Method for applying force to a living organism and apparatus for applying force to a living organism

Abstract

In the first embodiment of the present invention, provided is a method of applying a force to an organism, said method comprising: an air-liquid interface formation step for disposing an end of a channel in a liquid, in which the organism is immersed, to thereby form an air-liquid interface between the liquid and air within the channel or at the end thereof; a vector control step for controlling the vector of a force which is to be applied to the organism from the air-liquid interface; and a force application step for applying the force to the organism from the air-liquid interface.

Description

Technical Field

[0001] The present invention relates to a method of applying a force to a living body and a living body force applying device.

Background Art

[0002] In the research of cell biology and the like, among many cells in a culture vessel, specific cells are aspirated. Patent Document 1 discloses a system for assisting the aspiration operation for characteristic cells in a large number of cells. [Patent Document 1] Japanese Patent Application Laid-Open No. 2019-03,0263

[0003] [General Disclosure] In a first aspect of the present invention, a method of applying a force to a living body is provided. The method of applying a force to a living body may include a gas-liquid interface formation step of disposing an end portion of a flow path in a liquid in which the living body is immersed and forming bubbles that form a gas-liquid interface between the liquid and the gas in the flow path or at the end portion. The method of applying a force to a living body may include a vector control step of controlling a vector of the force applied to the living body from the gas-liquid interface. The method of applying a force to a living body may include a force application step of applying a force to the living body from the gas-liquid interface.

[0004] In a second aspect of the present invention, the vector control step may include controlling the magnitude of the force applied to the living body from the gas-liquid interface.

[0005] In a third aspect of the present invention, the vector control step may include controlling the magnitude of the force applied to the living body from the gas-liquid interface by controlling the gas pressure of the gas at the gas-liquid interface.

[0006] In a fourth aspect of the present invention, the gas-liquid interface formation step is performed by immersing an end portion of the flow path in a liquid and introducing gas supplied from a pump from the end portion into the liquid, and the vector control step may include controlling the gas pressure of the gas at the gas-liquid interface by controlling the pressurization of the pump.

[0007] In a fifth embodiment of the present invention, the gas-liquid interface formation step is performed by immersing the end of the flow channel in the liquid and introducing the gas into the liquid from the end, and the vector control step may include preparing a flow channel with an inner diameter corresponding to the magnitude of the force to be applied to the living organism.

[0008] In a sixth embodiment of the present invention, the vector control step may include controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the radius of curvature of the portion of the gas-liquid interface that comes into contact with the organism.

[0009] In a seventh embodiment of the present invention, the vector control step may include controlling the magnitude of the force applied to a living organism from the gas-liquid interface by controlling the surface tension between the gas and the liquid.

[0010] In an eighth embodiment of the present invention, the vector control step may include controlling the magnitude of the force applied to a living organism from the gas-liquid interface by controlling the moving acceleration of the gas-liquid interface.

[0011] In a ninth embodiment of the present invention, the gas-liquid interface formation step may be performed by immersing the end of the flow path in the liquid and introducing the gas into the liquid from the end, and the vector control step may include controlling the acceleration of movement of the gas-liquid interface by controlling the acceleration of movement of the flow path.

[0012] In a tenth embodiment of the present invention, the gas-liquid interface formation step is performed by introducing gas supplied from a pump into the liquid from the end, and the vector control step may include controlling the moving acceleration of the gas-liquid interface by controlling the pressurization acceleration of the pump.

[0013] In an eleventh embodiment of the present invention, the vector control step may include controlling the direction of the force applied to the living organism from the gas-liquid interface.

[0014] In a twelfth embodiment of the present invention, the vector control step may include controlling the direction of the force applied to the organism from the gas-liquid interface by controlling the orientation and direction of movement of the surface of the portion of the gas-liquid interface in contact with the organism.

[0015] In a thirteenth aspect of the present invention, the vector control step may include controlling the orientation of the surface of the portion of the gas-liquid interface that is in contact with the organism by controlling the radius of curvature of the portion of the gas-liquid interface that is in contact with the organism.

[0016] In a fourteenth embodiment of the present invention, the gas-liquid interface formation step is performed by immersing the end of the flow channel in the liquid and introducing the gas into the liquid from the end, and the vector control step may include controlling the orientation of the surface of the portion of the gas-liquid interface that comes into contact with the living organism by controlling the distance between the end and the bottom of a container holding the liquid.

[0017] In a fifteenth embodiment of the present invention, the gas-liquid interface formation step is performed by immersing the end of the flow channel in the liquid and introducing the gas into the liquid from the end, and the vector control step may include preparing a flow channel with an inner diameter corresponding to the direction of the force to be applied to the living organism.

[0018] In a sixteenth embodiment of the present invention, the vector control step may include controlling the direction of the force applied to the living organism from the gas-liquid interface from the gas side to the liquid side of the gas-liquid interface, or from the liquid side to the gas side of the gas-liquid interface.

[0019] In a 17th embodiment of the present invention, the vector control step may include control to move the gas-liquid interface in contact with a predetermined biological body.

[0020] Furthermore, in an eighteenth aspect of the present invention, a biological force application device for manipulating a living organism is provided. The biological force application device may include a liquid in which the living organism is immersed. The biological force application device may include a channel whose end is positioned in the liquid, and in which bubbles forming a gas-liquid interface between the liquid and gas are formed at the end or inside of the channel. The biological force application device may include a pump for supplying gas to the channel or for drawing gas from the channel. The biological force application device may include a vector control unit that controls the operation of the pump, thereby controlling the vector of force applied to the living organism from the gas-liquid interface, and applying force to the living organism through the gas-liquid interface.

[0021] A 19th embodiment of the present invention provides a biological loading device. The biological loading device may include a flow channel in which an end is positioned in a liquid contained in a container. The biological loading device may include a pump that introduces gas into the flow channel to form a gas-liquid interface at the end. The biological loading device may include a position control unit that controls the position of the container or the flow channel. The position control unit may move the flow channel or the container to a position at a first distance, which is a distance at which the gas-liquid interface can contact the bottom of the container, and then move the flow channel to a second position where the distance between the end and the bottom of the container is shorter than the first distance, with the gas-liquid interface formed at the end by the pump in contact with the bottom of the container.

[0022] It should be noted that the above summary of the invention does not enumerate all the necessary features of the present invention. Furthermore, subcombinations of these features may also constitute an invention. [Brief explanation of the drawing]

[0023] [Figure 1A] An example of the device configuration of the biological manipulation device 100 in this embodiment is shown. [Figure 1B] An example of the device configuration of the biological manipulation device 100 in this embodiment is shown. [Figure 2A] An example of a schematic diagram showing the structure of the nozzle 49 in this embodiment is shown. [Figure 2B]An example of a schematic diagram showing the structure of nozzle 49 in this embodiment is shown. [Figure 3A] An example of a schematic diagram showing the structure of nozzle 49 in this embodiment is shown. [Figure 3B] An example of a schematic diagram showing the structure of nozzle 49 in this embodiment is shown. [Figure 4] An example of a schematic diagram showing an example of a method for recovering an organism in this embodiment is shown. [Figure 5] An example of the specific configuration of information processing apparatus 170 in this embodiment is shown. [Figure 6] An example of the flow of a method for operating an organism in this embodiment is shown. [Figure 7A] An example of a GUI image displayed on output unit 160 in this embodiment is shown. [Figure 7B] An example of a GUI image displayed on output unit 160 in this embodiment is shown. [Figure 7C] An example of a GUI image displayed on output unit 160 in this embodiment is shown. [Figure 7D] An example of a GUI image displayed on output unit 160 in this embodiment is shown. [Figure 7E] An example of a GUI image displayed on output unit 160 in this embodiment is shown. [Figure 8] An example of the flow for replacing or adding the liquid of S600 in this embodiment is shown. [Figure 9A] An example of the flow for moving the relative position between nozzle 49 and the cell of S200 in this embodiment is shown. [Figure 9B] An example of the flow for moving the relative position between nozzle 49 and the cell of S200 in this embodiment is shown. [Figure 9C] An example of the flow for moving the relative position between nozzle 49 and the cell of S200 in this embodiment is shown. [Figure 10A] An example of the flow for forming bubbles of S300 in this embodiment is shown. [Figure 10B]An example of the flow for forming bubbles in S300 in this embodiment is shown. [Figure 11A] An example of the flow for performing the operation of S400 in this embodiment is shown below. [Figure 11B] This example shows how to recover cytoplasm and cell membranes from cells in this embodiment. [Figure 11C] This example illustrates how cells attach to and detach from air bubbles in this embodiment. [Figure 11D] An example of a schematic diagram illustrating the method for recovering cells in this embodiment is shown. [Figure 11E] An example of a cell culture obtained through subculturing in this embodiment is shown. [Figure 11F] An example of a cell culture obtained through subculturing in this embodiment is shown. [Figure 11G] An example of the analysis of the recovered cells in this embodiment is shown. [Figure 11H] An example of a schematic diagram showing the retained cells in this embodiment is shown. [Figure 11I] An example of compressed cells in this embodiment is shown. [Figure 12A] This is an example of a flow for removing air bubbles from S500 in this embodiment. [Figure 12B] This is an example of a flow for removing air bubbles from S500 in this embodiment. [Figure 13] This example shows a flow that controls the force vector applied by the gas-liquid interface 255 to the object being manipulated 35 in this embodiment. [Figure 14] This embodiment shows an example of a method for controlling the force vector applied from the gas-liquid interface 255 to the object being manipulated 35. [Figure 15] An example of a schematic diagram illustrating the control of the internal pressure of bubble 256 in this embodiment is shown. [Figure 16] An example of a schematic diagram showing the relationship between the inner diameter of the nozzle 49 and the radius of curvature r of the gas-liquid interface 255 in this embodiment is shown. [Figure 17]An example of a schematic diagram showing the relationship between the distance between the end portion 254 of the nozzle 49 and the bottom portion 25a of the container 25 and the radius of curvature r of the gas-liquid interface 255 in this embodiment is shown. [Figure 18] An example of a schematic diagram illustrating the direction of the force applied from the gas-liquid interface 255 to the object being manipulated 35 in this embodiment is shown. [Figure 19] An example of a schematic diagram illustrating the method 921 for controlling the inner diameter of the nozzle 49 in this embodiment is shown. [Figure 20] An example of a schematic diagram illustrating the method 922 for controlling the distance between the end portion 254 of the nozzle 49 and the bottom portion 25a of the container 25 in this embodiment is shown. [Figure 21] This example shows a schematic diagram illustrating the relationship between the inner diameter of the nozzle 49, the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25, the radius of curvature of the gas-liquid interface 255, the maximum force applied to the cells that are the target of manipulation 35, the magnitude of the downward force component (compressive force), the magnitude of the lateral force component (detachment force), the area of ​​the gas-liquid interface 255 to which the cells that are the target of manipulation 35 can adhere, the controllability of the nozzle 49, the number of target cells, and the cell adhesion relaxation treatment in this embodiment. [Figure 22] This figure illustrates the separation of the object to be operated on 35 from the gas-liquid interface 255 in this embodiment. [Figure 23] An example of a computer hardware configuration is shown. [Modes for carrying out the invention]

[0024] The present invention will be described below through embodiments, but these embodiments are not intended to limit the invention as defined in the claims. Furthermore, not all combinations of features described in the embodiments are necessarily essential to the solution of the invention. In addition, in the drawings, identical or similar parts may be given the same reference numeral to omit redundant descriptions.

[0025] Figure 1A shows an example of the configuration of the biological manipulation device 100 in this embodiment. The biological manipulation device 100 according to the present invention manipulates minute biological organisms such as cells using the interface between a gas and a liquid. For example, the biological manipulation device 100 can perform various operations on biological organisms, such as attaching a biological organism to the interface and then peeling off the biological organism that has adhered to the solid phase. The biological manipulation device 100 comprises a microscope unit 50, a camera 60, a camera 70, an operation unit 101, an output unit 160, an information processing unit 170, and an input unit 180.

[0026] The microscope unit 50 is a device for magnifying and observing or displaying the object to be manipulated 35 using a microscope. The object to be manipulated 35 is a living organism. The living organism may be an organic organism. For example, the living organism may be a cell. For example, the cell may be an animal cell or a plant cell. For example, the cell may be a living cell or a dead cell. Also, for example, the living organism may be a microscopic organism other than a cell. For example, the microscopic organism may be a microorganism, fungus, algae, living tissue, spheroid, etc. The living organism may also contain intracellular organelles.

[0027] The microscope unit 50 includes a light source 1 for fluorescence image observation, a dichroic mirror 2, a light 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 transmission image observation, 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.

[0028] The fluorescence image observation light source 1 is a light source used when observing the working object 35 as a fluorescence image. The working object 35 may be labeled with one or more types of fluorescent substances, or it may not be fluorescently labeled. The fluorescence image observation light source 1 shines light onto the working object 35 to excite or reflect it.

[0029] The light source 10 for transmission image observation is a light source used when observing the object to be operated 35 as a transmission image. The light source 10 for transmission image observation shines light that is transmitted through the object to be operated 35. The light that is transmitted through the object to be operated 35 may pass outside the nozzle or inside the nozzle.

[0030] The configuration of the microscope unit 50 other than that described above will be described later. Note that the microscope unit 50 may have a known configuration, not limited to the examples described above. For example, the configuration of the microscope unit 50 may have the configuration described in Japanese Patent Publication No. 7-13083 or Japanese Patent No. 3814869.

[0031] Camera 60 captures a fluorescence image of the object to be operated 35 and generates an image. The image data generated by camera 60 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, camera 60 may be a camera that captures fluorescence images, but is not limited to this. In the following description, camera 60 will be assumed to be a camera that captures fluorescence images.

[0032] Camera 70 captures a transmitted image of the object to be operated 35 and generates an image. The image data generated by 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, camera 70 may be a camera that captures transmitted images, but is not limited to this. In the following description, camera 70 will be assumed to be a camera that captures transmitted images.

[0033] Cameras 60 and 70 have an image sensor (not shown). Cameras 60 and 70 may be cooled cameras. A cooled camera is a camera that can suppress noise generated by heat by cooling the image sensor. The image sensor may be a CMOS image sensor (Complementary Metal Oxide Semiconductor) or a CCD image sensor (Charge Coupled Device). Cameras 60 and 70 may be housed in a different housing from the microscope unit 50.

[0034] The operating unit 101 manipulates the object to be manipulated 35 using the gas-liquid interface between the gas and the liquid. For example, the operating unit 101 manipulates living organisms (e.g., cells) in the liquid by forming bubbles in the liquid. The operating unit 101 includes all or at least some of the following: a nozzle actuator 40, a sample actuator 41, a flow path imaging camera 42, a light source 45, a light source 46, a pressure generation unit 47, a sensor unit 48, a nozzle 49, a flow path 51, a flow path exchange unit 53, a liquid storage unit 54, a sample lid 58, and a sample lid storage unit 59.

[0035] The nozzle actuator 40 mounts the nozzle 49 via the pressure generating unit 47 and moves the nozzle 49. As will be described later, 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 operable in any of the vertical, horizontal, and up-and-down directions. The nozzle actuator 40 may be operable only in the up-and-down direction. In this case, the vertical and horizontal movement of the nozzle actuator 40 may be controlled by the stage of the microscope unit 50. The nozzle actuator 40 may be operable only in the vertical and horizontal directions. In this case, the up-and-down movement of the nozzle actuator 40 may be controlled by the stage of the microscope unit 50. The nozzle actuator 40 may be fixed and not move. In this case, the vertical, horizontal, and up-and-down movement of the nozzle actuator 40 may be controlled by the stage of the microscope unit 50. The operation of the nozzle actuator 40 is controlled by the nozzle position control unit (not shown) of the bubble forming unit in the information processing device 170.

[0036] 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, or up-and-down directions. The stage may mount a transparent container 25 that houses the object to be operated 35. The container 25 may be a culture vessel filled with liquid. The sample actuator 41 may, but is not limited to, mount one or more containers and / or tubes. The operation of the sample actuator 41 is controlled by a stage position control unit (not shown) in the bubble formation unit of the information processing device 170. The stage may be provided in the operation unit 101 or in the microscope unit 50.

[0037] The flow channel imaging camera 42 images the tip of the nozzle 49. The flow channel imaging camera 42 may also image bubbles formed at the tip of the nozzle 49. The captured images may be sent to the image processing unit in the information processing device 170. Based on the captured images, the bubble formation 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 object to be operated 35. Instead of the flow channel imaging camera 42, a camera 60 or camera 70, etc., may be used to image 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 fluorescence image observation light source 1, the transmission image observation light source 10, light source 16, light source 17, light source 45 and light source 46 as illumination. Light sources 16 and 17 may be ring illuminations, but are not limited to these.

[0038] Light sources 45 and 46 illuminate the nozzle 49 and / or the object to be operated 35. Light sources 45 and 46 may, but are not limited to, ring illumination.

[0039] The pressure generating unit 47 generates 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 come into contact with the liquid, and supplies a preset amount of gas to that end. For example, the pressure generating unit 47 may have a syringe pump and an actuator that reciprocates the plunger of the syringe pump. The actuator may supply gas to the flow path 51 by pushing the plunger of the syringe pump toward the flow path 51, and the actuator may draw gas from the flow path 51 by pulling the plunger of the syringe pump toward the flow path 51. The pressure generating unit 47 is controlled by a bubble forming unit in the information processing device 170.

[0040] The liquid into which the object to be operated 35 is immersed may be, but is not limited to, a complete medium, a basic medium, or a buffer. A complete medium is a medium containing maintenance and growth factors necessary for the maintenance and proliferation of cells. A basic medium is a medium containing only a small amount of proteins, amino acids, or salts. A buffer is a liquid that maintains a pH and osmotic pressure suitable for cell survival. Known liquids, complete media, basic media, and buffers can be used.

[0041] The gas may be air. The gas may also contain moisture.

[0042] The sensor unit 48 has one or more sensors and detects the state of the nozzle 49 and the liquid and gas inside the nozzle 49. For example, the sensor unit 48 may detect the position, velocity 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 generation unit 47, and the position of the syringe pump plunger in the pressure generation unit 47. The sensor unit 48 may detect the ambient temperature and the temperature of the liquid inside the container 25. The sensor unit 48 may detect the humidity of the environment. The sensor unit 48 may also detect the pH of the liquid inside the container 25. The sensor unit 48 may detect the temperature and humidity of the gas inside the nozzle 49. The sensor unit 48 sends this information to the information processing device 170 (for example, the bubble forming unit 200 described later). Known sensors can be used in the sensor unit 48. The sensor unit 48 may be housed in a different housing from the pressure generation unit 47, or it may be housed inside the pressure generation unit 47.

[0043] The nozzle 49 is a device equipped with a flow path 51, which will be described later. The nozzle 49 may be rod-shaped or flat.

[0044] The flow path 51 is through which the liquid and gas being drawn in (intake) or discharged (supply) passes. The flow path 51 is provided inside the nozzle 49, penetrating the nozzle 49 in its longitudinal direction. A pressure generating unit 47 is connected to the other end of the flow path 51.

[0045] The flow path replacement unit 53 is a device for storing and disposing of the nozzles 49. When replacing the nozzles 49, the flow path replacement unit 53 may remove the nozzles 49 attached to the nozzle actuator 40 and dispose of them in the nozzle disposal unit (not shown) of the flow path replacement unit 53, and then attach a nozzle 49 stored in the nozzle storage unit (not shown) of the flow path replacement unit 53 to the nozzle actuator 40 in its place. The flow path replacement unit 53 may be omitted, in which case the nozzles 49 may be replaced by the operator.

[0046] The liquid storage unit 54 is a device for storing the liquid supplied to the container 25 and for recovering and disposing of the liquid from the container 25. When replacing the liquid, the liquid storage unit 54 may recover the liquid contained in the container 25 and dispose of it in the liquid disposal unit (not shown) of the liquid storage unit 54, and replenish the container 25 with the liquid stored in the liquid storage unit (not shown) of the liquid storage unit 54. Liquid replacement may involve replacing the same type of liquid. Liquid replacement may involve replacing different types of liquids. The liquid storage unit 54 may be omitted, in which case the liquid may be replaced by the operator.

[0047] 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 stored in the sample lid storage unit 59. The sample lid 58 may, if necessary, be taken out of the sample lid storage unit 59 and attached to the container 25 by a sample lid actuator (not shown), and removed from the container 25 and stored in the sample lid storage unit 59. In this case, the operation of the sample lid actuator may be controlled by a sample lid control unit (not shown) of the bubble forming 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 the container 25 and removed from the container 25 by the operator.

[0048] The output unit 160 outputs the processing results of the information processing device 170. For example, the output unit 160 outputs an image that has been processed internally by the information processing device 170 (for example, the image processing unit 300 described later). For example, the output unit 160 is a monitor connected to the information processing device 170.

[0049] The information processing device 170 exchanges commands and data with the microscope unit 50, camera 60, camera 70, operation unit 101, output unit 160, and input unit 180. For example, the information processing device 170 is connected to the microscope unit 50 and the operation unit 101, and controls the microscope unit 50 and the operation unit 101.

[0050] Specifically, the information processing device 170 switches the combination of the type of objective lens 6 placed in the optical path of the microscope unit 50 and / or the type of filter cube of the fluorescence filter. 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. Also, 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 use different light sources (light source 10 for transmission image observation and light source 1 for fluorescence image observation, respectively). For this reason, the internal components of the information processing device 170 (for example, the imaging control unit 171 described later) may switch one or more of the filter block, objective lens 6, and light source depending on whether to perform transmission image observation or at least one or more of one or more types of fluorescence image observation.

[0051] When performing fluorescence image observation, the information processing device 170 turns on the fluorescence image observation light source 1 and turns off the transmission image observation light source 10 in order to activate the optical path of the fluorescence image observation light source 1. When performing fluorescence image observation, the light emitted from the fluorescence image observation light source 1 illuminates the object to be operated 35 via the dichroic mirror 2, the optical deflector 3, the relay lens 4, the dichroic mirror 5, and the objective lens 6.

[0052] If the object to be operated 35 is fluorescently labeled, the fluorescent substance in the object to be operated 35 is excited and emits fluorescence. The fluorescence emitted from the object to be operated 35 reaches the light-receiving surface of the camera 60 via the objective lens 6, dichroic mirror 5, relay lens 4, light 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 object to be operated 35 is formed on the camera 60. Even if the object to be operated 35 is not fluorescently labeled, the object to be operated 35 can be observed using the light emitted from the light source 1 for fluorescence image observation that strikes the object to be operated 35 and reflects off it.

[0053] 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 in order to activate the optical path of the light source 10 for transmission image observation. When performing transmission image observation, the light emitted from the light source 10 illuminates the object to be worked on 35 via the bandpass filter 9, the focusing lens 8, and the condenser lens 7. The light that has passed through the object to be worked on 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 transmitted image of the object to be worked on 35 is formed on the camera 70. If the end of the nozzle 49 is difficult to see during fluorescence observation, transmission image observation may also be performed.

[0054] Furthermore, the information processing device 170 controls the relative positions of the nozzle 49 and the stage of the operation unit 101. In addition to controlling the microscope unit 50 and the operation unit 101, the information processing device 170 may receive images of the object to be manipulated 35 captured by the camera 60 and / or camera 70, and / or images captured by the flow path imaging camera 42 of the operation unit 101, and perform image processing such as generating a single composite image from multiple images. The information processing device 170 may also perform control of other operations of the biological manipulation device 100 and data processing as needed. The configuration of the information processing device 170 will be described later.

[0055] 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 object to be operated 35. The input unit 180 also inputs the amount of movement 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 biological manipulation device 100.

[0056] Figure 1B shows another example of the configuration of the biological manipulation device 100 in this embodiment. Figure 1B shows the biological manipulation device 100 when the microscope unit 50 is a phase-contrast microscope or a differential interference microscope. When the microscope unit 50 is a phase-contrast 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 10 for transmission image observation, a barrier filter 11, a projection lens 12, a light source 16, a light source 17, and a ring diaphragm 39. If 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 10 for transmission image observation, a barrier filter 11, a projection lens 12, a light source 16, a light source 17, a normal Ski prism 31, an analyzer (polarizer) 32, a polarizer (polarizer) 37, and a normal Ski prism 38. However, the microscope unit 50 is not limited to these and may have configurations other than those listed above. The description in Figure 1A may apply to the configuration of the biological manipulation device 100 other than the microscope unit 50.

[0057] Figures 2A and 2B are examples of schematic diagrams showing the structure of the nozzle 49 in this embodiment. In Figure 2A, the nozzle 49 includes a cylindrical portion 253 having a flow path 51. The cylindrical portion 253 may be a hollow cylinder. In this case, the shape of the cross-section of the cylindrical portion 253 perpendicular to the axial direction is circular. The flow path 51 may also be connected at one end to a pump 251 (for example, a syringe pump of the pressure generating unit 47). The pump 251 adjusts the pressure and / or volume of the bubbles by adjusting the amount of gas supplied to or drawn from the flow path 51, based on instructions from the information processing device 170 (for example, the bubble forming unit 200 described later).

[0058] In Figure 2B, if the end 254 of the cylindrical portion 253 that is not connected to the pump (not shown: for example, the syringe pump of the pressure generating unit 47) is placed in the liquid 261, the pump can supply gas to the flow path 51, thereby forming bubbles at the end 254. In this case, a gas-liquid interface 255 is formed at the boundary between the gas of the bubbles and the liquid 261. Note that the shape of the bubbles is not limited to spherical and may be deformed according to the shape of the end 254. Here, if gas is held at the end 254 of the flow path 51, a gas-liquid interface 255 is formed at the end 254 of the flow path 51. However, if both gas and liquid are present inside the flow path 51, a gas-liquid interface 255 may be formed inside the flow path 51 at the interface between the two.

[0059] When the gas-liquid interface 255 comes into contact with a living organism adhered to a solid phase, such as the inner bottom surface of a container 25 in liquid 261, moving the gas-liquid interface 255 allows the gas-liquid interface 225 to exert force on the living organism, causing it to detach from the solid phase and adhere to the gas-liquid interface 255. The movement of the gas-liquid interface 255 may be performed by the nozzle actuator 40 moving a nozzle 49 in which bubbles have been formed, 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 adhered and cultured. For example, the solid phase may be, but is not limited to, glass; resins such as polystyrene; 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 (for example, polymers whose hydrophilicity and cell adsorption properties can be controlled). In this embodiment, the gas-liquid interface 255 is formed by the interface between the gas and the liquid, but it is not limited to this and may be modified depending on the phase or substance in contact with the interface. Details of the method for detaching the biological organism from the solid phase will be described later.

[0060] The opening area of ​​the channel 51 at end 254 is not particularly limited, as long as it is large enough to manipulate living organisms. For example, the opening area may be larger than the contact area per living organism. The shape of end 254 is not particularly limited. Also, the inner diameter of the channel 51 may be the same along the entire length of the cylindrical portion 253.

[0061] Furthermore, the flow path 51 may be configured such that the pump 251 draws in the gas from the bubbles to which the organism is attached, thereby bringing the gas-liquid interface 225 into the flow path 51 and further recovering the organism. Alternatively, the nozzle 49 may be further equipped with another flow path separate from the flow path 51 for recovering the organism.

[0062] In the embodiments shown in Figures 2A and 2B, only one flow path 51 is formed within the nozzle 49, and only one pump 251 is connected to the flow path 51, resulting in a very simple and minimal configuration, which can reduce the maintenance and cost of the biological manipulation device 100.

[0063] Figures 3A and 3B are examples of schematic diagrams showing the structure of the nozzle 49 in other embodiments. While the examples in Figures 2A and 2B show the case where the flow path for forming bubbles and the flow path for collecting organisms are the same, the examples in Figures 3A and 3B show the case where the flow path for forming bubbles and the flow path for collecting organisms are different.

[0064] In Figure 3A, the cylindrical portion 253 of the nozzle 49 has a double structure consisting 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 inside 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.

[0065] Furthermore, the first channel 51a and the second channel 51b of the nozzle 49 may be connected at one end to the first pump 251a and the second pump 251b, respectively. For example, the pressure generating unit 47 may have a first pump 251a and a 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 in to the first channel 51a and the second channel 51b by actuators that receive instructions from the bubble forming unit 200. The shape of the cross-section of the cylindrical portion 253 perpendicular to the axial direction is donut-shaped in the first channel 51a and circular in the second channel 51b.

[0066] In Figure 3B, when the end 254 of the cylindrical portion 253 that is not connected to the first pump 251a and the second pump 251b is placed in the liquid 261, the first pump 251a can supply gas to the first flow path 51a, thereby forming bubbles at the end 254. In this case, a gas-liquid interface 255 is formed at the boundary between the gas of the bubbles and the liquid 261.

[0067] When the gas-liquid interface 255 comes into contact with a biological organism adhering to a solid phase in the liquid 261, the gas-liquid interface 255 can be moved to detach the biological organism from the solid phase and allow it to adhere to the gas-liquid interface 255. The second pump 251b may draw in the gas-liquid interface 225 into the channel 51 by sucking in the bubbles to which the biological organism has adhered via the second channel 51b, thereby recovering the biological organism.

[0068] In the above embodiment, the first pump 251a supplies gas to the first channel 51a to form bubbles, and the second pump 251b draws in gas through the second channel 51b to recover the organism. However, the second pump 251b may supply gas to the second channel 51b to form bubbles, and the first pump 251a may draw in gas through the first channel 51a to recover the organism. 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.

[0069] In the embodiments shown in Figures 3A and 3B, bubbles can be formed in one channel to attach organisms to the gas-liquid interface 255, and the attached organisms can be collected in the other channel simultaneously, thus shortening the time required to collect cells. In Figures 3A and 3B, embodiments are shown in which the cross-sectional shape of the cylindrical portion 253 perpendicular to the axial direction is donut-shaped in the first channel 51a and circular in the second channel 51b. However, the cross-sectional shape is not limited to donut-shaped or circular, and as long as there are two channels, the attachment and collection of organisms can be performed simultaneously.

[0070] Figure 4 is a schematic diagram showing an example of a method for recovering the target organism 35 in this embodiment. In 290a, the target organism 35 is cultured in a solid phase on the inner bottom surface of the container 25. The target organism 35 may be cultured in liquid 261. For example, the target organism 35 is adherent cells. For example, the liquid may be a complete culture medium.

[0071] In 290a, the pump 251 supplies gas to the flow path 51 of the nozzle 49, forming bubbles 256 at the end 254 of the nozzle 49. By bringing the bubbles 256 into contact with the object to be worked on 35, the gas-liquid interface 255 between the gas and the liquid 261 comes into contact with the object to be worked on 35. In this case, the pump 251 adjusts the supply and intake of gas to maintain the formed bubbles 256. This makes it easier to operate the object to be worked on 35 using the bubbles 256.

[0072] Next, in 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 object to be operated 35. Figure 4 shows the nozzle actuator 40 moving the nozzle 49 from left to right, but the direction in which the nozzle 49 moves is not limited as long as it is parallel to the surface of the solid phase. By moving the nozzle actuator 40, the object to be operated 35 can be detached from the solid phase. At this time, the detached object to be operated 35 adheres to the gas-liquid interface 255 of the bubble 256. The bubble forming unit 200 controls the pump 251 to adjust the pressure and / or volume of the supplied or sucked gas, thereby changing the size of the bubble 256 and allowing the object to be operated 35 within a desired range to be detached. Alternatively, instead of moving the nozzle 49 along the surface of the solid phase, the stage may be moved.

[0073] Next, at 290c, the pump 251 may draw in the gas in the flow path 51 to recover the object to be worked on 35 adhering to the gas-liquid interface 255. In this way, the bubbles 256 formed in the nozzle 49 can be used to selectively detach the object to be worked on 35 from the solid phase and recover it.

[0074] In the case of adherent cells that are strongly attached to the solid phase, the adhesion of the adherent cells may be relaxed beforehand before performing the method shown in Figure 4. The adhesion of adherent cells can be relaxed using known methods, as will be described later.

[0075] Figure 4 illustrates an example in which the gas-liquid interface 255 is moved by moving the nozzle 49 to detach and collect cells. However, the movement of the gas-liquid interface 255 is not limited to the above example. For example, after bringing the bubble 256 into contact with the object to be worked on 35, the volume of the bubble 256 may be increased. In this case, the contact surface between the bubble 256 and the solid phase will be widened, allowing the object to be selectively detached from the solid phase and collected. For example, after bringing the bubble 256 into contact with the object to be worked on 35, the nozzle actuator 40 may move the nozzle 49 closer to the solid phase. In this case as well, the bubble 256 is pressed against the solid phase, widening the contact surface between the bubble 256 and the solid phase, allowing the object to be selectively detached from the solid phase and collected.

[0076] Figure 5 shows an example of the specific configuration of the information processing device 170 in this embodiment. The information processing device 170 includes an imaging control unit 171, a recording unit 190, a bubble formation unit 200, a flow path control unit 250, a liquid control unit 260, and an image processing unit 300.

[0077] The imaging control unit 171 controls the light source 1 for fluorescence image observation, the objective lens 6, the fluorescence filter, the light source 10 for transmission image observation, the camera 42 for flow path imaging, the light source 45, the light source 46, the camera 60, and the camera 70, as described in Figures 1A and 1B. For example, when the imaging conditions for the object to be operated 35 are input to the input unit 180, the imaging control unit 171 makes the necessary adjustments for each image, according to the input imaging conditions, such as switching cameras, switching the type of objective lens 6 in the microscope unit 50, switching light sources, switching the type of fluorescence filter, the position of the stage, and the height of the objective lens 6. After the imaging control unit 171 has made the necessary adjustments, one or more of the cameras among the flow path imaging cameras 42, camera 60, and camera 70 take images of the object to be operated 35 or the nozzle 49 and generate images of the object to be operated 35 or the nozzle 49. One or more cameras send the generated image data to the image processing unit 300. Furthermore, the generated image data may be recorded in the recording unit 190 and / or output to the output unit 160.

[0078] The recording unit 190 may be, but is not limited to, memory, an internal hard disk drive, or an external recording medium. The information processing device 170 has a central processing unit (CPU), and the information processing device 170 is realized by the CPU executing a computer program recorded in the recording unit 190.

[0079] The bubble forming unit 200 controls the pressure and volume of the bubbles formed in the flow path 51, the supply and intake of gas, the movement of the nozzle 49, and the movement of the stage. The bubble forming unit 200 may include all or part of the nozzle position control unit, stage position control unit, volume control unit, supply control unit, and intake control unit.

[0080] The nozzle position control unit controls the nozzle actuator 40 and controls the movement of the nozzle 49, the airflow in the bubbles associated with the movement of the nozzle 49, and the movement of the gas-liquid interface 255 associated with the movement 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.

[0081] The stage position control unit controls the sample actuator 41 and controls the movement of the stage on which the container 25 containing the object to be operated 35 is mounted, the airflow in the bubbles accompanying the movement of the stage, and the movement of the gas-liquid interface 255 accompanying the movement of the stage. The stage position control unit also receives position information of the stage and the object to be operated 35 from the sensor unit 48 or the sample actuator 41.

[0082] The volume control unit controls the actuator of the pressure generation unit 47 to supply or draw gas from the syringe pump, thereby controlling the pressure and / or volume of the bubbles formed in the flow path 51. The volume control unit also receives information on the pressure and / or volume of the bubbles from the nozzle actuator 40, the pressure generation unit 47, or the sensor unit 48.

[0083] Furthermore, if the nozzle 49 includes a gas supply channel for supplying (injecting) gas and a gas recovery channel for recovering (inhaling) gas, the volume control unit or the air supply control unit controls the first pump 251a connected to the gas supply channel, thereby controlling the volume of gas supplied to the gas supply channel. The volume control unit or the air supply control unit receives information on the amount of gas supplied to the gas supply channel from the nozzle actuator 40, the pressure generation unit 47, or the sensor unit 48.

[0084] Furthermore, if the nozzle 49 includes a gas supply channel for supplying (injecting) gas and a gas recovery channel for recovering (inhaling) gas, the volume control unit or intake control unit controls the second pump 251b connected to the gas recovery channel, thereby controlling the amount (volume) of gas drawn in from the gas recovery channel. The volume control unit or intake control unit receives information on the amount of gas drawn in from the gas recovery channel from the nozzle actuator 40, the pressure generation unit 47, or the sensor unit 48.

[0085] The flow path control unit 250 controls the storage, installation, and disposal of the nozzle 49. The flow path control unit 250 receives instructions from the operator regarding the installation 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 exchange unit 53 to take the nozzle 49 from the flow path storage unit of the flow path exchange unit 53, install the nozzle 49 on the nozzle actuator 40, or remove the nozzle 49 that is installed on the nozzle actuator 40 and dispose of it in the flow path disposal unit of the flow path exchange unit 53.

[0086] The liquid control unit 260 controls the storage, replenishment, and disposal of liquid. The liquid control unit 260 receives instructions from the operator regarding operations related to the replenishment and disposal of liquid from 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 container 25 with the liquid stored in the liquid storage unit 54, or to retrieve the liquid contained in the container 25 and dispose of it in the liquid disposal unit of the liquid storage unit 54.

[0087] The image processing unit 300 receives images captured by the flow channel imaging camera 42, camera 60, and camera 70 from these cameras. The image processing unit 300 may use several 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 the fluorescence image captured by camera 60 and the transmission image captured by 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 them to the output unit 160.

[0088] 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) can be positive, zero, or negative. The smaller the value of the difference (E1-E2), the more organisms adhere to the bubbles. Here, since the value of the interfacial free energy E1 is constant, there is little room for control by the energy control unit. Therefore, by controlling 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.

[0089] Figure 6 shows an example of a flow chart of a method for manipulating a living organism in this embodiment. The living organism 35 to be manipulated in this embodiment can be manipulated by performing the processes S100 to S680 in Figure 6. For the sake of explanation, the processes S100 to S680 will be described in order, but at least some of these processes may be executed in parallel, or the steps may be rearranged without departing from the spirit of the present invention.

[0090] First, in S100, the sample actuator 41 receives the organism to be operated on 35. For example, in S100, the sample actuator 41 places a container 25 containing the organism to be operated on 35 along with a liquid on the stage. The lid of the container 25 may be removed in order to operate on the organism to be operated on 35. The lid may be replaced by an actuator that replaces lids, or it may be replaced by the operator. After the sample actuator 41 receives the organism to be operated on 35, the information processing device 170 proceeds to S120.

[0091] Next, in S120, camera 60 or camera 70 captures a wide observation field including the object to be operated 35 and generates an image. The imaging control unit 171 sets the observation method to low-magnification transmission imaging and sends an instruction to camera 70 to capture the observation field. The imaging control unit 171 may also set the observation method to fluorescence imaging and send an instruction to camera 60 to capture the observation field. The imaging control unit 171 may receive input of imaging conditions from the operator via the input unit 180. Camera 60 or camera 70 captures 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 camera 60 or camera 70 has captured the observation field, the imaging control unit 171 proceeds to processing in S140.

[0092] Next, in S140, the information processing device 170 receives input from the operator via the input unit 180 regarding the object to be operated 35 and the type of operation. The object to be operated 35 may be a single cell, a group of cells (colony), the cytoplasm and / or cell membrane of a cell, or a spheroid, but is not limited to these. The type of operation may be the retrieval of the object to be operated 35, the removal of the object to be operated 35, the retention of the object to be operated 35, or the compression of the object to be operated 35, but is not limited to these.

[0093] Figure 7A is an example of a GUI (Graphical User Interface) image displayed on the output unit 160, showing the observation field captured by camera 60 or camera 70. In Figure 7A, the cells aaa, bbb, and ccc to be manipulated are specified as the target cells 35 via the input unit 180. As shown in Figure 7A, the organism to be manipulated 35 can be arbitrarily specified via the input unit 180. As shown in Figure 7A, removal areas and / or protection areas may be provided in the observation field so that removal areas and / or protection areas are selected in the GUI image. By providing removal areas, the risk of cells other than those to be recovered being recovered can be reduced. Also, by providing protection areas, the risk of accidentally removing recovered cells when removing cells in the removal areas can be reduced.

[0094] Figure 7B is an example of a GUI image displayed on the output unit 160, where the collection and transfer destinations for the target cells aaa, bbb, and ccc (35 cells) are specified as A1, A2, and A3 of a 12-well plate, respectively. As shown in Figure 7B, the transfer destination can be arbitrarily specified via the input unit 180. For example, the transfer destination may be the same plate, a different plate, a petri dish, a microtest tube, a PCR tube, or a conical tube.

[0095] Figure 7C is an example of a GUI image that displays a table on the output unit 160 listing the ID number of the cell to be manipulated 35, the x and y coordinates of the cell to be manipulated 35 on the sample actuator 41, the size of the cell to be manipulated 35, and the destination of the cell to be manipulated 35. In the table shown in Figure 7C, the ID number, x and y coordinates, size, and destination are shown as items, but the items to be displayed are not limited to these. In this way, by specifying the cell to be manipulated 35 and the destination via the input unit 180, the image processing unit 300 may output the table to the output unit 160.

[0096] Figure 7D is an example of a GUI screen displayed on the output unit 160 for selecting the type of operation to be performed on the target cell 35. The input unit 180 receives instructions from the operator regarding what kind of operation to perform on the target cell 35 and inputs them into the information processing device 170. For example, as shown in the display area 111, the operation on the cell may be subculturing, or it may be holding or moving the cell, but it is not limited to these. For example, as shown in the display area 112, the operation on the cell may be compressing the cell to observe the deeper parts of the cell, but it is not limited to this. In Figure 7D, the type of operation is shown as being selected by radio buttons, but the method of selection is not limited to radio buttons.

[0097] Figure 7E shows another example of a GUI screen displayed on the output unit 160 for selecting the type of operation on the target cell 35. For example, as shown in display area 113, the type of operation on the 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 pull-down menus and radio buttons.

[0098] The input unit 180 may send instructions input by the operator to the information processing unit 170 based on the screens shown in Figures 7A to 7E. After the information processing unit 170 receives the instructions, the imaging control unit 171 proceeds to process S160.

[0099] In addition, in S160, if the object to be operated 35 is an adherent cell that is strongly attached to the solid phase, an additional step may be performed to relax the adhesion of the adherent cell beforehand. In this case, in the step of receiving the operating conditions in S140, the information processing device 170 may receive input regarding whether or not to perform the adhesion relaxation process.

[0100] The adhesion of adherent cells can be relaxed using known methods. For example, the adhesion of adherent cells may be relaxed by removing the liquid (e.g., culture medium), washing with a buffer, and then treating the adherent cells with an adhesion relaxation solution. For example, the adhesion relaxation solution may be a protease solution, a metal ion-free solution, or a chelating agent solution. As an example, the adhesion relaxation solution is a trypsin-EDTA solution. The adhesion of adherent cells may be relaxed by the liquid control unit 260 or by the operator. After treating the adherent cells with the adhesion relaxation solution and weakening the adhesion, the process may proceed to S160. Note that if a step is performed by the operator, not limited to the relaxation of adherent cell adhesion, the process may start again from the sample reception step in S100. Alternatively, instead of treatment with an adhesion relaxation solution, the adhesion may be weakened using a substrate that relaxes adhesion. For example, the substrate that relaxes adhesion may be one whose adhesion is relaxed in response to temperature or light irradiation.

[0101] Next, in S160, the information processing device 170 receives input from the operator via the input unit 180 regarding the liquid replacement or addition process. For example, if the operator wants to adjust the adhesion force between the biological object 35 to be operated on and the bubbles, the information processing device 170 may receive input to perform the liquid replacement or addition process. If the information processing device 170 receives an instruction to perform the liquid replacement or addition process, the information processing device 170 may proceed to S600. If the information processing device 170 receives an instruction not to perform the liquid replacement or addition process, the information processing device 170 may proceed to S180.

[0102] In S600, the liquid control unit 260 replaces the liquid in the container 25 containing the object to be operated 35 or adds another liquid to the liquid in the container 25. In S600, the step of replacing or adding liquid includes steps S610 to S630, as shown in Figure 8.

[0103] First, in S610, the information processing device 170 receives input from the operator via the input unit 180 regarding whether or not to remove the liquid. If the information processing device 170 is instructed to remove the liquid, the process proceeds to S615. If the information processing device 170 is instructed not to remove the liquid, the process proceeds to S620.

[0104] In S615, the liquid control unit 260 controls the liquid storage unit 54 to remove the liquid. For example, the liquid control unit 260 instructs the liquid storage unit 54 to collect a predetermined amount of liquid from the container 25 and dispose of it in the liquid disposal unit of the liquid storage unit 54. At this time, the liquid storage unit 54 may collect and dispose of the entire amount of liquid. Alternatively, the liquid storage unit 54 may collect and dispose of a portion of the liquid (for example, half the amount). After the liquid storage unit 54 has collected the liquid, the liquid control unit 260 proceeds to process S620.

[0105] In S620, the liquid control unit 260 instructs the liquid storage unit 54 to add the adhesion adjusting reagent to the container 25. The adhesion adjusting reagent is a reagent that adjusts the adhesion between the organism and the bubbles. For example, the adhesion adjusting reagent may change the concentration of inorganic salts and / or amphiphilic substances in the liquid. As an example, the adhesion adjusting reagent may be a buffer containing or not containing at least one of calcium ions or 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 adjusting reagent stored in the liquid storage section of the liquid storage unit 54.

[0106] In the example above, we described an example of replenishing the adhesion-adjusting reagent in container 25. However, instead of adding the adhesion-adjusting reagent to container 25, the adhesion between the organism and the bubbles may be adjusted by removing inorganic salts or amphiphilic substances contained in the liquid by attaching them to a filter or the like.

[0107] In S630, the information processing device 170 receives instructions from the operator via the input unit 180 regarding whether to repeat the above series of operations. If the information processing device 170 is instructed 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 container 25. If the information processing device 170 is instructed not to repeat the series of operations, the information processing device 170 proceeds to S180. Note that the steps and substeps in S600 may be performed by the operator, in which case the process may start again from the sample reception step in S100.

[0108] 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. The flow path control unit 250, in accordance with the instruction, retrieves the nozzle 49 from the flow path storage unit of the flow path exchange unit 53 and sends an instruction to the flow path exchange unit 53 to attach the nozzle 49 to the nozzle actuator 40. At this time, a suitable nozzle 49 may be selected depending on the size of the object to be operated 35, the type of operation, etc. The selection of the nozzle 49 may be specified by the operator via the input unit 180, or it may be automatically specified by the flow path control unit 250. After the nozzle actuator 40 has attached the nozzle 49, the flow path control unit 250 proceeds to S200. If the nozzle 49 is already attached to the nozzle actuator 40, or if the nozzle actuator 40 and the nozzle 49 are integrally formed, and it is not necessary to attach the nozzle 49 to the nozzle actuator 40, then step S180 may be omitted.

[0109] Next, in S200, the nozzle actuator 40 moves the relative position between the nozzle 49 and the object to be operated 35. For example, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move the relative position between the nozzle 49 and the object to be operated 35. In S200, the step of moving the relative position includes steps S210 to S225 as shown in Figure 9A, steps S230 to S256 as shown in Figure 9B, or steps S260 to S282 as shown in Figure 9C.

[0110] Figure 9A shows an example of a flow in which the relative position between the nozzle 49 and the target object 35 is moved based on an image taken of the position of the end 254 of the nozzle 49.

[0111] First, in S210, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 to a preset position. The nozzle actuator 40 may be an actuator that controls the x, y, and z positions. Here, the z position may be a position in the vertical direction (also called the direction along gravity, up and down, or z direction), the x position may be a position in any x direction perpendicular to the z direction (also called the vertical direction), and the y position may be a position in the y direction perpendicular to the x and z directions (also called the horizontal direction).

[0112] 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 container 25, then moving the focus of the camera 60 and / or camera 70 upward by an arbitrary distance, and then having the nozzle actuator 40 align the tip of the nozzle 49 with the focus of the camera 60 and / or camera 70. For example, the arbitrary distance may be less than or equal to the radius of the 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 container 25 is less than or equal to the radius of the bubble formed, the bubble will contact the bottom surface, and the organism located on the bottom surface can be manipulated using the bubble interface. In this case, the x and y positions of the nozzle 49 can be set using the nozzle actuator 40 or the sample actuator 41 based on images of the object to be manipulated 35 captured using the camera 60 and / or camera 70. Alternatively, the position of the nozzle 49 may be set using a flow path imaging camera 42 instead of, or in conjunction with, the camera 60 and / or camera 70.

[0113] Alternatively, the z-position of the nozzle 49 may be adjusted by using the flow path imaging camera 42 to image the tip of the nozzle 49 and the bottom surface of the container 25 from the side of the nozzle 49 to set the z-position. Furthermore, the shape of the bubbles or the liquid volume in the flow path 51 may be confirmed by imaging 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 forming unit 200 proceeds to process S215.

[0114] Next, in S215, the channel imaging camera 42 captures an image of the end portion 254 of the nozzle 49. The 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 it to the bubble formation unit 200.

[0115] Next, in S220, the bubble forming unit 200 determines whether the position of the nozzle 49 is different from a preset position based on the image of the captured end portion 254 of the nozzle 49. For example, the bubble forming unit 200 calculates the positional difference between the image of the captured end portion 254 of the nozzle 49 and the image of the end portion 254 of the nozzle 49 at a preset xyz position (i.e., the initial position), and if the difference is greater than or equal to a threshold, it determines that the position of the nozzle 49 is different from the initial position.

[0116] If the bubble forming unit 200 determines that the position of the nozzle 49 is different from its initial position, it proceeds to process S225; otherwise, it proceeds to process S300.

[0117] In S225, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40 in order 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. For example, the bubble forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S220. The nozzle actuator 40 receives the instruction and proceeds to S210. In the second and subsequent S210s, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move by an amount corresponding to the amount of movement.

[0118] Figure 9B shows an example of a flow in which the relative position between the nozzle 49 and the object to be operated 35 is moved based on the load sensed by the nozzle actuator 40.

[0119] First, in S230, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move 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 located above the 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 forming unit 200 proceeds to S235.

[0120] 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 forming 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 rapidly. Therefore, by measuring the value of the load sensed by the nozzle actuator 40, the bubble forming 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 downwards. Alternatively, the nozzle actuator 40 may send the value of the load it senses to the bubble forming unit 200 instead of the sensor unit 48.

[0121] Next, in S240, the bubble forming unit 200 determines whether the measured load value is less than or equal to the set load. If the measured load value is less than or equal to the set load, the bubble forming unit 200 proceeds to S242; otherwise, it proceeds to S245. As described above, the bubble forming unit 200 calculates the difference between the set load and the measured load, and if the difference value is greater than or equal to the threshold, it determines that the nozzle 49 has not reached the bottom of the container 25.

[0122] In S242, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40 in order to move the nozzle 49 to a preset position, and sends an instruction to the nozzle actuator 40 to move by the determined amount. For example, the bubble forming unit 200 may determine an amount of movement corresponding 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 onward, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move by an amount corresponding to the amount of movement.

[0123] In S245, the bubble forming unit 200 sets the initial z position of the nozzle 49. For example, the bubble forming unit 200 may set the initial z position of the nozzle 49 without moving it after the last S230. Alternatively, the bubble forming 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 that position as the initial z position. This results in the initial z position of the nozzle 49 being located a predetermined distance above the bottom surface. For example, the arbitrary distance may be less than or equal to the radius of the bubble formed at the end 254 of the nozzle 49. After the bubble forming unit 200 sets the initial z position of the nozzle 49, the bubble forming unit 200 proceeds to S250.

[0124] Next, in S250, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 to a preset xyz position (i.e., the initial position). The movement of the nozzle 49 may be on the xy plane. In this case, the z position is controlled based on the load value sensed by the nozzle actuator 40. In addition to movement on the xy plane, the movement of the nozzle 49 may also include movement in the z direction as needed. After the nozzle actuator 40 has moved the nozzle 49 to the preset xyz position, the bubble forming unit 200 proceeds to S252.

[0125] Next, in S252, the flow path imaging camera 42 captures an image of the end portion 254 of the nozzle 49. The flow path 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 it to the bubble formation unit 200.

[0126] Next, in S254, the bubble forming unit 200 determines whether the position of the nozzle 49 is different from a preset position based on the image of the captured end portion 254 of the nozzle 49. For example, the bubble forming unit 200 calculates the positional difference between the image of the captured end portion 254 of the nozzle 49 and the image of the end portion 254 of the nozzle 49 at a preset xyz position (i.e., the initial position). If the difference is greater than or equal to a threshold, the bubble forming unit 200 determines that the position of the nozzle 49 is different from the initial position.

[0127] If the bubble forming unit 200 determines that the position of the nozzle 49 is different from the initial position, it proceeds to process S256; otherwise, it proceeds to process S300.

[0128] In S256, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40 in order 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. For example, the bubble forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S254. The nozzle actuator 40 receives the instruction, and the bubble forming unit 200 proceeds to S250. In S250 from the second time onward, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move by an amount corresponding to the amount of movement.

[0129] Figure 9C shows an example of a flow that moves the relative position between the nozzle 49 and the object to be operated 35 based on the internal pressure of the bubble formed at the end 254 of the nozzle 49.

[0130] First, in S260, the bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles at the end 254 of the nozzle 49. Prior to forming bubbles, the bubble-forming unit 200 may send an instruction to the nozzle actuator 40 to move the end 254 of the nozzle 49 into the liquid. The bubble-forming step and substep in S260 may be the same as the step and substep in S300 described later.

[0131] Next, in S262, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move 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 located above the 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 forming unit 200 proceeds to S264.

[0132] In addition, in S262, the tip of the nozzle 49 may detect the liquid surface and control its z position based on the internal pressure value measured by the sensor unit 48. When the bubble forming unit 200 maintains the internal pressure of the nozzle to be above or below atmospheric pressure and the tip of the nozzle 49 reaches the liquid surface, the value of the internal pressure measured by the sensor unit 48 changes due to the external force caused by the deformation of the gas-liquid interface upon contact with the liquid surface. Therefore, the bubble forming unit 200 can determine whether the tip of the nozzle 49 has reached the liquid surface by measuring the internal pressure value of the bubble. As a result, even if the position to which the nozzle 49 is moved is not set in advance, the bubble forming unit 200 can send an instruction to the nozzle actuator 40 for z position control with the liquid surface as the reference position, and move the position of the nozzle 49.

[0133] Next, in S264, the sensor unit 48 measures the internal pressure of the formed bubble and sends the measured value of the bubble's internal pressure to the bubble forming unit 200. When the bubble reaches the bottom of the container 25, its shape deforms due to interaction with the bottom, and the bubble's internal pressure changes rapidly. Therefore, the bubble forming unit 200 can determine whether the bubble has reached the bottom of the container 25 by measuring the value of the bubble's internal pressure. As another example of internal pressure measurement, the nozzle actuator 40 may move the nozzle 49 downwards while the sensor unit 48 measures the internal pressure, or the bubble forming unit 200 may control the pressure, or the pressure generation unit 47 may be activated. By operating simultaneously in this way, bottom detection can be accelerated, and the pressure change process during the bubble formation process can be used as a detection indicator. Alternatively, instead of the sensor unit 48, the nozzle actuator 40 may measure the internal pressure of the bubble and send the measured value of the internal pressure to the bubble forming unit 200. Furthermore, the value of the internal pressure of the bubble measured by the sensor unit 48 or the nozzle actuator 40 may be stored in the recording unit 190 beforehand. In this case, the bubble forming unit 200 may determine whether the bubble has reached the bottom of the container 25 by comparing the value of the internal pressure stored in the storage unit 190 with the internal pressure of the bubble measured by the sensor unit 48 or the nozzle actuator 40.

[0134] Next, in S266, the bubble forming unit 200 determines whether the measured internal pressure of the bubble is within a preset range. If the measured internal pressure is outside the preset range, the bubble forming unit 200 proceeds to S268; otherwise, it proceeds to S270. As described above, the bubble forming unit 200 calculates the absolute difference between the set internal pressure and the measured internal pressure of the bubble. If the difference is greater than or equal to a threshold, the bubble forming unit 200 determines that the nozzle 49 has reached the bottom of the container 25.

[0135] In S268, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40 in order to move the nozzle 49 to a preset position, and sends an instruction to the nozzle actuator 40 to move by the determined amount. For example, the bubble forming unit 200 may determine an amount of movement corresponding 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 onward, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move by an amount corresponding to the amount of movement.

[0136] In S270, the bubble forming unit 200 controls the pressure generating unit 47 to remove bubbles from the end 254 of the nozzle 49. The bubble removal step and substep in S270 may be the same as the steps and substeps in S500 described later.

[0137] Next, in S272, the bubble forming unit 200 sets the initial z position of the nozzle 49. The step in S272 may be the same as the step in S245. After the bubble forming unit 200 sets the initial z position of the nozzle 49, the bubble forming unit 200 proceeds to the process in S274.

[0138] Next, in S274, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 to a preset xyz position (i.e., the initial position). The movement of the nozzle 49 may be on the xy plane, and may also include movement in the z direction as needed. After the nozzle actuator 40 moves the nozzle 49 to the preset xyz position, the bubble forming unit 200 proceeds to S276.

[0139] Next, in S276, the flow path imaging camera 42 captures an image of the end portion 254 of the nozzle 49. The flow path 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 it to the bubble formation unit 200.

[0140] Next, in S280, the bubble forming unit 200 determines whether the position of the nozzle 49 is different from a preset position based on the image of the captured end portion 254 of the nozzle 49. For example, the bubble forming unit 200 calculates the positional difference between the image of the captured end portion 254 of the nozzle 49 and the image of the nozzle end portion 254 at a preset xyz position (i.e., the initial position). If the difference is greater than or equal to a threshold, the bubble forming unit 200 may determine that the position of the nozzle 49 is different from the initial position.

[0141] If the bubble forming unit 200 determines that the position of the nozzle 49 is different from the initial position, it proceeds to process S282; otherwise, it proceeds to process S300.

[0142] In S282, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40. For example, the bubble forming unit 200 determines the amount of movement of the nozzle actuator 40 in order 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. For example, the bubble forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S280. The nozzle actuator 40 receives the instruction, and the bubble forming unit 200 proceeds to S274. In S274 from the second time onward, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move by an amount corresponding to the amount of movement.

[0143] 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 bubbles. Prior to forming bubbles, the bubble-forming unit 200 may instruct 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 Figure 10A, or steps S370 to S392, as shown in Figure 10B.

[0144] Figure 10A shows an example of a flow that enlarges the gas-liquid interface 255 based on an image taken of the position of the end 254 of the nozzle 49.

[0145] In S320, the bubble forming unit 200 sends an instruction to the pressure generating unit 47 connected to the flow path 51 to expand the gas-liquid interface 255 at the tip of the flow path 51 (form bubbles). For example, the bubble forming unit 200 sends an instruction to the actuator of the pressure generating unit 47 to push the plunger of the syringe pump by a preset distance, or to push the plunger of the syringe pump until a preset pressure is reached. As a result, 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 (bubbles are formed). After the gas-liquid interface 255 has expanded (bubbles have been formed), the bubble forming unit 200 proceeds to S330.

[0146] Next, in S330, the channel imaging camera 42 captures an image of the bubbles formed at the end 254 of the nozzle 49. The 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 it to the bubble formation unit 200.

[0147] Next, in S340, the bubble forming unit 200 determines, based on the image of the captured bubble, whether the shape of the formed bubble differs from a preset bubble shape. The bubble forming unit 200 predicts the shape of the bubble formed at the end 254 of the nozzle 49 from information such as the internal pressure inside the nozzle 49, the inner diameter of the end 254 of the nozzle 49, the wettability of the nozzle 49 (liquid contact angle), the type of liquid, and the type of gas. For example, the bubble forming unit 200 may determine whether the shape of the formed bubble differs from the preset bubble shape by comparing the image of the captured bubble with the bubble shape predicted from the above information.

[0148] If the shape of the formed bubbles differs from the set bubble shape, the bubble forming unit 200 proceeds to S342; otherwise, it proceeds to S400.

[0149] In S342, the bubble forming unit 200 determines the amount of movement of the plunger of the syringe pump in the pressure generating unit 47. For example, the bubble forming unit 200 determines the amount of movement of the plunger of the syringe pump in the pressure generating unit 47 (for example, the distance to push or pull the plunger of the syringe pump) in order to form bubbles at the tip of the nozzle 49 in a predetermined shape. The bubble forming unit 200 sends an instruction to the pressure generating unit 47 to operate by the determined amount. For example, the bubble forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S340.

[0150] The amount of movement may be the amount of movement of the actuator of the pressure generating unit 47, or it 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 process S320. In the second and subsequent S320, the pressure generating unit 47 performs an amount of movement corresponding to the amount of movement.

[0151] Figure 10B shows an example of a flow that expands the gas-liquid interface 255 based on the internal pressure in the nozzle 49.

[0152] Step S370 may be the same as step S320. After completing S370, the bubble forming unit 200 proceeds to S380.

[0153] Next, in S380, the sensor unit 48 measures the internal pressure inside the nozzle 49 and sends the measured value of the internal pressure inside the nozzle 49 to the bubble forming unit 200. Alternatively, instead of the sensor unit 48, the nozzle actuator 40 may measure the internal pressure inside the nozzle 49 and send the measured value of the internal pressure to the bubble forming unit 200.

[0154] Next, in S390, the bubble forming unit 200 determines whether the measured internal pressure value in the nozzle 49 is within a preset internal pressure range. If the measured internal pressure value is outside the preset internal pressure range, the bubble forming unit 200 proceeds to S392; otherwise, it proceeds to S400. For example, the bubble forming unit 200 calculates the difference between the preset internal pressure and the measured internal pressure in the nozzle 49, and if the difference is greater than or equal to a threshold, it may determine that the set internal pressure has not been reached.

[0155] In S392, the bubble forming unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generating unit 47 (for example, the distance the syringe pump plunger is pushed or pulled) in order to achieve the set internal pressure in the nozzle 49. The bubble forming unit 200 sends an instruction to the pressure generating unit 47 to operate by the determined amount. For example, the bubble forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S390.

[0156] The amount of movement may be the amount of movement of the actuator of the pressure generating unit 47, or it 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 process S370. In S370 from the second time onward, the pressure generating unit 47 performs an operation corresponding to the amount of movement.

[0157] In S400, the operation unit 101 performs an operation on the target 35. For example, the bubble formation unit 200 sends an instruction to the operation unit 101 to perform an operation on the target 35 based on the instruction received via the input unit 180. In S400, the step of performing an operation includes steps S410 to S460, as shown in Figure 11A. For example, the operation may be the removal of unwanted cells, the recovery or movement of cell membranes and / or cell membranes, the recovery of cells, the retention of cells, or the compression of cells.

[0158] Figure 11A shows an example of a flow chart for performing an operation on the target organism 35. Figure 11A illustrates an example where the target organism 35 is a cell. Note that the target organism 35 is not limited to a cell; it may be any other living organism.

[0159] First, in S410, if the system was instructed in S140 to remove unnecessary cells, the information processing device 170 proceeds to S412. In S410, if the system was instructed in S140 not to remove unnecessary cells, the information processing device 170 proceeds to S420.

[0160] In step S412, the bubble-forming unit 200 removes cells by attaching them to the formed bubbles.

[0161] For example, the bubble forming unit 200 instructs the nozzle actuator 40 to move the nozzle 49 in the xyz direction to the location where the target cells are located. After the nozzle actuator 40 has moved the nozzle 49 to the target position, the bubble forming unit 200 may move the nozzle 49 and / or the stage to bring the bubble's gas-liquid interface 255 into contact with the cells.

[0162] For example, the nozzle actuator 40 or the sample actuator 41 identifies the location of the target cells from the image captured by the camera 60 or camera 70, and moves to align the center of the nozzle 49 with the target position. After the cells have come into contact with the gas-liquid interface 255 of the bubble, the nozzle actuator 40 collects the target cells into the flow path 51, as shown in 290a to 290c of Figure 4, and the liquid storage unit 54 may discard these cells into the liquid waste section of the liquid storage unit 54.

[0163] As another example, the bubble-forming unit 200 may form a gas-liquid interface 255 of a predetermined size in step and substep S300, and control the expansion of the gas-liquid interface 255 to cause cells to adhere to and detach from the gas-liquid interface 255. After the liquid control unit 260 removes unwanted cells, the bubble-forming unit 200 proceeds to process S500.

[0164] In S420, if the bubble-forming unit 200 was instructed to collect the cytoplasm and / or cell membrane in S140, it proceeds to S422; otherwise, it proceeds to S430.

[0165] In S422, the bubble-forming unit 200 uses the formed bubbles to separate the cytoplasm and / or cell membrane, and collect them by attaching them to the bubbles. For example, the bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles at the tip of the channel 51 and attaches 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 portions and attach 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, expands the bubbles, or controls the nozzle actuator 40 to move the nozzle 49 toward the cells and press the bubbles against the cells to compress them. Subsequently, the bubble-forming unit 200 moves to separate the cytoplasm and / or cell membrane portions from the cells at the gas-liquid interface, thereby separating the portion of the cells that has bulged outward due to the compression. 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 from the object to be operated 35.

[0166] For example, the bubble-forming unit 200 may instruct the nozzle actuator 40 to move the nozzle 49 from its initial position to the location where the target cells are located in the xyz direction. After the nozzle actuator 40 has moved the nozzle 49 to the target 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 cells. As an example, the bubble-forming unit 200 may identify the location of the target cells from an image captured by the camera 60 or camera 70, and control the nozzle actuator 40 to move the nozzle 49 so that its center aligns with the target position. Here, the identification of the location of the target cells may be performed by the operator. In this case, the bubble-forming unit 200 may receive input from the operator regarding the location of the target cells from the input unit 180 and identify the location.

[0167] It is known that cell membranes have areas that exhibit relatively soft physical properties and areas that exhibit relatively hard physical properties, due to differences in the composition of lipids that make up the membrane components. The bubble-forming unit 200 controls the nozzle actuator 40 or the pressure generating unit 47 to compress the cells with bubbles. Here, the compression of the cells with bubbles may be achieved by the bubble-forming unit 200 forming a gas-liquid interface 255 of a predetermined size in step and substep S300, and controlling the expansion of the gas-liquid interface 255 to cause the cells to adhere to the gas-liquid interface 255 and compress them. Next, by utilizing the fact that the relatively soft parts of the cell membrane bulge outward, the bulged parts may be attached to the gas-liquid interface 255 and moved away from the cells to detach the cell membrane, and the cell membrane may be collected in the channel 51 while still attached to the gas-liquid interface 255. Furthermore, when the cell membrane is cut in this manner, it expands as it is pushed from the inside by the cytoplasm, so the detached cell membrane contains cytoplasmic components inside, and these cytoplasmic components can also be collected in the channel 51. After the nozzle actuator 40 has detached the necessary cytoplasm and / or cell membrane portions, the bubble forming unit 200 proceeds to process S434.

[0168] Figure 11B shows how cytoplasm and cell membranes are recovered from cells according to this embodiment. The bubble-forming unit 200 controls the pressure-generating unit 47 and the nozzle actuator 40 to bring the relative position of the HeLa cells (human cervical cancer cells) cultured on the solid phase of the container 25 closer to the gas-liquid interface 255 of the bubbles (802a). Next, the bubble-forming unit 200 compresses the cells by controlling the gas-liquid interface 255 to press against them (802b). At this time, it was observed that the soft part of the cell membrane bulged outward due to the compression (arrow in 802b). Next, the gas-liquid interface 255 is moved away from the cells to separate the cytoplasm and cell membrane in the bulged part (arrow in 802c). Finally, the separated cytoplasm and cell membrane are attached to the gas-liquid interface 255 and recovered (802d). Furthermore, when performing the operation shown in Figure 11B, the operation may be carried out by enlarging the gas-liquid interface 255, by moving the nozzle 49, or by moving the stage.

[0169] Next, in S434, the bubble-forming unit 200 determines whether the instruction received in S140 subsequently includes the collection of the cytoplasm and / or cell membrane of the target 35. If the determination is positive, the bubble-forming unit 200 proceeds to S435; otherwise, it proceeds to S500.

[0170] 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. At this time, the object to be operated 35 may be recovered simultaneously with the removal of the bubbles. For example, the object to be operated 35 may be recovered into the liquid present in the flow path 51. The bubble removal step and substep in S435 may be the same as the step and substep in S500, and their details will be described later.

[0171] Next, in S436, the bubble forming unit 200 controls the pressure generating unit 47 and the nozzle actuator 40 to draw in gas in order to form a new bubble at the end 254 of the nozzle 49. The bubble forming unit 200 instructs 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 generating unit 47 may pull the plunger of the syringe pump to draw in the required amount of gas.

[0172] Next, in S437, the bubble-forming unit 200 controls the pressure generating unit 47 to form new bubbles at the end 254 of the nozzle 49. The steps and substeps of S437 may be the same as those described in S300. After the pressure generating unit 47 has formed bubbles at the end 254 of the nozzle 49, the bubble-forming unit 200 proceeds to step S420.

[0173] In S430, the bubble-forming unit 200 determines whether it has been instructed to collect cells in S140. If the determination is positive, the bubble-forming unit 200 proceeds to S432; otherwise, the bubble-forming unit 200 proceeds to S440.

[0174] In S432, the bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles and cause cells to adhere to the bubbles. The bubble-forming unit 200 may, if necessary, detach the cells attached to the bubbles from the solid phase.

[0175] For example, the bubble forming unit 200 instructs the nozzle actuator 40 to move the nozzle 49 from its initial position to the location where the target cells are located in the x, y, and z directions. 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 bubbles 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 to bring the gas-liquid interface 255 of the bubbles into contact with the cells.

[0176] As an example, the nozzle actuator 40 or the sample actuator 41 identifies the location of the target cells from the image captured by the camera 60 or camera 70, and moves the nozzle 49 to align its center with the target position. After the nozzle actuator 40 or the sample actuator 41 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 path 51 and form bubbles at the tip of the flow path 51. Next, 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 bubbles into contact with the cells. For example, after the cells have come into contact with the gas-liquid interface 255 of the bubbles, the nozzle actuator 40 may detach the cells as needed by moving the gas-liquid interface 255.

[0177] The bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles, and after cells are attached to the bubbles, the bubble-forming unit 200 proceeds to S434. The steps from S434 onward may be as previously described. In this case, the continuous recovery in step S434 is not limited to cytoplasm only or cells only, but may include both cytoplasm and cells.

[0178] Figure 11C shows how, according to this embodiment, cultured cells attach to and detach from bubbles. 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 bring the relative position of the cells and the nozzle 49 closer together so that the HeLa cells cultured on the solid phase of the container 25 can come into contact with the gas-liquid interface 255 of the bubbles (804a). Next, the cells come into contact with the gas-liquid interface 255 of the bubbles (804b). Next, the nozzle actuator 40 moves the gas-liquid interface 255 (804c), causing the cells to attach to the gas-liquid interface 255 and detach (804d). Note that when performing the operation shown in Figure 11C, the operation may be performed by enlarging the gas-liquid interface 255, by moving the nozzle 49, or by moving the stage.

[0179] Figure 11D shows a schematic diagram of the case where steps S430→S432→S434→S435→S436→S437 are repeated. When continuously collecting cytoplasm and / or cell membranes, and when continuously collecting cells, the procedure may be carried out as shown in the schematic diagram of Figure 11D. In 810a, after the nozzle actuator 40 places the nozzle 49 into the liquid, the bubble forming unit 200 controls the pressure generating unit 47 to supply air to a syringe pump (not shown) to form bubbles at the end 254 of the nozzle 49. Next, cells adhering to the bottom surface of the container 25 are detached by adhering them to the gas-liquid interface 255 of the bubbles, and collected into the flow path 51 by drawing gas into the syringe pump. Next, in 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 path 51. Next, in 810c, after the nozzle actuator 40 places the nozzle 49 into the liquid, the syringe pump supplies air to the end 254 of the nozzle 49 to form new bubbles, and the cells adhering to the bottom surface of the container 25 are collected by adhering them to the gas-liquid interface 255 of the bubbles. In this way, the bubble-forming unit 200 controls the pressure generation unit 47 and the nozzle actuator 40, allowing two cell groups to be continuously collected into the flow path 51 without mixing via gas. A photograph showing two cell groups collected with air in between using this method is also shown. Note that although Figure 11D explains the case where the nozzle 49 is moved, the operation in Figure 11D may be performed by moving the stage instead of moving the nozzle 49.

[0180] Figure 11E shows the process of collecting established cell lines and subculturing the collected cells according to this embodiment. The bubble-forming unit 200 controls the pressure-generating unit 47 and the nozzle actuator 40 to attach, detach, and collect the established cell lines HeLa cells (human cervical cancer cells, 820a), HT29 cells (human colorectal cancer cells, 820b), and KatoIII cells (human gastric signet ring cell carcinoma cells, 820c) from the solid phase of the container 25 using the gas-liquid interface 255 of the bubbles, and release them into the culture medium in another container for 1.5 days (820a and 820b) and 2 days (820c). After subculturing, it was confirmed that all cells had proliferated. In other words, according to this embodiment, even when established cell lines are detached using the gas-liquid interface 255 of the bubbles, it was possible to proliferate the cells without damaging their viability. When performing the operation shown in Figure 11E, the operation may be carried out by moving the nozzle 49, or by moving the stage.

[0181] Figure 11F shows the recovery of iPS cells and the subsequent subculturing of the recovered iPS cells according to this embodiment. The bubble-forming unit 200 controls the pressure-generating unit 47 and the nozzle actuator 40 to attach, detach, and recover the cultured iPS cell colonies from the solid phase of the container 25 using the gas-liquid interface 255 of the bubbles. These colonies are then released into a liquid culture medium in another container and cultured for 4 days before the transmission image is observed. As a comparative example, iPS cells subculturized using a conventional cell subculturing method (mechanical passage) were used. As a result, no morphological differences were observed between the iPS cells of this embodiment (830a) and the iPS cells of the comparative example (830b). After culturing for 10 days, 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 subculturized three times and cultured for 30 days before being stained with alkaline phosphatase again. 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 undifferentiated iPS cells that maintain self-renewal ability express high levels of alkaline phosphatase. In other words, with this embodiment, even when iPS cells were detached and collected using the gas-liquid interface 255, it was possible to proliferate the iPS cells while maintaining their undifferentiated state without affecting the maintenance of their undifferentiated ability. Note that when performing the operation shown in Figure 11F, the operation may be performed by moving the nozzle 49 or by moving the stage.

[0182] Figure 11G shows the process of recovering and analyzing cell lines according to this embodiment. HeLa cells, which are cell lines, were detached and recovered from the solid phase of the container 25 using the gas-liquid interface 255 of the bubbles. The bubble-forming unit 200 controlled the pressure generation unit 47 and the nozzle actuator 40 to select 1, 4, and 8 HeLa cells from the solid phase and detach them using the gas-liquid interface 255 of the bubbles, and these cells were recovered together with 7.5 nL of liquid culture medium (835a). Next, the recovered HeLa cells were released into 12.5 μL of cell lysis reagent and the cells were lysed (835b). cDNA was synthesized from β-actin mRNA in the cell lysis solution containing the HeLa cells, and a PCR reaction was performed (835c). As a result, an amount of cDNA roughly proportional to the number of recovered cells was detected. In other words, according to this embodiment, it was possible to recover one cell or any number of cells using the gas-liquid interface 255 and perform molecular biological analysis. When performing the operation shown in Figure 11G, the operation may be carried out by moving the nozzle 49, or by moving the stage.

[0183] Next, in S440, the bubble-forming unit 200 determines whether it has been instructed in S140 to hold and image the cells. If the determination is positive, the bubble-forming unit 200 proceeds to S442; otherwise, it proceeds to S450.

[0184] In S442, the bubble-forming unit 200 controls the process to attach cells to the formed bubbles and to retain the attached cells. For example, the bubble-forming unit 200 instructs the nozzle actuator 40 to move the nozzle 49 from its initial position to the location where the target cells are located in the x, y, and z directions. After the nozzle actuator 40 moves the nozzle 49 to the target position, the bubble-forming unit 200 controls the pressure generation unit 47 to supply gas to the flow path 51 and form bubbles at the tip of the flow path 51. 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 bubbles into contact with the cells. As an example, the nozzle actuator 40 or the sample actuator 41 identifies the location of the target cells from the image captured by the camera 60 or camera 70 and moves the nozzle 49 to align its center with the target position. After the cells come into contact with the gas-liquid interface 255 of the bubble, the bubble-forming unit 200 proceeds to process S444.

[0185] Here, 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 operator regarding the location of the target cell from the input unit 180 and determine the location. In addition, although the above example describes the case where the tip of the bubble is brought into contact with the cell, contact between the bubble and the cell may also be made 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 be moved so that the center of the nozzle 49 is aligned with the vicinity of the target cell. Furthermore, 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 moving the gas-liquid interface 255.

[0186] Next, in S444, the imaging control unit 171 instructs camera 60 or camera 70 to image the cells held in the bubbles. Camera 60 or 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 it to the bubble formation unit 200.

[0187] After the camera 60 or camera 70 has imaged the cells it has captured, the bubble formation unit 200 proceeds to process S500.

[0188] Figure 11H is a schematic diagram showing how culture cells are held at the gas-liquid interface 255 of a bubble and observed. Suspended cells or weakly attached cells move freely in the culture medium due to slight vibrations, making them difficult to observe using a microscope or other instruments. In 840a, the bubble-forming unit 200 controls the nozzle actuator 40 to insert the end 254 of the nozzle 49 into the liquid culture medium in the container 25 where the suspended cells (target cells 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, creating a gas-liquid interface 255. The bubble-forming unit 200 controls the nozzle actuator 40 or the pressure generating unit 47 to attach the suspended cells, which will be the target cells 35, to the formed gas-liquid interface 255. Next, in 840b, the pressure generating unit 47 controls the internal pressure of the bubble to temporarily hold the cells at the gas-liquid interface 255 of the bubble. Next, at 840c, the pressure generating unit 47 reduces the bubble by drawing gas from the flow path 51. 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 reducing the bubble, and the held cells can be observed using a microscope or the like. In this way, this embodiment allows cells to be held at the gas-liquid interface 255 of the bubble, enabling observation of the cells without moving them.

[0189] Furthermore, in the case of adherent cells scattered on the solid phase of container 25, it is necessary to move the stage to observe them in a wide field of view using a microscope or the like. In 842a, the bubble-forming unit 200 controls the nozzle actuator 40 to insert the end 254 of the nozzle 49 into the liquid culture medium of container 25 in which the adherent cells (operation target 35) are cultured. Next, the pressure generating unit 47 supplies gas to the flow channel 51 to form a bubble at the tip of the nozzle 49, 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, causing the cells to adhere to the gas-liquid interface 255 and then detach. 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 reduces the bubble by drawing gas from the flow channel 51. Cells held in this state can be observed simultaneously using a microscope or other instrument because they are located in a narrow area on the same z-axis plane. Thus, this embodiment allows for a reduction in the field of view to be observed by holding the cells at the gas-liquid interface of the bubble.

[0190] As an example of such observation techniques, KatoIII cells, which are suspension cells, were scattered on a solid phase, and some of the cells were attached to the gas-liquid interface 255 (844a). Subsequently, the bubble-forming unit 200 controlled the internal pressure of the bubbles via the pressure-generating unit 47, thereby shrinking the bubbles and holding the cells at the gas-liquid interface 255. When the held cells were focused on, the surrounding cells were out of focus (844b). At this time, when the stage was moved, the surrounding cells moved, but the cells held at the gas-liquid interface 255 did not, making it easy to observe the held cells with a microscope (844c).

[0191] In S450, the bubble-forming unit 200 determines whether it has been instructed in S140 to compress and image the cells. If the determination is positive, the bubble-forming unit 200 proceeds to S452; otherwise, it proceeds to S460.

[0192] In step S452, the bubble-forming unit 200 controls the pressure-generating unit 47 and the nozzle actuator 40 to compress the cells using bubbles. For example, the bubble-forming unit 200 controls the pressure-generating unit 47 to form bubbles at the tip of the channel 51 and brings these bubbles into contact with the cells to be manipulated 35. When performing the operation shown in Figure 11H, the operation may be performed by moving the nozzle 49 or by moving the stage.

[0193] For example, the bubble forming unit 200 sends an instruction to the nozzle actuator 40 to move the nozzle 49 from its initial position to the location where the target cells are located in the x, y, and z directions. For example, the bubble forming unit 200 identifies the location of the target cells from the image captured by the camera 60 or camera 70, and controls the nozzle actuator 40 to move the nozzle 49 so that the center of the nozzle 49 aligns with the target position.

[0194] Here, 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 operator regarding the location of the target cell from the input unit 180 and determine the location. Contact between the bubble and the cell may be made 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 be moved so that the center of the nozzle 49 is aligned 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 be moved so that the center of the nozzle 49 is aligned near the target cell. In this case, the bubble can be formed beside the cell, and the nozzle 49 can be moved to gradually compress the cell from the side.

[0195] 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 to the flow path 51, thereby forming 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 stage may be moved to bring the gas-liquid interface 255 into contact with the cell. The bubble forming unit 200 may also be controlled to expand the gas-liquid interface 255 via the pressure generating unit 47 to bring the cell into contact with the gas-liquid interface 255.

[0196] As an example, the bubble-forming unit 200 operates the plunger of the syringe pump of the pressure-generating unit 47 with a preset amount of movement to form a bubble of a preset volume, and then moves the nozzle 49 and / or stage using the nozzle actuator 40 or the sample actuator 41 so that the nozzle 49 is positioned so that the bubble compresses the cells. Next, the bubble-forming unit 200 may control the pressure-generating unit 47 to expand the bubble formed at the tip of the flow path 51, or control the nozzle actuator 40 to move the nozzle 49 toward the cells and press the bubble against the cells, thereby compressing them.

[0197] As an example, the bubble-forming unit 200 may, after moving the nozzle 49 very close to the cell, control the pressure-generating unit 47 to form a bubble of a predetermined volume at the tip of the channel 51, thereby compressing the cell.

[0198] Next, in S454, the imaging control unit 171 instructs camera 60 or camera 70 to image the compressed cells. Camera 60 or 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 it to the output unit 160.

[0199] Alternatively, or in addition to the above, the sensor unit 48 or the nozzle actuator 40 may measure the pressure exerted by the bubbles on the cells and send the measured pressure value to the bubble-forming unit 200. The camera 60 or camera 70 may repeatedly capture images while changing the pressure exerted by the bubble-forming unit 200 on the cells. The pressure exerted on the cells can be changed by the bubble-forming unit 200 controlling the pressure generation unit 47 to change the internal pressure and / or volume of the bubbles.

[0200] As an example, the bubble-forming unit 200 may move the nozzle 49 very close to the cell, then control the pressure-generating unit 47 to form a bubble at the tip of the channel 51, and change the internal pressure and / or volume of the bubble to maintain or change the pressure that compresses the cell, thereby compressing the entire cell or various parts of the cell. It is expected that different parts of the cell will have different stiffness depending on the composition and distribution of the cell membrane or intracellular organelles. In this way, the composition and distribution of the cell membrane or intracellular organelles in the cell can be analyzed using the pressure and / or observed image during compression by the bubble as indicators. By compressing the cell, the thickness of the cell is reduced, allowing for clear observation of structures deep within the cell, and the lateral spreading causes closely spaced structures to separate and be observed individually. By observing the cell while compressing it, information on the force applied to the cell and the amount of morphological change inside and outside the cell can be obtained, making it possible to analyze information on the cell's mechanics. After the camera 60 or camera 70 images the compressed cell, the bubble-forming unit 200 proceeds to process S500.

[0201] Figure 11I shows how, according to this embodiment, a cell line was compressed using air bubbles to observe the deep interior of the cell. The nucleus and cytoplasm of a spheroid (850a) formed from living HT29 cells were stained, and by compressing it with air bubbles, it was possible to observe structures deep within the cell, such as the nucleus (850b). Comparing the thicker central portion in particular, the nucleus is not visible in the central part of 850a before compression, but it can be confirmed in the central part of 850b after compression. Conventionally, in order to observe structures deep within a cell, cells have been fixed with formalin or methanol, and the cells have been thinly sliced ​​for observation, or observation has been performed using a special microscope specialized for deep observation. According to this embodiment, structures deep within a cell can be observed in a living state without the need for a special microscope. Furthermore, in the spheroid before compression (850a), the nuclei were closely packed together, making it difficult to recognize individual nuclei. However, in the spheroid observed after compression (850b), the spheroid expanded in both the longitudinal and transverse directions, creating sufficient spacing between the nuclei, allowing for independent recognition of the nuclei. Conventionally, microscopic systems with improved optical systems and fluorescent labeling methods have been researched and developed to independently recognize two or more closely spaced organelles inside a cell; these are called super-resolution microscopes. While these microscopy techniques increase resolution, they narrow the field of view and increase imaging time. According to this embodiment, two or more closely spaced organelles inside a cell can be independently recognized in a living state, without the use of a special microscope, while maintaining the field of view and with a short imaging time. In addition, it is possible to reconstruct the three-dimensional structure of the original cell from the observed image and mechanical information of the compressed cell.

[0202] In S460, the bubble-forming unit 200 controls the operation unit 101 so that the necessary operations other than those in S410 to S450 of the instructions received in S140 are performed on the target 35. For example, the operations may be evaluation of cell adhesion or induction of cell differentiation, but these will be described later. After completing S460, the bubble-forming unit 200 proceeds to S500.

[0203] 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 bubbles. Here, the recovery of the object to be operated 35 may be performed simultaneously when reducing or removing bubbles. In S500, the step of reducing the gas-liquid interface 255 includes steps S510 to S544 as shown in Figure 12A, or steps S560 to S594 as shown in Figure 12B.

[0204] Figure 12A shows an example of a flow that reduces the gas-liquid interface 255 based on an image taken of the position of the end portion 254 of the nozzle 49.

[0205] In S510, the bubble forming unit 200 controls the pressure generating unit 47 to perform a suction operation on the flow path 51, drawing in the gas-liquid interface 255 from the tip of the flow path 51. At this time, liquid is also drawn in simultaneously. The liquid drawn in may be used to separate the object to be operated 35 from the gas-liquid interface 255. The liquid drawn in may be a liquid contained in the container 25 (for example, a culture medium), or another liquid stored in the liquid storage unit 54.

[0206] For example, the bubble-forming unit 200 sends an instruction to the pressure-generating unit 47 to pull the plunger of the syringe pump by a preset distance, or to pull the plunger of the syringe pump until a preset pressure is reached. Upon receiving instructions from the volume control unit or intake control unit within the bubble-forming unit 200, the pressure-generating unit 47 draws in gas. As a result, the gas-liquid interface 255 is reduced (bubbles are removed), and the gas-liquid interface 255 is drawn into the flow path 51. After the gas-liquid interface 255 is reduced (bubbles are removed), the bubble-forming unit 200 proceeds to process S520.

[0207] Next, in S520, the flow path 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 it to the bubble formation unit 200.

[0208] Next, in S530, the bubble forming unit 200 determines, based on the image of the captured end portion 254 of the nozzle 49, whether the position of the gas-liquid interface 255 taken in by the flow channel 51 is different from a position pre-set in the bubble forming unit 200. If the positions are different, the bubble forming unit 200 proceeds to S532; otherwise, it proceeds to S540. For example, the bubble forming unit 200 calculates the difference between the position of the gas-liquid interface 255 calculated based on the image of the captured end portion 254 of the nozzle 49 and a pre-set position. If the difference is greater than or equal to a threshold, the bubble forming unit 200 may determine that the position of the gas-liquid interface 255 taken in by the flow channel 51 is different from the set position.

[0209] In S532, the bubble-forming unit 200 determines the amount of movement of the plunger of the syringe pump in the pressure-generating unit 47. For example, the bubble-forming unit 200 determines the amount of movement of the plunger of the syringe pump in the pressure-generating unit 47 (for example, the distance to push or pull the plunger of the syringe pump) in order to capture the gas-liquid interface 255 up to a preset position of the gas-liquid interface 255. The bubble-forming unit 200 sends an instruction to the pressure-generating unit 47 to operate by the determined amount. For example, the bubble-forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S530.

[0210] The amount of movement may be the amount of movement of the actuator of the pressure generating unit 47, or it 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 process S510. In S510 from the second time onward, the pressure generating unit 47 performs an operation corresponding to the amount of movement.

[0211] In S540, if the instructions received in S140 include detaching cells from the interface (for example, cell retrieval), the bubble-forming unit 200 proceeds to S542; otherwise, the bubble-forming unit 200 proceeds to S640.

[0212] In S542, the bubble forming unit 200 instructs 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 it upward by a predetermined distance, the bubble forming unit 200 proceeds to S544.

[0213] Next, in S544, the bubble-forming 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 attached to the gas-liquid interface 255 detach from the gas-liquid interface 255 and move into the liquid. For example, the bubble-forming unit 200 moves the gas-liquid interface 255 at high speed by causing the pressure generating unit 47 to rapidly reciprocate the plunger of the syringe pump, thereby repeatedly supplying and drawing in air within the flow path 51. Alternatively, the bubble-forming unit 200 may also move the gas-liquid interface 255 in the flow path 51 at high speed by causing the nozzle actuator 40 to reciprocate the nozzle 49 at high speed in the vertical direction (±z direction) and / or in the vertical and horizontal directions (±xy direction).

[0214] The bubble-forming unit 200 may cause the gas-liquid interface 255 to move at high speed in place (where S542 was performed), or it may cause the nozzle actuator 40 to place the nozzle 49 into the liquid at the specified destination before the movement is performed. The bubble-forming unit 200 can appropriately detach cells attached to the gas-liquid interface 255 by controlling the liquid movement speed based on information such as the internal pressure inside the nozzle 49 received from the sensor unit 48. Alternatively, the bubble-forming unit 200 may vibrate the gas-liquid interface 255 by forming an electromagnetic field inside the nozzle 49.

[0215] Furthermore, the bubble-forming unit 200 may control the process to detach the cells from the gas-liquid interface 255 by bringing the bubbles into contact with the filter. The bubble-forming unit 200 may also detach the cells from the gas-liquid interface 255 by controlling the liquid storage unit 54 to add a liquid that reduces the free energy of the interface. In addition, the bubble-forming unit 200 may control the nozzle actuator 40 and the pressure generating unit 47 to form bubbles at the tip of the nozzle 49 at the designated destination, and detach the cells by rubbing them against the bottom surface of the container 25 at the designated destination. The bubble-forming unit 200 may also control the pressure generating unit 47 to increase the internal pressure of the bubbles to push out and detach the cells. Alternatively, the liquid at the destination may be changed to 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 process S640.

[0216] Figure 12B shows an example of a flow that reduces the gas-liquid interface 255 based on the internal pressure in the nozzle 49.

[0217] In S560, 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. The step in S560 may be the same as the step in S510. Next, the bubble-forming unit 200 proceeds to the process in S570.

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

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

[0220] In S582, the bubble-forming unit 200 determines the amount of movement of the plunger of the syringe pump of the pressure generating unit 47 (for example, the distance the syringe pump plunger is pushed or pulled) in order to achieve the set internal pressure in the nozzle 49. The bubble-forming unit 200 sends an instruction to the pressure generating unit 47 to operate by the determined amount. For example, the bubble-forming unit 200 may determine an amount of movement corresponding to the magnitude of the difference calculated in S580.

[0221] The amount of movement may be the amount of movement of the actuator of the pressure generating unit 47, or it 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 process S560. In the second and subsequent S560s, the pressure generating unit 47 performs an amount of movement corresponding to the amount of movement.

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

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

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

[0225] If there are other objects to be operated on 35 in S650, the bubble-forming unit 200 proceeds to S660. If there are no other objects to be operated on 35 in S650, the bubble-forming unit 200 proceeds to S680.

[0226] In step S660, if it is necessary to replace the nozzle 49 when operating another target 35, the bubble forming unit 200 proceeds to step S670; otherwise, it proceeds to step S200.

[0227] In S670, the flow path control unit 250 instructs the flow path exchange unit 53 to remove the nozzle 49 attached to the nozzle actuator 40 and dispose of it in the nozzle disposal unit of the flow path exchange unit 53. Alternatively, the nozzle may be stored with the cells still inside without releasing them, and the cells may be analyzed afterward. In this case, the nozzle 49 may be stored in the nozzle storage unit of the flow path exchange unit 53 instead of being discarded. After the flow path exchange unit 53 discards the nozzle 49, the process proceeds to S180.

[0228] In S680, the nozzle 49 may be disposed of using the same procedure as in S670. The flow path replacement unit 53 disposes of the nozzle 49, and the flow ends.

[0229] The above flowchart describes, as examples of operations on target 35, the removal of unwanted cells, cytoplasm and / or cell membrane recovery, cell recovery and passage, cell retention, and cell compression. Several other examples of operations are also possible.

[0230] One example of the procedure involves applying a culture substrate or drug to the solid phase at the bottom of container 25 and evaluating the adhesion of these culture substrates or drugs to cells. Since the adhesion to cells can be evaluated using indicators such as the internal pressure of the air bubbles when detaching the cells, the movement speed of the nozzle, and the load, the effectiveness of the culture substrate or drug on cell adhesion can be evaluated.

[0231] Another example of the operation is cell sorting. Following the flow described above, pressure is applied to the cells using bubbles. Depending on the type of cell, the cell membrane, intracellular components, or physical properties may differ. Therefore, after the bubble formation unit 200 controls the nozzle actuator 40 and / or the pressure generation unit 47 and starts, stops, and releases the pressure on the cells with bubbles, the process of change in cell shape and the shape of the cells may differ. In addition, depending on the type of cell, some cells may rupture when compressed. These can be used as indicators to sort the cells.

[0232] Another example of the procedure is to observe the changes in shape of cells during the compression process. Compression is applied to the cells using bubbles according to the flow described above. For example, during the compression process, the pressure applied to the cells may be varied, and the entire cell or different parts of the cell may be compressed to image the changes in the cell's shape.

[0233] Another example of this operation is the rupture or severance of cells by compression. By applying significant pressure to cells, they can be ruptured or severed. Rupture or severance of cells allows for the recovery of the cell membrane, cytoplasm, and / or organelles, and also allows for the severance of connections between cells (for example, synapses, which are connections between nerve cells).

[0234] Another example of the operation is the induction of cell differentiation. Osteoblasts, muscle cells, and vascular endothelial progenitor cells are known to be differentiated by mechanical stimulation. Differentiation can be induced in these cells by applying pressure using bubbles from the pressure generating unit 47, according to the flow described above.

[0235] Another example of the operation is gene introduction into cells. Following the flow described above, the bubble-forming unit 200 uses bubbles to attach vesicle-like objects, such as cell membranes, to the gas-liquid interface 255, bringing them into contact with the cell membrane. This allows the contents of the vesicle to be taken up by the cell through membrane fusion. At this time, by encapsulating genes within the vesicle, the genes can be taken up into the cell. In addition to genes, other macromolecules can also be taken up into the cell through the pores. Furthermore, following the flow described above, when the bubble-forming unit 200 uses bubbles to compress the cell at the gas-liquid interface 255, tiny gaps are more likely to form in a part of the membrane during the process of cell deformation. At this time, by adding genes to the cell culture medium, the genes can be taken up into the cell through these gaps. In addition to genes, other macromolecules can also be taken up into the cell through these gaps.

[0236] Another example of operation is integration with external devices such as cell culture devices like fermenters or cell analysis devices like cell sorters. Following the flow described above, the bubble-forming unit 200 may use bubbles to take in cells into the channel 51 and release them at a designated location on the integrated external device, thereby moving the cells. Alternatively, the channel 51 may be directly connected to the external device, allowing the cells taken into the channel 51 to be sent to the external device for cell movement.

[0237] Another example of the operation is the operation of emulsions. An emulsion is a droplet in an oil-liquid or 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 is arranged around the droplet or oil droplet, forming a monolayer at the interface. Such monolayers are also found in some organelles within cells, such as endosomes and lipid droplets. Following the flow described above, the bubble-forming unit 200 may use bubbles to attach the emulsion to the gas-liquid interface 255, or further operations may be performed.

[0238] Methods for detaching and / or recovering cells include using a special substrate that reacts to temperature and light to locally denature the substrate and detach the cells, and using ultrasound to detach cells. However, these methods require means for recovering the cells. The method of the present invention, however, does not require a special substrate and provides both means for detaching and recovering cells. Furthermore, one method for recovering detached cells is to recover them using a liquid flow that aspirates the liquid, such as with an aspirator. However, this method may recover cells and a large amount of liquid simultaneously, or may involve cells other than the target cells. However, the method of the present invention allows for the recovery of cells attached to the gas-liquid interface by incorporating the gas-liquid interface into the nozzle. This enables the easy recovery of target cells with a very small amount of liquid, without involving cells other than the target cells. In addition, it is known that applying a strong liquid flow when aspirating liquid can adversely affect cells, but such adverse effects can be avoided by using the method of the present invention.

[0239] In the method for recovering the object to be operated 35 shown in Figure 4, the details of the method for detaching the object to be operated 35 from the bottom 25a of the container 25 will be described below. Conventionally, when recovering the object to be operated 35, the force vector applied to the object to be operated 35 at the gas-liquid interface 255 was not controlled. In this embodiment, the gas-liquid interface 255 controls the force vector applied to the object to be operated 35, thereby detaching the object to be operated 35 from the inner bottom surface of the container 25 and facilitating the recovery of the object to be operated 35.

[0240] Furthermore, by implementing the method in this embodiment, it is possible not only to detach and recover the object to be manipulated 35 from the bottom 25a of the container 25, but also to perform other operations such as compressing the object to be manipulated 35 to recover the cytoplasm and / or cell membrane, compressing the object to be manipulated 35 to apply stimulation, compressing the object to be manipulated 35 to cause it to burst or cut, compressing the object to be manipulated 35 to observe it, moving the object to be manipulated 35 to concentrate it in a certain area, or detaching the object to be manipulated 35 from the gas-liquid interface 255.

[0241] Figure 13 shows an example of a flow in this embodiment that controls the force vector applied by the gas-liquid interface 255 to the object being worked on 35. In this embodiment, by performing the processes from S710 to S730 in this order, the object being worked on 35 is detached from the inner bottom surface of the container 25, making it easier to recover the object being worked on 35. However, the processes from S710 to S730 do not have to be performed in this order. For example, the process of S720 may be performed before the process of S710, or the processes of S710 and S720 may be performed simultaneously. Also, S730 may be performed simultaneously with S720.

[0242] First, in S710, the end 254 of the nozzle 49 is placed in the liquid 261 in which the object to be operated 35 is immersed, and gas is supplied to the flow path 51 by a pump, thereby forming bubbles 256 in the flow path 51 of the nozzle 49 or at the end 254 of the nozzle 49. By forming bubbles 256, a gas-liquid interface 255 between the liquid 261 and the gas is formed. The step of forming bubbles 256 in the flow path 51 of the nozzle 49 or at the end 254 of the nozzle 49 can be carried out by the method shown in the description of Figure 2B or Figure 4 above.

[0243] Next, in S720, the force vector applied from the gas-liquid interface 255 to the object being manipulated 35 is controlled. In this embodiment, the bubble-forming unit 200 functions as a vector control unit that controls the force vector applied from the gas-liquid interface 255 to the object being manipulated 35.

[0244] Figure 14 shows an example of a method for controlling the force vector applied from the gas-liquid interface 255 to the object being manipulated 35 in this embodiment. As shown in Figure 14, the method for controlling the force vector applied from the gas-liquid interface 255 to the object being manipulated 35 includes a method 910 for controlling the magnitude of the force applied from the gas-liquid interface 255 to the object being manipulated 35, and a method 920 for controlling the direction of the force applied from the gas-liquid interface 255 to the object being manipulated 35.

[0245] The method 910 for controlling the magnitude of the force applied from the gas-liquid interface 255 to the object being manipulated 35 includes a method 911 for controlling the internal pressure (gas pressure) of the bubbles 256 that form the gas-liquid interface 255, and a method 912 for controlling the acceleration of the movement of the gas-liquid interface 255. Here, the method 911 for controlling the internal pressure of the bubbles 256 and the method 912 for controlling the acceleration of the movement of the gas-liquid interface 255 can be used in combination.

[0246] Figure 15 shows an example of a schematic diagram illustrating the control of the internal pressure of the bubble 256 in this embodiment. Figures 15(a) and (b) show the case where the bubble 256 presses the object to be operated 35 against the wall W, and the object to be operated 35 pushes back with a force of magnitude F. Figure 15(a) shows the case where the force applied by the internal pressure of the bubble 256 is less than F, and Figure 15(b) shows the case where the force applied by the internal pressure of the bubble 256 is greater than F. Using Figures 15(a) and (b), a method 911 for controlling the internal pressure of the bubble 256 will be explained.

[0247] As shown in Figure 15(a), when there is a wall W in the direction in which the object to be manipulated 35 is being pushed, if the force applied by the internal pressure of the bubble 256 is smaller than the force F that the object to be manipulated 35 pushes back, the bubble 256 cannot push the object to be manipulated 35, and the object to be manipulated 35 falls into the bubble 256. On the other hand, as shown in Figure 15(b), when the force applied by the internal pressure of the bubble 256 is larger than the force F that the object to be manipulated 35 pushes back, the bubble 256 can push the object to be manipulated 35, and the object to be manipulated 35 can be crushed and deformed.

[0248] As described above, when there is a wall W in the direction in which the object 35 is being pushed, it is not possible to push the object 35 with a force greater than the internal pressure of the bubble 256. Therefore, the internal pressure of the bubble 256 defines the maximum value of the force that pushes the object 35 when there is a wall W in the direction in which the object 35 is being pushed. Thus, the internal pressure of the bubble 256 is controlled in order to control the maximum value of the force that the gas-liquid interface 255 applies in the direction in which the object 35 is being pushed.

[0249] In Figure 15, a wall W is assumed for illustrative purposes, but this can also be applied to the case where the object to be manipulated 35 is adhered to the bottom 25a of the container 25. When the object to be manipulated 35 is pushed from the side by the gas-liquid interface 255 of the bubble 256, if the maximum force applied by the internal pressure of the bubble 256 is less than the force with which the object to be manipulated 35 is adhered, the bubble 256 will not be able to push the object to be manipulated 35, and the object to be manipulated 35 will be drawn into the bubble 256. On the other hand, if the maximum force applied by the internal pressure of the bubble 256 is greater than the force with which the object to be manipulated 35 is adhered, the bubble 256 can push the object to be manipulated 35, and the object to be manipulated 35 can be pushed and detached.

[0250] Returning to Figure 14, methods for controlling the internal pressure of the bubble 256 include a method 913 for controlling the internal pressure of the bubble 256 using a pressure generating unit 47, a method 914 for controlling the radius of curvature at the point where the gas-liquid interface 255 and the object being worked on 35 come into contact, and a method 915 for controlling the interfacial free energy E2 of the gas-liquid interface 255. These methods 913 to 915 can be used in combination.

[0251] In method 913, where the pressure generating unit 47 controls the internal pressure of the bubble 256, the end 254 of the flow path 51 of the nozzle 49 is immersed in the liquid 261, and gas supplied from the syringe pump is introduced into the liquid 261 from the end 254 of the flow path 51. Then, the pressure generating unit 47 connected to the nozzle 49 causes the plunger of the syringe pump to reciprocate, thereby supplying gas to the flow path 51 of the nozzle 49 to pressurize it, or drawing gas from the flow path 51 of the nozzle 49 to depressurize it, thereby adjusting the internal pressure (gas pressure) of the bubble 256.

[0252] Equation 1 below is a formula used to explain method 914 for controlling the internal pressure of bubble 256 using the radius of curvature of the gas-liquid interface 255, and method 915 for controlling it using the interfacial free energy E2 of the gas-liquid interface 255. In Equation 1 below, Pin represents the internal pressure of bubble 256, Pout represents the liquid pressure, ΔP represents the absolute value of the difference between the internal pressure of bubble 256 and the liquid pressure, E2 represents the interfacial free energy (surface tension) of the gas-liquid interface 255, and r represents the radius of curvature of the gas-liquid interface 255. When Pout is constant, the last equation of Equation 1 is obtained. [Formula 1] ΔP = |Pout - Pin| = E2 / r Therefore, Pin = E² / r

[0253] From Equation 1, it can be seen that the internal pressure of bubble 256 is proportional to the interfacial free energy E2 of the gas-liquid interface 255 and inversely proportional to the radius of curvature r of the gas-liquid interface 255. Therefore, the internal pressure of bubble 256 can be controlled by controlling the interfacial free energy E2 of the gas-liquid interface 255 and the radius of curvature r at the point of contact between the gas-liquid interface 255 and the object being manipulated 35.

[0254] The following describes a method 914 for controlling the radius of curvature r of the gas-liquid interface 255. As shown in Figure 14, the method 914 for controlling the radius of curvature r of the gas-liquid interface 255 includes a method 918 for controlling the inner diameter of the nozzle 49 and a method 919 for controlling the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25. These methods 918 and 919 can be used in combination. Note that the method 914 for controlling the radius of curvature r of the gas-liquid interface 255 may be performed before the process S710 in Figure 13.

[0255] Figure 16 shows an example of a schematic diagram illustrating the relationship between the inner diameter of the nozzle 49 and the radius of curvature r of the gas-liquid interface 255 in this embodiment. Figure 16(a) shows a nozzle 49a with a large inner diameter R1, and Figure 16(b) shows a nozzle 49b with a small inner diameter R2. The inner diameter of the nozzle 49 refers to the inner diameter of the hollow cross-section of the flow path 51 of the nozzle 49. As shown in Figure 16(a), when a nozzle 49a with a large inner diameter R1 is used, the radius of curvature r of the gas-liquid interface 255 becomes large, and as shown in Figure 16(b), when a nozzle 49b with a small inner diameter R2 is used, the radius of curvature r of the gas-liquid interface 255 becomes small. Although Figure 16 shows an example where the nozzle 49 is cylindrical, i.e., the cross-sectional shape of the nozzle 49 is circular, the cross-sectional shape of the nozzle 49 does not have to be circular. In such cases, the maximum or average inner diameter of the nozzle 49 correlates with the radius of curvature r of the gas-liquid interface 255.

[0256] From the above, the method 918 for controlling the inner diameter of the nozzle 49 allows for the control of the radius of curvature r of the gas-liquid interface 255 by preparing a nozzle 49 with an inner diameter corresponding to the maximum value of the pressing force to be applied to the target object 35, and therefore, the maximum value of the pressing force to be applied to the target object 35 can be controlled.

[0257] Figure 17 shows an example of a schematic diagram illustrating the relationship between the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 and the radius of curvature r of the gas-liquid interface 255 in this embodiment. Figure 17(a) shows the case where the distance L1 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is long, and Figure 17(b) shows the case where the distance L2 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is short. As shown in Figure 17(a), when the distance L1 is long, the bubbles 256 formed at the end 254 of the nozzle 49 are not sufficiently crushed, so the radius of curvature r of the gas-liquid interface 255 is large, and as shown in Figure 17(b), when the distance L2 is short, the bubbles 256 formed at the end 254 of the nozzle 49 are sufficiently crushed, so the radius of curvature r of the gas-liquid interface 255 is small. Furthermore, when the distance L2 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is short, the relationship between the distance L2 and the radius of curvature r of the gas-liquid interface 255 may be measured in advance and stored in the recording unit 190.

[0258] From the above, the method 919 for controlling the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 allows for the control of the radius of curvature r of the gas-liquid interface 255 by controlling the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25, and therefore allows for the control of the maximum force applied to the object to be operated 35.

[0259] In the method 915 for controlling the interfacial free energy E2 of the bubble 256, as shown in Equation 1 above, the internal pressure of the bubble 256 and the interfacial free energy E2 of the gas-liquid interface 255 are proportional. It can be seen that increasing the interfacial free energy E2 of the gas-liquid interface 255 between the gas and the liquid 261 increases the internal pressure of the bubble 256, and decreasing the interfacial free energy E2 decreases the internal pressure of the bubble 256. This makes it possible to control the magnitude of the force applied from the gas-liquid interface 255 to the object being manipulated 35.

[0260] The interfacial free energy E2 of the gas-liquid interface 255 can be controlled by the energy control unit by changing the type and composition of the solute. For example, adding an inorganic salt as a solute to the liquid can increase the interfacial free energy E2, while adding a polar organic compound or surfactant as a solute can decrease the interfacial free energy E2.

[0261] Furthermore, the magnitude of the interfacial free energy E2 at the gas-liquid interface 255 may be controlled by adding other liquids to the liquid in the container 25. If the solution is a complete culture medium, buffer solution, basic culture medium, or water may be added as the other liquid. In this case, the concentration of the solute in the complete culture medium changes by adding the other liquid. Therefore, the interfacial free energy E2 at the gas-liquid interface 255 can be adjusted.

[0262] The interfacial free energy E2 at the gas-liquid interface 255 can be controlled by the energy control unit by changing the type and composition of the gas.

[0263] Furthermore, the magnitude of the interfacial free energy E2 may be adjusted by removing inorganic salts or polar organic compounds from the solution. Removal of inorganic salts may be performed by adding a chelating agent such as EDTA to the solution. Removal of polar organic compounds or surfactants may be performed by adding a substrate such as a filter, column, or beads to the solution, and the substrate adsorbing the polar organic compounds or surfactants in the solution.

[0264] The following describes method 912 for controlling the moving acceleration of the gas-liquid interface 255. The force applied to the object being manipulated 35 is the product of the mass of the object being manipulated 35 and the moving acceleration of the gas-liquid interface 255 to which the object being manipulated 35 is attached. In other words, the force applied to the object being manipulated 35 attached to the gas-liquid interface 255 is proportional to the moving acceleration of the gas-liquid interface 255 to which the object being manipulated 35 is attached. Therefore, by controlling the moving acceleration of the gas-liquid interface 255, the magnitude of the force applied from the gas-liquid interface 255 to the object being manipulated 35 can be controlled. Originally, the sum of the products of the mass and acceleration at each minute part of the object being manipulated 35 becomes the force applied to the object being manipulated 35, and by further considering the normal force and frictional force applied to the object being manipulated 35, the accurate applied force can be calculated. On the other hand, since the object being manipulated 35 is very small, even by ignoring these effects and using the simplified approach described above, results that match theory and effect can be obtained.

[0265] Returning to Figure 14, the method 912 for controlling the moving acceleration of the gas-liquid interface 255 includes a method 916 for controlling the acceleration of the nozzle 49 and a method 917 for controlling the pressurizing acceleration of the syringe pump. These methods 916 and 917 can be used in combination.

[0266] In method 916 for controlling the acceleration of the nozzle 49, the acceleration of the gas-liquid interface 255 is controlled by controlling the acceleration of the flow path 51 of the nozzle 49 in which the bubbles 256 are formed. The bubble forming unit 200 determines the acceleration of the nozzle actuator 40 that operates the nozzle 49 and sends an instruction to the nozzle actuator 40 to operate at the determined acceleration. The nozzle actuator 40 operates the nozzle 49 at the determined acceleration.

[0267] In a method 917 for controlling the pressurization acceleration of a syringe pump, the movement acceleration of a gas-liquid interface 255 is controlled by controlling the acceleration of gas supply or intake from the syringe pump. In this case, a pressure generation unit 47 of a bubble formation unit 200 controls a plunger to control the acceleration of supplying gas from the syringe pump or the acceleration of taking in gas into the syringe pump. Thereby, the pressurization acceleration of bubbles 256 formed at an end portion 254 of a nozzle 49 is controlled to control the movement acceleration of the gas-liquid interface 255.

[0268] FIG. 18 shows an example of a schematic diagram for explaining the direction of the force applied from the gas-liquid interface 255 to an operation target 35 in the present embodiment. The lower diagram of FIG. 18 shows a partially enlarged view of the vicinity of the operation target 35 in the upper diagram of FIG. 18. A method 920 for controlling the direction of the force applied from the gas-liquid interface 255 to the operation target 35 will be described using FIG. 18. As shown in FIG. 18, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 is along a straight line connecting a contact point P where the gas-liquid interface 255 and the operation target 35 contact and a center point Q of a circle assuming that the curve of the gas-liquid interface 255 at the contact point P is a circle, and is determined by whether the gas-liquid interface 255 moves from the gas side toward the liquid side or from the liquid side toward the gas side. Therefore, in the vector control stage of S720, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 can be controlled by controlling the orientation and the movement direction of the surface at the contact point P where the gas-liquid interface 255 and the operation target 35 contact. In the present embodiment, in the vector control stage of S720, forming and maintaining bubbles 256 at an end portion 254 of the nozzle 49 may be included.

[0269] In the example shown in FIG. 18, since the gas-liquid interface 255 is pushing the operation target 35 downward to the right, a force is applied from the gas-liquid interface 255 to the operation target 35 in the direction of D1. That is, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 is from the gas side to the liquid side of the gas-liquid interface 255. However, when the gas-liquid interface 255 pulls the operation target 35 while the operation target 35 is attached to the gas-liquid interface 255, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 is, contrary to the above, from the liquid side to the gas side of the gas-liquid interface 255. Further, in the example shown in FIG. 18, the explanation is based on the premise that the gas-liquid interface 255 is stationary. However, when the gas-liquid interface 255 is moving in a specific direction, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 is determined by the direction of the surface at the contact point P where the gas-liquid interface 255 and the operation target 35 contact and the moving direction of the gas-liquid interface 255. For example, when the gas-liquid interface 255 is moving to the right in FIG. 18, the direction of the force applied from the gas-liquid interface 255 to the operation target 35 has a larger rightward component and is a slightly lateral direction compared to the direction D1.

[0270] Returning to FIG. 14, the method 920 for controlling the direction of the force applied from the gas-liquid interface 255 to the operation target 35 includes a method 921 for controlling the inner diameter of the nozzle 49, a method 922 for controlling the distance between the end portion 254 of the nozzle 49 and the bottom portion 25a of the container 25, and a method 923 for adjusting the traveling direction of the gas-liquid interface 255. These methods 921 to 923 can be used in combination.

[0271] FIG. 19 shows an example of a schematic diagram for explaining the method 921 for controlling the direction of the force by controlling the inner diameter of the nozzle 49 in the present embodiment. FIG. 19(a) shows the case where the nozzle 49a with a thick inner diameter R1 is used, and FIG. 19(b) shows the case where the nozzle 49b with a thin inner diameter R2 is used. The middle diagrams in FIGS. 19(a) and (b) show partial enlarged views near the operation target 35 in the upper diagrams of FIGS. 19(a) and (b), and the lower diagrams in FIGS. 19(a) and (b) show experimental images of the operation target 35.

[0272] As shown in Figure 19(a), when a nozzle 49a with a wide inner diameter R1 is used, the radius of curvature r at the contact point P of the bubble 256 becomes large, and the line connecting the center point Q of the circle to the contact point is relatively downward. Therefore, the gas-liquid interface 255 contacts the object being worked on 35 from a slightly upward direction. Thus, when the gas-liquid interface 255 moves from the gas side towards the liquid side, the direction in which the gas-liquid interface 255 applies force to the object being worked on 35 is in the direction of D1. In this case, as shown in the experimental image in the lower part of Figure 19(a), the object being worked on 35 goes under the bubble 256 without detaching and is compressed. On the other hand, as shown in Figure 19(b), when a nozzle 49b with a narrow inner diameter R2 is used, the radius of curvature r at the contact point P of the bubble 256 becomes small, and the line connecting the center point Q of the circle to the contact point is relatively lateral. Therefore, the gas-liquid interface 255 contacts the object being worked on 35 from a slightly lateral direction. Therefore, when the gas-liquid interface 255 moves from the gas side towards the liquid side, the direction in which the gas-liquid interface 255 applies force to the object being manipulated 35 is in the direction of D2. In this case, as shown in the experimental image in the lower part of Figure 19(b), the object being manipulated 35 is separated from the bottom 25a of the container 25. The direction of the force that the gas-liquid interface 255 exerts on the object being manipulated 35 is also determined by the relative sizes of the inner diameter of the nozzle 49 and the diameter of the object being manipulated 35. For example, if the inner diameter of the nozzle 49 and the diameter of the object being manipulated 35 are approximately the same, the direction of the force that the gas-liquid interface 255 exerts on the object being manipulated 35 is relatively lateral.

[0273] As described above, when a nozzle 49a with a large inner diameter R1 is used, a force is applied in the direction D1 that crushes the object to be worked on 35 from above, and when a nozzle 49b with a small inner diameter R2 is used, a force is applied to the object to be worked on 35 in a slightly lateral direction D2, making it possible to separate it from the bottom 25a. As described above, by preparing a nozzle 49 with an inner diameter corresponding to the magnitude of the force to be applied to the object to be worked on 35, the radius of curvature r at the contact point P where the gas-liquid interface 255 and the object to be worked on 35 come into contact can be controlled, and the orientation of the surface and the direction of movement of the surface at the contact point P where the gas-liquid interface 255 and the object to be worked on 35 come into contact can be controlled, thereby controlling the direction of the force applied to the object to be worked on 35. Note that the method 921 for controlling the inner diameter of the nozzle 49 may be performed before the process S710 in Figure 13.

[0274] Figure 20 shows an example of a schematic diagram illustrating a method 922 for controlling the direction of force by controlling the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 in this embodiment. Figure 20(a) shows the case where the distance L1 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is long, and Figure 20(b) shows the case where the distance L2 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is short. The middle diagrams in Figures 20(a) and (b) show magnified views of the area around the target object 35 in the upper diagrams of Figures 20(a) and (b), and the lower diagrams in Figures 20(a) and (b) show experimental images of the target object 35.

[0275] As shown in Figure 20(a), when the distance L1 is long, the bubble 256 is not sufficiently compressed, the radius of curvature r at the contact point P of the bubble 256 is large, and the line connecting the center point Q of the circle is relatively downward. Therefore, the gas-liquid interface 255 contacts the object being manipulated 35 from a slightly upward direction. Thus, when the gas-liquid interface 255 moves from the gas side towards the liquid side, the direction in which the gas-liquid interface 255 applies force to the object being manipulated 35 is in the direction of D1. In this case, as shown in the experimental image in the lower part of Figure 20(a), the object being manipulated 35 does not detach and goes under the bubble 256, and is compressed. On the other hand, as shown in Figure 20(b), when the distance L2 is short, the bubble 256 is sufficiently compressed, the radius of curvature r at the contact point P of the bubble 256 is small, and the line connecting the center point Q of the circle is relatively lateral. Therefore, the gas-liquid interface 255 contacts the object being manipulated 35 from a slightly lateral direction. Therefore, when the gas-liquid interface 255 moves from the gas side towards the liquid side, the direction in which the gas-liquid interface 255 applies force to the object being manipulated 35 is in the direction of D3. In this case, as shown in the experimental image in the lower part of Figure 20(b), the object being manipulated 35 is separated from the bottom 25a of the container 25.

[0276] As described above, when the distance L1 between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is long, force is applied by crushing the object to be operated 35 from above, and when the distance L2 is short, force can be applied to the object to be operated 35 from slightly to the side. Therefore, the nozzle position control unit can move the flow path 51 or the container 25 to a position at a first distance, which is a distance at which the gas-liquid interface 255 can contact the bottom 25a of the container 25, and move the flow path 51 to a second position where the distance between the end 254 and the bottom 25a of the container 25 is shorter than the first distance, while the gas-liquid interface 255 formed at the end 254 by the syringe pump is in contact with the bottom 25a of the container 25. As described above, by controlling the distance between the end portion 254 of the nozzle 49 and the bottom portion 25a of the container 25, the radius of curvature r at the contact point P where the gas-liquid interface 255 and the object to be operated 35 come into contact can be controlled, and the orientation of the surface and the direction of movement of the surface at the contact point P can be controlled, thereby controlling the direction of the force applied to the object to be operated 35.

[0277] In method 923 for adjusting the direction of movement of the gas-liquid interface 255, the nozzle actuator 40 adjusts the direction of movement of the gas-liquid interface 255, thereby moving the nozzle 49 in a desired direction to control the direction of the force applied to the object 35. For example, by moving the nozzle 49 to the right while the gas-liquid interface 255 and the object 35 are in contact, the direction of the force applied by the gas-liquid interface 255 to the object 35 can be made slightly to the right. Also, by moving the nozzle 49 downwards while the gas-liquid interface 255 and the object 35 are in contact, the direction of the force applied by the gas-liquid interface 255 to the object 35 can be made slightly downward. The direction of movement of the gas-liquid interface 255 can also be adjusted by changing the pressure of the syringe pump to move the gas-liquid interface 255.

[0278] This completes the control of the force vector applied from the gas-liquid interface 255 to the object being manipulated 35, as shown in S720 of Figure 13. Next, the process moves to the stage of applying force to the object being manipulated 35, as shown in S730 of Figure 13. In the stage of applying force to the object being manipulated 35 in S730, force is applied to the object being manipulated 35 based on the force vector to be applied to the object being manipulated 35 determined in S720. The force applied to the object being manipulated 35 is performed by the nozzle actuator 40 of the bubble forming unit 200 operating the nozzle 49 and / or stage to move the gas-liquid interface 255 relative to the bottom 25a of the container 25. Alternatively, the pressure generating unit 47 connected to the nozzle 49 may reciprocate the plunger of the syringe pump to supply or draw air to the bubbles 256, thereby applying force to the object being manipulated 35.

[0279] Figure 21 shows an example of a schematic diagram illustrating the relationship between the inner diameter of the nozzle 49, the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25, the radius of curvature of the gas-liquid interface 255, the maximum force applied to the cells that are the target of manipulation 35, the magnitude of the downward force component (compression force), the magnitude of the lateral force component (detachment force), the area of ​​the gas-liquid interface 255 to which the cells that are the target of manipulation 35 can adhere, the controllability of the nozzle 49, the number of target cells, and the cell adhesion relaxation treatment in this embodiment. Regarding the compression force and detachment force, it is assumed that the cells are pushed across the gas-liquid interface 255 while the target of manipulation 35 is in contact with the bottom 25a. As shown in Figure 21, if the inner diameter of the nozzle 49 is large and the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is long, when attempting to push the cells 35 that are the target of manipulation, the component of the compressive force on the cells is large and the component of the detachment force is small, resulting in the cells being compressed from above. Therefore, in order to detach the cells, it is necessary to first apply a strong cell adhesion relaxation treatment to relax the cell adhesion.

[0280] On the other hand, if the inner diameter of the nozzle 49 is large and the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is short, when attempting to push the cells 35 that are the target of manipulation, the component of the compressive force on the cells is small and the component of the detachment force is large, resulting in pushing the cells laterally. In this case, a weak cell adhesion relaxation treatment is sufficient, or even unnecessary, to detach the cells.

[0281] Furthermore, even when the inner diameter of the nozzle 49 is small and the distance between the end 254 of the nozzle 49 and the bottom 25a of the container 25 is short, when attempting to press the cells 35 that are the target of manipulation, the component of the compressive force on the cells is small and the component of the detachment force is large, resulting in the cells being pressed laterally. Therefore, to detach the cells, a weak cell adhesion relaxation treatment is sufficient beforehand, or a cell adhesion relaxation treatment may not be necessary at all.

[0282] As described above, by controlling the force vector applied by the gas-liquid interface 255 to the object 35, for example, a lateral force can be effectively applied to the object 35 to detach it from the bottom 25a of the container 25, making it easier to retrieve the object 35. Furthermore, by increasing the downward force component applied to the object 35 during the process of moving the gas-liquid interface 255 so that the cells are pushed, the object 35 can be compressed from above, allowing for observation of the cells, which are the object 35, under pressure from above. At this time, the maximum force applied to the object 35 becomes the internal pressure of the bubble 256. Therefore, for example, by monitoring the internal pressure of the bubble 256 while observing, the force applied to the object 35 and the changes in the observed image can be easily captured in real time. Moreover, if it is desired to observe the object 35 under pressure from directly above, the gas-liquid interface 255 can be brought into contact with the object 35 from directly above to compress it. In this case, the bubble 256 is controlled so that the area near its apex contacts a specific object 35.

[0283] The vector control step in S720 may also include control to move the gas-liquid interface 255 so that it comes into contact with a predetermined target object 35. In this case, the bubble-forming unit 200 obtains the horizontal position (XY position) of the gas-liquid interface 255 and the target target object 35 from the image captured by the camera 60 or camera 70, and obtains the vertical position (Z position) of the gas-liquid interface 255 and the target target object 35 from the image captured by the flow path imaging camera 42 to determine the relative positional relationship between the gas-liquid interface 255 and the target target object 35. Subsequently, based on the determined relative positional relationship, the bubble-forming unit 200 moves the nozzle actuator 40 to move the nozzle 49 and / or stage to bring the gas-liquid interface 255 into contact with the target target object 35. Alternatively, the operator may input the amount of movement of the nozzle 49 and / or stage based on the determined relative positional relationship to move the nozzle 49 and / or stage to bring the gas-liquid interface 255 into contact with the target target object 35.

[0284] Alternatively, the vector control step S720 may be performed by installing a miniature pressure gauge at the bottom 25a of the container 25 containing the liquid 261 and detecting the pressure from the gas-liquid interface 255 with the miniature pressure gauge. In this case, the sensor unit 48 detects the pressure generated at the bottom 25a of the container 25, and the sensor unit 48 sends this information to the bubble-forming unit 200. The bubble-forming unit 200 may perform feedback control of the pressure generation unit 47 based on the detected pressure.

[0285] Furthermore, the bubble-forming unit 200 may acquire contact images of the gas-liquid interface 255 and the object to be operated 35 using at least one of the cameras 60, 70 and the flow path imaging camera 42, and understand the contact conditions between the gas-liquid interface 255 and the object to be operated 35 to estimate the direction of the force that the gas-liquid interface 255 applies to the object to be operated 35. Based on the estimated direction of the force, the bubble-forming unit 200 may move the nozzle 49 and / or the stage to perform feedback control so that the direction of the force is in a predetermined direction.

[0286] For example, if a contact image like the one shown in the lower part of Figure 19(a) is acquired and the gas-liquid interface 255 moves from the gas side towards the liquid side, it can be inferred that the direction of the force applied by the gas-liquid interface 255 to the object being worked on 35 is in the direction of D1. Here, if we want to apply a lateral force to the object being worked on 35 to separate it from the bottom 25a of the container 25, the direction of D1 has a slightly larger downward component, which would compress the object being worked on 35 from above, so it is not a desirable direction. In this case, by moving the nozzle 49 to the right in Figure 19(a) while the gas-liquid interface 255 and the object being worked on 35 are in contact, the direction of the force applied by the gas-liquid interface 255 to the object being worked on 35 can be made slightly to the right, bringing it closer to the direction of D2 in the lower part of Figure 19(b). This makes it possible to effectively apply a lateral force to the object being worked on 35 and separate it from the bottom 25a of the container 25. Furthermore, in the state shown in Figure 19(b), by moving the nozzle 49 to the right, the direction of the force applied by the gas-liquid interface 255 to the target object 35 can be shifted further to the right from the direction of D2.

[0287] Figure 22 illustrates the detachment of the object to be manipulated 35 from the gas-liquid interface 255 in this embodiment. The lower figures in Figures 22(a) and (b) show experimental images of the upper figures in Figures 22(a) and (b). By controlling the force vector applied by the gas-liquid interface 255 to the object to be manipulated 35, the object to be manipulated 35 attached to the gas-liquid interface 255 can be detached. As shown in Figures 22(a) and (b), the bubble forming unit 200 controls the pressure generating unit 47 to draw in gas, thereby moving the gas-liquid interface 255 upward in Figures 22(a) and (b), and thus detaching the object to be manipulated 35 from the gas-liquid interface 255. In the lower figure of Figure 22(a), the object to be manipulated 35, which was attached to the gas-liquid interface 255, is detached from the gas-liquid interface 255 in the lower figure of Figure 22(b).

[0288] Furthermore, with the object to be manipulated 35 attached to the gas-liquid interface 255, the orientation of the surface at the contact point and the direction of movement of the surface may be controlled to apply a force to the object to be manipulated 35 in a specific direction (moving it from the liquid side towards the gas side). When the force pulling the object to be manipulated 35 is greater than the force that causes the object to be manipulated 35 to adhere to the gas-liquid interface 255, the object to be manipulated 35 will detach from the gas-liquid interface 255. For example, the pressure generating unit 47 connected to the nozzle 49 can pull the plunger of the syringe pump to draw in air, moving the gas-liquid interface 255 to which the object to be manipulated 35 is attached from the liquid side towards the gas side, thereby detaching the object to be manipulated 35 from the gas-liquid interface 255. Alternatively, the pressure generating unit 47 can reciprocate the plunger of the syringe pump to effectively apply a pulling force to the object to be manipulated 35, thereby detaching the object to be manipulated 35 from the gas-liquid interface 255. In addition, the nozzle 49 may be moved to vibrate the gas-liquid interface 255 to which the object to be manipulated 35 is attached. This effectively applies a pulling force to the object being manipulated 35, allowing the object being manipulated 35, which was attached to the gas-liquid interface 255, to be detached from the gas-liquid interface 255.

[0289] Figure 23 shows an example of the hardware configuration of a computer 1900 that functions as an information processing device 170. The computer 1900 according to this embodiment includes a CPU peripheral unit having a CPU 2000, RAM 2020, graphics controller 2075, and display device 2080 which are interconnected by a host controller 2082; an input / output unit having a communication interface 2030, hard disk drive 2040, and 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, flexible disk drive 2050, and input / output chip 2070 which are connected to the input / output controller 2084.

[0290] The host controller 2082 connects the RAM 2020 with the CPU 2000 and the graphic controller 2075 that access the RAM 2020 at a high transfer rate. The CPU 2000 operates based on programs stored in the ROM 2010 and the RAM 2020, and controls each part. The graphic controller 2075 acquires the image data generated by the CPU 2000 or the like on the frame buffer provided in the RAM 2020, and causes it to be displayed on the display device 2080. Instead of this, the graphic controller 2075 may internally include a frame buffer that stores the image data generated by the CPU 2000 or the like. The display device 2080 can display various information (for example, images, position information of the operation target 35, etc.) generated inside the information processing device 170.

[0291] The input / output controller 2084 connects the host controller 2082 with the communication interface 2030, the hard disk drive 2040, and the 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. Also, the communication interface functions as hardware for performing 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 a program or data from the CD-ROM 2095 and provides it to the hard disk drive 2040 via the RAM 2020.

[0292] Furthermore, the I / O controller 2084 is connected to the ROM 2010, the flexible disk drive 2050, and the relatively slow I / O devices of the I / O chip 2070. The ROM 2010 stores the boot program that the computer 1900 runs when it starts up, and / or programs that depend on the computer 1900's hardware. The flexible disk drive 2050 reads programs or data from the flexible disk 2090 and provides them to the hard disk drive 2040 via the RAM 2020. The I / O chip 2070 connects the flexible disk drive 2050 to the I / O controller 2084 and also connects various I / O devices to the I / O controller 2084 via, for example, a parallel port, serial port, keyboard port, mouse port, etc.

[0293] The program provided to the hard disk drive 2040 via RAM2020 is stored on a recording medium such as a flexible disk 2090, CD-ROM 2095, or IC card and provided by the user. The program is read from the recording medium, installed on the hard disk drive 2040 in the computer 1900 via RAM2020, and executed by the CPU 2000.

[0294] The program installed on the computer 1900, which causes the computer 1900 to function as an information processing device 170, includes a bubble formation module, an energy control module, and an operation module. These programs or modules may interact with the CPU 2000, etc., to cause the computer 1900 to function as a bubble formation unit 200, a liquid control unit 260, etc.

[0295] The information processing described in these programs is read by the computer 1900 and functions as a specific means, such as a bubble forming unit 200 or a liquid control unit 260, in which the software and the various hardware resources described above work together. Then, by performing calculations or processing of information according to the purpose of use of the computer 1900 in this embodiment, a specific information processing device 170 tailored to the purpose of use is constructed.

[0296] For example, when computer 1900 communicates with an external device, the CPU 2000 executes a communication program loaded onto RAM 2020 and instructs the communication interface 2030 to perform communication processing based on the processing content described in the communication program. The communication interface 2030, under the control of the CPU 2000, reads transmission data stored in a transmission buffer area on a storage device such as RAM 2020, hard disk drive 2040, flexible disk 2090, or CD-ROM 2095 and sends it to the network, or writes received data received from the network to a reception buffer area on the storage device. In this way, the communication interface 2030 may transfer transmission and reception data to and from the storage device using the DMA (Direct Memory Access) method, or alternatively, the CPU 2000 may transfer transmission and reception data by reading data from the source storage device or communication interface 2030 and writing the data to the destination communication interface 2030 or storage device.

[0297] Furthermore, the CPU 2000 reads all or necessary parts of files or databases stored in external storage devices such as the hard disk drive 2040, CD-ROM drive 2060 (CD-ROM 2095), and flexible disk drive 2050 (flexible disk 2090) into the RAM 2020 via DMA transfer, and performs various processing on the data in the RAM 2020. Then, the CPU 2000 writes the processed data back to the external storage device via DMA transfer, etc. In this process, the RAM 2020 can be considered to temporarily hold the contents of the external storage device, so in this embodiment, the RAM 2020 and the external storage device are collectively referred to as memory, recording unit, or storage device, etc.

[0298] Here, the storage device stores information necessary for information processing by the information processing device 170, such as video data, as needed, and supplies it to each component of the information processing device 170 as needed.

[0299] In this embodiment, various types of information such as programs, data, tables, and databases are stored on such a storage device and are subject to information processing. The CPU 2000 can also hold a portion of the RAM 2020 in cache memory and perform reading and writing operations on the cache memory. Even in this configuration, the cache memory performs a part of the RAM 2020's functions; therefore, in this embodiment, unless otherwise specified, the cache memory is included in the RAM 2020, memory, and / or storage device.

[0300] Furthermore, the CPU2000 performs various operations on the data read from RAM2020, including various calculations, information processing, conditional judgments, and information retrieval / replacement as specified by the program's instruction sequence, and writes the data back to RAM2020. For example, when the CPU2000 performs a conditional judgment, it determines whether the various variables shown in this embodiment satisfy conditions such as being greater than, less than, greater than or equal to, less than or equal to, or equal to, compared with other variables or constants. If the condition is met (or not met), it branches to a different instruction sequence or calls a subroutine.

[0301] Furthermore, the CPU2000 can search for information stored in files or databases within the storage device. For example, if multiple entries are stored in the storage device, each corresponding to the attribute value of a second attribute, the CPU2000 can search among the multiple entries stored in the storage device for an entry whose attribute value of the first attribute matches a specified condition, and by reading the attribute value of the second attribute stored in that entry, it can obtain the attribute value of the second attribute associated with the first attribute that satisfies the predetermined condition.

[0302] The programs or modules described above may be stored on an external recording medium. In addition to the flexible disk 2090 and CD-ROM 2095, other recording media that can be used include optical recording media such as DVDs or CDs, magneto-optical recording media such as MOs, tape media, and semiconductor memory such as IC cards. Alternatively, a storage device such as a hard disk or RAM installed on a server system connected to a dedicated communication network or the Internet may be used as a recording medium, and the program may be provided to the computer 1900 via the network.

[0303] In this disclosure, the information processing device 170 is shown to have a CPU 2000 as a processor, but the type of processor is not particularly limited. For example, a GPU, ASIA, FPGA, etc. can be used as appropriate as the processor. Also, in this disclosure, the information processing device 170 is 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, other storage devices such as solid-state drives may be used instead of, or together with, the hard disk drive 2040.

[0304] 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 or improvements can be made to the above embodiments. It will be clear from the claims that such modified or improved forms may also be included in the technical scope of the present invention.

[0305] It should be noted that the execution order of operations, procedures, steps, and stages in the devices, systems, programs, and methods shown in the claims, specifications, and drawings is not explicitly stated as "before," "in advance," etc., and that these can be performed in any order unless the output of a previous process is used in a later process. Even if the operation flow in the claims, specifications, and drawings is described using phrases such as "first," "next," etc. for convenience, it does not mean that it is essential to perform the operations in that order.

[0306] (Note) [Item 1] A gas-liquid interface formation step involves placing the end of a channel in a liquid in which a living organism is immersed, and forming a gas-liquid interface between the liquid and the gas within or at the end of the channel. A vector control stage that controls the force vector applied to a living organism from the gas-liquid interface, The force application stage involves applying force to a living organism from the gas-liquid interface, A method of applying force to a living organism that possesses these characteristics. [Item 2] The vector control stage involves controlling the magnitude of the force applied to the organism from the gas-liquid interface. The method described in item 1. [Item 3] The vector control step involves controlling the magnitude of the force exerted on the organism from the gas-liquid interface by controlling the gas pressure of the gas at the gas-liquid interface. The method described in item 2. [Item 4] The gas-liquid interface formation step is performed by immersing the end of the channel in the liquid and introducing the gas supplied from the pump into the liquid from the end. The vector control step includes controlling the gas pressure of the gas at the gas-liquid interface by controlling the pressurization of the pump. The method described in item 3. [Item 5] The gas-liquid interface formation step is performed by immersing the end of the channel in the liquid and introducing the gas into the liquid from the end. The vector control step includes preparing a channel with an inner diameter corresponding to the magnitude of the force to be applied to the organism. The method described in item 3 or 4. [Item 6] The vector control step involves controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the radius of curvature of the portion of the gas-liquid interface in contact with the organism. The method described in any one of items 3 through 5. [Item 7] The vector control step involves controlling the magnitude of the force exerted on the organism from the gas-liquid interface by controlling the surface tension between the gas and the liquid. The method described in any one of items 3 through 6. [Item 8] The vector control stage involves controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the acceleration of the gas-liquid interface. The method described in any one of items 2 through 7. [Item 9] The gas-liquid interface formation step is performed by immersing the end of the channel in the liquid and introducing the gas into the liquid from the end. The vector control step includes controlling the acceleration of the gas-liquid interface by controlling the acceleration of the flow path. The method described in item 8. [Item 10] The gas-liquid interface formation step is performed by introducing gas supplied from the pump into the liquid from the end. The vector control step includes controlling the acceleration of the gas-liquid interface by controlling the pressurization acceleration of the pump. The method described in item 8 or 9. [Item 11] The vector control step includes detecting the pressure from the gas-liquid interface using a pressure sensor located at the bottom of the container holding the liquid. The method described in item 1. [Item 12] The vector control step involves controlling the direction of the force applied to the organism from the gas-liquid interface. The method described in item 1. [Item 13] The vector control step includes controlling the direction of the force applied to the organism from the gas-liquid interface by controlling the orientation and direction of movement of the surface of the part of the gas-liquid interface in contact with the organism. The method described in item 12. [Item 14] The vector control step includes controlling the orientation of the surface of the portion of the gas-liquid interface in which the organism comes into contact, by controlling the radius of curvature of that portion of the gas-liquid interface. The method described in item 13. [Item 15] The gas-liquid interface formation step is performed by immersing the end of the channel in the liquid and introducing the gas into the liquid from the end. The vector control step involves controlling the orientation of the surface of the portion of the gas-liquid interface that comes into contact with the organism by controlling the distance between the end and the bottom of the container holding the liquid. The method described in item 13 or 14. [Item 16] The gas-liquid interface formation step is performed by immersing the end of the channel in the liquid and introducing the gas into the liquid from the end. The vector control step includes preparing a channel with an inner diameter corresponding to the direction of the force to be applied to the organism. The method described in any one of items 13 to 15. [Item 17] The vector control step includes controlling the direction of the force applied to the living organism from the gas-liquid interface from the gas side to the liquid side, or from the liquid side to the gas side of the gas-liquid interface. The method described in item 12. [Item 18] The vector control step includes controlling the movement of the gas-liquid interface so that it comes into contact with a predetermined biological body. The method described in any one of items 1 through 17. [Item 19] A biological force application device for manipulating living organisms, The liquid in which the living organism is immersed, A flow channel whose end is positioned in a liquid, wherein a liquid-gas interface is formed at the end or inside of the flow channel, A pump that supplies gas into a flow path or draws gas from a flow path, A vector control unit controls the operation of the pump, thereby controlling the force vector applied to the living organism from the gas-liquid interface, and applies force to the living organism at the gas-liquid interface. A biological force-applying device equipped with the following features. [Item 20] The vector control unit controls the magnitude of the force applied to the living organism from the gas-liquid interface. A biological load-bearing device as described in item 19. [Item 21] The vector control unit controls the magnitude of the force applied to the living organism from the gas-liquid interface by controlling the gas pressure at the gas-liquid interface. A biological load-adding device as described in item 20. [Item 22] The vector control unit controls the magnitude of the force applied to the living organism from the gas-liquid interface by controlling the acceleration of movement at the gas-liquid interface. A biological load-bearing device as described in any one of items 19 to 21. [Item 23] The vector control unit controls the direction of the force applied to the living organism from the gas-liquid interface. A biological load-bearing device as described in any one of items 19 to 22. [Item 24] A computer program that contains instructions internally, When an instruction is executed by a processor or programmable circuit, A processor or programmable circuit A gas-liquid interface formation step involves placing the end of a channel in a liquid in which a living organism is immersed, and forming a gas-liquid interface between the liquid and the gas within or at the end of the channel. A vector control stage that controls the force vector applied to a living organism from the gas-liquid interface, The force application stage involves applying force to a living organism from the gas-liquid interface, Controlling actions including Computer program. [Item 25] A channel with its end positioned in the liquid contained in the container, A pump that introduces gas into a flow path and forms a gas-liquid interface at its end, It comprises a position control unit that controls the position of a container or flow path, The position control unit moves the flow path or container to a position where it is at a first distance, which is a distance at which the gas-liquid interface can come into contact with the bottom of the container. With the gas-liquid interface formed at the end by the pump in contact with the bottom of the container, the flow path is moved to a second position where the distance between the end and the bottom of the container is shorter than the first distance. Biological body manipulation device. [Explanation of symbols]

[0307] 1. Light source for fluorescence image observation 2 Dichroic Mirrors 3 Optical deflector 4 Relay Lens 5 Dichroic Mirrors 6. Objective lens 7. Condenser lens 8. Focusing lens 9 Bandpass filter 10 Light source for transmission image observation 11 Barrier filter 12 Projection Lens 13 Barrier Filter 14 Projection lens 15 pinholes 16 light source 17 Light source 25 Container 25a bottom 35. Target of Operation 40 Nozzle Actuators 41 Sample Actuator 42 Camera for fluid flow imaging 45 Light source 46 Light source 47 Pressure generation unit 48 Sensor section 49, 49a, 49b nozzles 50 Microscope Section 51 Flow channels 51a First channel 51b Second channel 53 Flow channel replacement section 54 Liquid storage section 60 Cameras 70 Cameras 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 Devices 171 Imaging Control Unit 180 Input section 190 Records Department 200 Bubble-forming section 250 Flow control unit 251 pump 251a Pump No. 1 251b Pump No. 2 253 Cylindrical part 253a Outer cylinder 253b Inner cylinder 254 End 255 Air-liquid interface 256 bubbles 260 Liquid Control Unit 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 Input / Output Chip 2075 Graphics Controller 2080 display device 2082 Host Controller 2084 Input / Output Controller 2090 Flexible Disk 2095 CD-ROM

Claims

1. In a container containing a liquid containing a living organism, the tip of a tubular channel having an opening at its tip is immersed in the liquid, and a gas-liquid interface is formed in the channel or at the tip of the channel between the liquid and a gas in contact with the liquid (gas formation step). In a state where the gas-liquid interface is in contact with the living organism, a vector control step is performed to control the force vector, which is the direction and magnitude of the force acting on the living organism due to the surface tension of the gas-liquid interface and the pressure of the gas, by performing at least one of the following: a change in the volume of the gas due to the operation of the pump, or a movement in the relative position of the flow path and the container. A force application step in which force is applied to the living organism from the gas-liquid interface, Equipped with, The vector control step includes controlling the direction of the force applied to the organism from the gas-liquid interface by manipulating the distance between the tip of the flow path and the bottom of the container to change the curvature of the gas-liquid interface, or by manipulating the direction of movement of the relative positions. Methods of applying force to living organisms.

2. The vector control step includes controlling the magnitude of the force applied to the organism from the gas-liquid interface. The method according to claim 1.

3. The vector control step includes controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the gas pressure of the gas at the gas-liquid interface. The method according to claim 2.

4. The gas-liquid interface formation step is performed by immersing the tip of the flow path in the liquid and introducing the gas supplied from the pump into the liquid from the tip. The vector control step includes controlling the gas pressure of the gas at the gas-liquid interface by controlling the pressurization of the pump. The method according to claim 3.

5. The gas-liquid interface formation step is performed by immersing the tip of the flow channel in the liquid and introducing gas into the liquid from the tip. The vector control step includes preparing a channel with an inner diameter corresponding to the magnitude of the force to be applied to the organism. The method according to claim 3 or 4.

6. The vector control step includes controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the radius of curvature of the portion of the gas-liquid interface that contacts the organism. The method according to any one of claims 3 to 5.

7. The vector control step includes controlling the magnitude of the force applied to the living organism from the gas-liquid interface by controlling the surface tension between the gas and the liquid. The method according to any one of claims 3 to 6.

8. The vector control step includes controlling the magnitude of the force applied to the organism from the gas-liquid interface by controlling the moving acceleration of the gas-liquid interface. The method according to any one of claims 2 to 7.

9. The gas-liquid interface formation step is performed by immersing the tip of the flow channel in the liquid and introducing gas into the liquid from the tip. The vector control step includes controlling the acceleration of movement of the gas-liquid interface by controlling the acceleration of movement of the flow path. The method according to claim 8.

10. The gas-liquid interface formation step is performed by introducing gas supplied from the pump into the liquid from the tip. The vector control step includes controlling the acceleration of the gas-liquid interface by controlling the pressurization acceleration of the pump. The method according to claim 8 or 9.

11. The vector control step includes controlling the direction of the force applied to the organism from the gas-liquid interface by controlling the orientation and direction of movement of the surface of the portion of the gas-liquid interface in contact with the organism. The method according to any one of claims 1 to 10.

12. The vector control step includes controlling the orientation of the surface of the portion of the gas-liquid interface that the organism contacts by controlling the radius of curvature of the portion of the gas-liquid interface that the organism contacts, The method according to claim 11.

13. The gas-liquid interface formation step is performed by immersing the tip of the flow channel in the liquid and introducing gas into the liquid from the tip. The vector control step includes controlling the orientation of the surface of the portion of the gas-liquid interface that comes into contact with the biological organism by controlling the distance between the tip and the bottom of the container holding the liquid. The method according to claim 11 or 12.

14. The gas-liquid interface formation step is performed by immersing the tip of the flow channel in the liquid and introducing gas into the liquid from the tip. The vector control step includes preparing a channel with an inner diameter corresponding to the direction of the force to be applied to the organism. The method according to any one of claims 11 to 13.

15. The vector control step includes controlling the direction of the force applied to the living organism from the gas-liquid interface from the gas side of the gas-liquid interface to the liquid side, or from the liquid side to the gas side of the gas-liquid interface. The method according to claim 10 or claim 11.

16. The vector control step includes controlling the movement of the gas-liquid interface so that it comes into contact with a predetermined biological body. The method according to any one of claims 1 to 15.

17. A flow path for positioning the tip portion in the liquid contained in a container that holds a liquid containing a living organism, A pump connected to the aforementioned flow path, for supplying a gas that comes into contact with the liquid to the flow path, or for drawing a gas that comes into contact with the liquid from the flow path to form a gas-liquid interface at the tip, The system comprises a position control unit that moves the relative position of the container or the flow path, The pump or the position control unit, while the gas-liquid interface is in contact with the living organism, performs at least one of the following: a change in the volume of the gas due to the operation of the pump, or a movement in the relative position between the flow path and the container, and controls the direction of the force vector applied to the living organism from the gas-liquid interface by manipulating the distance between the tip of the flow path and the bottom of the container, or the direction of the movement of the relative position, thereby controlling the direction of the force vector applied to the living organism from the gas-liquid interface. Biological body manipulation device.

Images