 I / O Controller 2090 flexible disk 2095 CD-ROM

Claims

[Claim 1] a bubble formation stage in which the organisms form bubbles in the liquid in which they are immersed; a bio-attachment step of attaching the bio-organisms to the bubbles; an airflow control step of generating an airflow in the air bubbles and manipulating the position of the organism by the airflow; A method for operating an organism comprising:

Images