Method and apparatus for manipulating living organisms

Abstract

According to a first aspect of the present invention, provided is an operation method for a living body, comprising: a gas-liquid interface forming step for immersing an end part of a channel in a liquid in which the living body is immersed and forming, inside the channel or on the end part, a gas-liquid interface on which a restoration force works with respect to a minute movement of the interface; and an operation step for operating the living body by using the gas-liquid interface.

Description

Technical Field

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

Background Art

[0002] In cell biology research and the like, it is common practice to aspirate specific cells from among a large number of cells in a culture vessel. Patent Document 1 discloses a cell aspiration support system that aspirates target cells using a chip. [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2016-000007

[0003] [General Disclosure] In a first aspect of the present invention, there is provided a method for manipulating an organism. The method for manipulating an organism may comprise a gas-liquid interface forming step of immersing an end of a flow channel in a liquid in which an organism is immersed, and forming a gas-liquid interface in the flow channel or at the end, on which a restoring force acts against minute interface movement. Further, the method for manipulating an organism may comprise an operation step of manipulating the organism using the gas-liquid interface.

[0004] In a second aspect of the present invention, the gas-liquid interface forming step of the method for manipulating an organism may comprise a step of discharging or aspirating gas from the end.

[0005] In a third aspect of the present invention, the gas-liquid interface forming step of the method for manipulating an organism may comprise discharging gas from the end and maintaining the gas-liquid interface at the end, on which a restoring force acts against minute volume changes.

[0006] In a fourth aspect of the present invention, in the gas-liquid interface forming step of the method for manipulating an organism, a gas volume V (m³) occupied by the gas continuous from the gas-liquid interface to the inside of the flow channel may satisfy the following formula with respect to a radius r_h (m) of the end of the flow channel: V ≦ a × r h 4 + b × r h 3 However, a = 2.65 × 10 8 , and b = 2.59 × 10 2 It satisfies the condition.

[0007] In a fifth embodiment of the present invention, the method for manipulating the biological organism may further include a volume reduction step, prior to the gas-liquid interface formation step, which reduces the volume of gas occupied by the gas that extends from the end to the interior of the flow path.

[0008] In a sixth embodiment of the present invention, the volume reduction step of the method for manipulating the biological organism may include taking the liquid into the flow channel and partitioning the gas inside the flow channel with the taken-in liquid.

[0009] In a seventh embodiment of the present invention, the volume reduction step of the method for manipulating the biological organism may include filling the channel with a filler material that blocks a portion of the space inside the channel.

[0010] In an eighth aspect of the present invention, the flow path of the method for manipulating the biological organism may include a space formed inside a nozzle and a pump connected to the nozzle. The volume reduction step may include reducing the capacity of the pump to 10% or less of its maximum capacity.

[0011] In a ninth embodiment of the present invention, the flow channel of the method for manipulating the biological organism may be provided with a partitioning member that divides the space inside the flow channel.

[0012] In a tenth embodiment of the present invention, the cross-sectional shape of the end of the channel of the method for manipulating the biological organism may be a shape having a protrusion inward.

[0013] In an eleventh embodiment of the present invention, at the end of the channel of the method for manipulating the living organism, an angle is formed between the inner surface of the channel and the end surface of the channel member forming the channel, and the angle of this angle may be in the range of 90 ± 5 degrees.

[0014] In a twelfth embodiment of the present invention, the contact angle between the flow channel member forming the flow channel of the method for manipulating the biological organism and the liquid by the droplet method may be 90 degrees or less.

[0015] In a thirteenth embodiment of the present invention, the flow channel of the method for manipulating the biological organism may have a tip portion provided on the end side. The flow channel may have an inner portion connected to the tip portion and having a flow channel diameter different from that of the tip portion. The flow channel may form a step at the connection point between the tip portion and the inner portion.

[0016] In a fourteenth aspect of the present invention, the flow channel of the method for manipulating the biological organism may comprise a portion with a wider flow channel diameter and a portion with a narrower flow channel diameter. The flow channel diameter of the narrower portion of the flow channel diameter may be one-tenth or less of the flow channel diameter of the wider portion of the flow channel diameter.

[0017] In a fifteenth embodiment of the present invention, the method for manipulating the biological organism may further include a surface tension reduction step, which reduces the surface tension of the liquid between the gas and the biological organism, prior to the gas-liquid interface formation step.

[0018] In a sixteenth aspect of the present invention, the method for manipulating the biological organism may further include a detection step for detecting a first pressure applied to the gas-liquid interface. The detection step may detect a change in the first pressure when gas is continuously released into the flow path, and determine a second pressure capable of forming the gas-liquid interface based on the changed value of the first pressure.

[0019] In a 17th aspect of the present invention, the method for manipulating the organism may further include a detection step of detecting a first pressure applied to the gas-liquid interface. The method for manipulating the organism may detect, based on a change in the first pressure, that the end of the flow path has come into contact with the liquid, or that the gas-liquid interface formed at the end of the flow path has come into contact with the bottom of the container in which the organism is cultured.

[0020] In a eighteenth aspect of the present invention, the method for manipulating an organism may further include, before the gas-liquid interface forming step, a moving step of moving the flow channel and the organism such that the relative positions thereof are brought closer to each other.

[0021] Furthermore, in a nineteenth aspect of the present invention, there is provided an organism manipulation device for manipulating an organism. The organism manipulation device may include a flow channel having an end portion immersed in a liquid in which the organism is immersed. The organism manipulation device may include a gas-liquid interface manipulation unit that forms, inside or at an end of the flow channel, a gas-liquid interface on which a restoring force acts against minute interface movement, and manipulates the organism by the gas-liquid interface.

[0022] In a twentieth aspect of the present invention, the gas-liquid interface manipulation unit of the organism manipulation device may discharge gas from the end portion into the liquid. The gas-liquid interface on which a restoring force acts against a minute volume change may be maintained at the end portion.

[0023] In a twenty-first aspect of the present invention, in the organism manipulation device, a gas volume V (m³) occupied by the gas continuous from the gas-liquid interface to the inside of the flow channel may satisfy the following formula with respect to a radius r_h (m) of an end portion of the flow channel:[« V≦a×r h 4 + b×r h 3 , provided that a=2.65×10 8 and b=2.59×10 2 are satisfied.

[0024] Furthermore, in a 22nd aspect of the present invention, a biological manipulation device for manipulating living organisms is provided. The biological manipulation device may include a pump that introduces gas into the flow path to form a gas-liquid interface at the end. The biological manipulation device may include a pressure detection unit that detects a first pressure applied to the gas-liquid interface. The biological manipulation device may include a storage unit that stores a second pressure capable of maintaining the gas-liquid interface. The pump may change the amount of gas introduced based on the change in the first pressure and control the amount of gas introduced into the flow path so as to maintain the first pressure at or below the second pressure.

[0025] 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]

[0026] [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 the nozzle 49 in this embodiment is shown. [Figure 3A] An example of a schematic diagram showing the structure of the nozzle 49 in this embodiment is shown. [Figure 3B] An example of a schematic diagram showing the structure of the nozzle 49 in this embodiment is shown. [Figure 4] An example of a schematic diagram illustrating an example of a method for recovering biological organisms in this embodiment is shown. [Figure 5] An example of the specific configuration of the information processing device 170 in this embodiment is shown. [Figure 6] An example of a flow chart for manipulating a living organism in this embodiment is shown. [Figure 7A]An example of a GUI image displayed on the output unit 160 in this embodiment is shown. [Figure 7B] An example of a GUI image displayed on the output unit 160 in this embodiment is shown. [Figure 7C] An example of a GUI image displayed on the output unit 160 in this embodiment is shown. [Figure 7D] An example of a GUI image displayed on the output unit 160 in this embodiment is shown. [Figure 7E] An example of a GUI image displayed on the output unit 160 in this embodiment is shown. [Figure 8] An example of a flow for replacing or adding liquid S600 in this embodiment is shown. [Figure 9A] This example shows a flow that moves the relative position between the nozzle 49 of S200 and the cells in this embodiment. [Figure 9B] This example shows a flow that moves the relative position between the nozzle 49 of S200 and the cells in this embodiment. [Figure 9C] This example shows a flow that moves the relative position between the nozzle 49 of S200 and the cells in this embodiment. [Figure 10A] An example of the flow for forming bubbles in 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 13A] This is a diagram illustrating the stabilization formula for the gas-liquid interface. [Figure 13B] This diagram shows the relationship between the angle θm between the meniscus and the nozzle surface at the gas-liquid interface and the right-hand side of the stabilization equation. [Figure 13C] This is a diagram illustrating a method for stabilizing air bubbles. [Figure 13D] Here is an example of a method for reducing the volume within the nozzle's flow path. [Figure 13E] Here is an example of a method for reducing the volume within the nozzle's flow path. [Figure 13F] This diagram illustrates the relationship between dimensionless volume and dimensionless curvature when the shape of the nozzle end is changed. [Figure 13G] The relationship between the rate of change in Laplace pressure and the volume of bubbles when the chamfer angle at the end of the nozzle is changed is shown. [Figure 13H] This figure shows the relationship between dimensionless volume and dimensionless curvature when the contact angle of the nozzle surface is changed. [Figure 13I] This diagram illustrates the creation of a step in the nozzle. [Figure 13J] An example of a nozzle with a stepped design is shown. [Figure 13K] This diagram shows how bubbles are stably formed by a tiny volume of gas. [Figure 13L] This diagram shows the relationship between the radius of the channel end and the maximum stable volume of the bubble. [Figure 14] An example of a computer hardware configuration is shown. [Modes for carrying out the invention]

[0027] 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.

[0028] 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 biological organisms such as cells using the interface between gas and 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, a gas-liquid interface manipulation unit 101, an output unit 160, an information processing unit 170, and an input unit 180.

[0029] 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 living organism other than a cell. For example, the living organism may be a microorganism, fungus, algae, living tissue, spheroid, etc. The living organism may also contain organelles within a cell.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The gas-liquid interface operating unit 101 operates the object to be operated 35 using the gas-liquid interface between the gas and the liquid. For example, the gas-liquid interface operating unit 101 operates a living organism (e.g., a cell) in the liquid by forming bubbles in the liquid. The gas-liquid interface 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. Here, the gas-liquid interface operating unit 101 may form a gas-liquid interface 255 inside or at the end of the flow path 51, in which a restoring force acts against minute interface movement. For example, the gas-liquid interface operating unit 101 may release gas into the liquid from the end 254 of the nozzle 49, maintaining a gas-liquid interface 255 at the end 254 where a restoring force acts against minute interface movement. The formation of bubbles is stabilized by the restoring force acting against minute interface movement, and the mechanism for this will be described later.

[0038] 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.

[0039] 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 within the information processing device 170. The stage may be provided in the gas-liquid interface operation unit 101 or in the microscope unit 50.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The gas may be air. The gas may also contain moisture.

[0045] 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.

[0046] 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.

[0047] The flow path 51 is through which the liquid and gas to be drawn in (inhaled) or discharged (supplied) passes. The flow path 51 is provided inside the nozzle 49 and penetrates the longitudinal direction of the nozzle 49. The end 254 of the flow path 51 may be immersed in the liquid in which the object to be operated 35 is immersed. A pressure generating unit 47 is connected to the other end of the flow path 51.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The information processing device 170 exchanges commands and data with the microscope unit 50, camera 60, camera 70, gas-liquid interface 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 gas-liquid interface operation unit 101, and controls the microscope unit 50 and the gas-liquid interface operation unit 101.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] When performing transmission image observation, the information processing device 170 turns on the transmission image observation light source 10 and turns off the fluorescence image observation light source 1 to activate the optical path of the transmission image observation light source 10. When performing transmission image observation, the light emitted from the transmission image observation light source 10 illuminates the object to be operated 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 operated 35 reaches the light-receiving surface of the camera 70 via the objective lens 6, the dichroic mirror 5, the barrier filter 11, and the projection lens 12. At this time, a transmission image of the object to be operated 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.

[0057] Furthermore, the information processing device 170 controls the relative positions of the nozzle 49 and the stage of the gas-liquid interface operation unit 101. In addition to controlling the microscope unit 50 and the gas-liquid interface operation unit 101, the information processing device 170 may receive images of the object to be operated 35 captured by the camera 60 and / or camera 70, and / or images captured by the flow path imaging camera 42 of the gas-liquid interface operation unit 101, and perform image processing such as generating a single composite image from multiple images. The information processing device 170 may also control other operations of the biological manipulation device 100 and perform data processing as needed. The configuration of the information processing device 170 will be described later.

[0058] 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.

[0059] 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 (which may include a phase plate), a condenser lens 7, a focusing lens 8, a bandpass filter 9, a light source 10 for observing transmitted images, 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 include configurations other than those listed above. For example, a phase-contrast microscope may include a normal Ski prism 31, and a differential interference microscope may include a ring aperture 39. The description in Figure 1A may apply to the configuration of the biological manipulation device 100 other than the microscope unit 50.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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).

[0114] 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 be in contact with 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.

[0115] Alternatively, the position of the nozzle 49 may be set using the flow path imaging camera 42 instead of, or in combination with, the cameras 60 and / or 70. For example, 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. 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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. For example, the bubble forming unit 200 may send an instruction to the nozzle actuator 40 to move so that the relative position between the nozzle 49 and the target of operation 35 is brought closer. In this case, the nozzle actuator 40 can be moved so that the relative position between the nozzle 49 (flow path 51) and the target of operation 35 is brought closer.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] Next, in S252, 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.

[0128] 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.

[0129] If the bubble forming unit 200 determines that the position of the nozzle 49 is different from its initial position, it proceeds to process S256; otherwise, it proceeds to process S300.

[0130] 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. For example, the bubble forming unit 200 may send an instruction to the nozzle actuator 40 to move so that the relative position between the nozzle 49 and the target of operation 35 is brought closer. In this case, the nozzle actuator 40 can be moved so that the relative position between the nozzle 49 (flow path 51) and the target of operation 35 is brought closer.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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. The volume control unit 200 maintains the internal pressure of the nozzle to be above or below atmospheric pressure, and when 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 volume control 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 volume control 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.

[0135] 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 volume control 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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. In this case, the z position is controlled based on the internal pressure value measured 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 moves the nozzle 49 to the preset xyz position, the bubble forming unit 200 proceeds to S276.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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. For example, the bubble forming unit 200 may send an instruction to the nozzle actuator 40 to move so that the relative position between the nozzle 49 and the target of operation 35 is brought closer. In this case, the nozzle actuator 40 can be moved so that the relative position between the nozzle 49 (flow path 51) and the target of operation 35 is brought closer.

[0145] 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.

[0146] 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.

[0147] 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 is released from the end 254, causing the gas-liquid interface 255 at the tip of the flow path 51 to expand (bubbles are formed). After the gas-liquid interface 255 has expanded (bubbles have been formed), the bubble forming unit 200 proceeds to S330.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] Figure 10B shows an example of a flow that expands the gas-liquid interface 255 based on the internal pressure in the nozzle 49.

[0154] Step S370 may be the same as step S320. After completing S370, the bubble forming unit 200 proceeds to S380.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] In S400, the gas-liquid interface operation unit 101 performs an operation on the target object 35. For example, the bubble formation unit 200 sends an instruction to the gas-liquid interface operation unit 101 to perform an operation on the target object 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.

[0160] 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.

[0161] 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.

[0162] In step S412, the bubble-forming unit 200 removes cells by attaching them to the formed bubbles.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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 generation 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] After the camera 60 or camera 70 has imaged the cells it has captured, the bubble formation unit 200 proceeds to process S500.

[0190] 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.

[0191] 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.

[0192] 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).

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In S460, the bubble-forming unit 200 controls the gas-liquid interface manipulation unit 101 so that the necessary operations other than those in S410-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.

[0205] 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.

[0206] 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 254 of the nozzle 49.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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).

[0216] 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.

[0217] 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.

[0218] Figure 12B shows an example of a flow that reduces the gas-liquid interface 255 based on the internal pressure in the nozzle 49.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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 operation corresponding to the amount of movement.

[0224] 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.

[0225] 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.

[0226] 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 adhered 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] The above flowchart describes, as examples of operations on target 35, the removal of unwanted cells, cytoplasm and / or cell membrane retrieval, cell retrieval and passage, cell retention, and cell compression. Several other examples of operations are also possible.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] Another example of the operation includes rupture or cleavage of cells by compression. Applying a large pressure to cells allows the cells to be ruptured or cleaved. By rupturing or cleaving cells, cell membranes, cytoplasm, and / or organelles, etc. can be recovered, and connections between cells (e.g., synapses, which are connections between nerve cells) can be cut.

[0236] Another example of the operation includes induction of cell differentiation. It is known that differentiation of osteoblasts, muscle cells, vascular endothelial progenitor cells, and the like is induced by applying mechanical stimulation. Differentiation can be induced for these cells by the pressure generating unit 47 applying compression using air bubbles in accordance with the flow described above.

[0237] Another example of the operation includes gene introduction into cells. In accordance with the flow described above, the bubble forming unit 200 uses bubbles to adhere a vesicle-shaped object such as a cell membrane to the gas-liquid interface 255 and bring it into contact with the cell membrane, whereby the contents of the vesicle are incorporated into the cell via membrane fusion. At this time, by encapsulating a gene in the vesicle in advance, the gene can be incorporated into the cell. In addition, not only genes but also other macromolecules can be incorporated into cells through pores. Furthermore, in accordance with the flow described above, when the bubble forming unit 200 compresses cells at the gas-liquid interface 255 using bubbles, minute gaps are easily generated in a part of the membrane during the deformation of the cells. At this time, by adding a gene to the cell culture medium, the gene can be incorporated into the cell through the gaps. In addition, not only genes but also other macromolecules can be incorporated into cells through the gaps.

[0238] Another example of the operation includes cooperation with external devices such as cell culture devices like fermenters and cell analysis devices like cell sorters. In accordance with the flow described above, cells may be moved by the bubble forming unit 200 using bubbles to take cells into the flow path 51 and discharge the cells to a specified position of the linked external device. Alternatively, since the flow path 51 is directly connected to the external device, the cells taken into the flow path 51 may be sent to the external device to move the cells.

[0239] As another example of an operation, manipulation of an emulsion can be mentioned. An emulsion is droplets in an oil liquid or oil droplets in an aqueous solution. In order to stabilize the formed emulsion, the emulsion may be caused to contain an amphiphilic substance such as a surfactant. Surfactants and the like are arranged so as to surround the droplets or oil droplets, and form a monomolecular film at the interface. Such monomolecular films are also found in some intracellular organelles, such as endosomes and lipid droplets. According to the flow described above, the bubble forming unit 200 may cause the emulsion to adhere to the gas-liquid interface 255 using bubbles, or may further perform manipulation.

[0240] As methods for detaching and / or recovering cells, there are methods in which a special substrate that reacts to temperature or light is used to locally denature the substrate to detach cells, and methods in which cells are detached using ultrasound, but these methods require a means for recovering cells. However, the method of the present invention does not require a special substrate, and includes both means for detaching cells and means for recovering cells. Further, as a means for recovering detached cells, there is a method of recovering cells by a liquid flow that sucks liquid, such as an aspirator, but this method has problems such as recovering cells together with a large amount of liquid, and there is a possibility of entraining cells other than the target cells. However, in the method of the present invention, by incorporating the gas-liquid interface into a nozzle, cells adhering to the gas-liquid interface can be recovered, and the target cells can be easily recovered with a very small amount of liquid without entraining cells other than the target cells. Further, it is known that applying a strong liquid flow when sucking liquid adversely affects cells, but such adverse effects can be avoided by using the method of the present invention.

[0241] As described above, the method of manipulating the manipulation target 35 using the gas-liquid interface 255 has been described. Here, by stably controlling the formed gas-liquid interface 255, the manipulation target 35 can be manipulated easily and stably. Hereinafter, a method for stably controlling the gas-liquid interface 255 will be described in detail.

[0242] First, we will explain the theory for stably controlling the gas-liquid interface 255. When the gas-liquid interface of a bubble is stably maintained, the internal pressure P of the bubble i And the Laplace pressure P of the meniscus (gas-liquid interface 255) m They are in balance. Laplace pressure P m This is the pressure difference between the gas phase within the bubble and the liquid phase of liquid 261. In other words, the following equation 1 holds true. [Formula 1]

number

[0243] Furthermore, the conditions for a restoring force to act in response to minute volume changes are met. Without a restoring force, the bubble would continue to expand, making it difficult to maintain or control. Specifically, the bubble is stable if equation 2 below holds true. Here, V represents the volume of the bubble. In the following explanation, this equation 2 will be referred to as the stabilization equation. [Formula 2]

number

[0244] Let's explain the left-hand side of the stabilization equation (Equation 2) above. If we assume that the change in internal pressure of the bubble in response to a small volume change is a quasi-static adiabatic process, then the following Equation 3 holds true. Here, κ represents the specific heat ratio. [Formula 3]

number

[0245] In equation 3 above, if we let V approach 0, or if V diverges to infinity, [Equation 4]

number

[0246] Therefore, the left-hand side of the stabilization formula (Mathematical Formula 2) can take negative real values from 0. Accordingly, in the stabilization formula (Mathematical Formula 2), as the volume V of the bubble is reduced as much as possible, the left-hand side of the stabilization formula (Mathematical Formula 2) becomes smaller, so the stabilization formula (Mathematical Formula 2) is more likely to be satisfied, and the bubble is maintained stably. Thus, it is shown that in order to maintain bubbles stably, reducing the bubble volume V is preferable.

[0247] Next, the right-hand side of the stabilization formula (Mathematical Formula 2) will be described with reference to FIG. 13A. In FIG. 13A, let γ be the surface tension between the gas and the liquid 261, and r be the radius of the flow path 51 of the nozzle 49 h , and let θ be the angle formed between the end 254 of the nozzle 49 and the meniscus (gas-liquid interface 255) m . In this case, P on the right-hand side of the stabilization formula (Mathematical Formula 2) m and V (described as V m ) are expressed as a function of θ m by the following Mathematical Formula 5. [Mathematical Formula 5] [Math.]

[0248] Therefore, the right-hand side of the stabilization formula (Mathematical Formula 2) is expressed as a function of θ m by the following Mathematical Formula 6. [Mathematical Formula 6] [Math.]

[0249] FIG. 13B is a graph representing the above Mathematical Formula 6, where the horizontal axis represents θ m and the vertical axis represents the right-hand side of the stabilization formula (Mathematical Formula 2), that is, Mathematical Formula 6. According to FIG. 13B, the right-hand side of the stabilization formula (Mathematical Formula 2) is 0 or more when θ m is 90 degrees or less, and is less than 0 when θ m exceeds 90 degrees. Therefore, when θ m is 90 degrees or less, the right-hand side of the stabilization formula (Mathematical Formula 2) is 0 or more, and the left-hand side of the stabilization formula (Mathematical Formula 2) is 0 or less as shown in Mathematical Formula 4, so the stabilization formula (Mathematical Formula 2) is always satisfied.

[0250] As described above, the angle θ between the end portion 254 of the nozzle 49 and the meniscus (gas-liquid interface 255) m If the temperature is below 90 degrees, the bubbles will remain stable and easier to control. Furthermore, the smaller the volume V of the bubbles is, the more stable the bubbles will remain and the easier they will be to control.

[0251] Also, θ m When the temperature exceeds 90 degrees, the stabilization formula (Equation 2) no longer holds true, which means that the internal pressure P of the bubble... i The Laplace pressure P of the meniscus (gas-liquid interface 255) m As it grows larger, the bubble expands rapidly due to the pressure difference. At this time, the internal pressure P of the bubble increases due to the expansion of the bubble's volume V. i The pressure drops sharply. The internal pressure P of such a bubble i By detecting changes in this, it is also possible to experimentally determine the pressure at which a bubble is stably maintained. For example, compressing the volume V of the bubble and the internal pressure P of the bubble i As the pressure increases, the moment the bubble becomes unstable, the internal pressure P of the bubble increases due to the expansion of the bubble's volume V. i Because it drops rapidly, the internal pressure P of the bubble i The internal pressure P of the bubble just before it drops. i This allows us to determine that this is the pressure at which the bubble can be stably maintained. This method for detecting the pressure at which the bubble can be stably maintained may be performed before the step of forming the bubble (enlarging the gas-liquid interface 255), that is, before performing the steps and substeps of S300, and the pressure at which the bubble can be stably maintained may be used as the set internal pressure. This method for detecting the pressure at which the bubble can be stably maintained, based on the internal pressure of the bubble, is also valid when the bubble shape is not hemispherical, such as when the bubble is in contact with the bottom surface.

[0252] Next, Figure 13C is a diagram illustrating a method for stabilizing the gas-liquid interface 255 based on the above description. The method for stabilizing the gas-liquid interface 255 described below may, but is not limited to, being performed before the step of forming bubbles (enlarging the gas-liquid interface 255), that is, before the steps and substeps of S300. For example, the method for stabilizing the gas-liquid interface 255 may be performed in the middle of the step and substeps (substeps of S300) of forming bubbles. In methods related to flow channel structures such as 911, which will be described later, it may be performed in advance before performing S100.

[0253] For example, let's consider a case where a method for stabilizing the gas-liquid interface 255 is performed before step S300. The method for stabilizing the gas-liquid interface 255 may be performed automatically by the biological manipulation device 100. In this case, the information processing device 170 may first receive input from the operator via the input unit 180 regarding whether or not to stabilize the gas-liquid interface 255. If the information processing device 170 is instructed to stabilize the gas-liquid interface 255, the information processing device 170 will perform one or more of the methods shown in 910 to 933 in Figure 13C. Otherwise, the information processing device 170 will proceed to S300. Note that the method for stabilizing the gas-liquid interface 255 may be performed by the operator rather than automatically by the biological manipulation device 100. For example, if the method for stabilizing the gas-liquid interface 255 is performed by the operator immediately before step S300, the process may resume from S300.

[0254] Figures 13D and 13E illustrate a method for reducing the volume of gas occupied by the gas continuous from bubble 256 to the interior of the flow path 51, as shown in 910 of Figure 13C. As explained above, the bubble is maintained stably by reducing the volume of gas continuous with the bubble. For example, if the flow path 51 includes the space formed inside the nozzle 49 and the syringe pump connected to the nozzle 49, the flow path volume may be reduced to 10% or less, 8% or less, or even 5% or less of the maximum capacity by the syringe pump, but is not limited to these.

[0255] Let's give an example of how to perform 910. As one example, in 701 of Figure 13D, 911 reduces the volume of the flow path 51 in the nozzle 49 by making it extremely narrow, thereby reducing the volume of gas continuous with the bubble and maintaining the bubble stably. For example, if the length of the flow path 51 is 150 mm, the bubble can be maintained stably by making the flow path diameter 100 μm or less, 75 μm or less, and further 50 μm or less. In this case, the method of 911 can be performed by pre-installing the nozzle 49 with the extremely narrow flow path 51 onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle in S180. The step of attaching the nozzle 49 may be performed in the same way as in S180. The nozzle 49 with the extremely narrow flow path 51 may be stored in advance in the nozzle storage section of the flow path exchange section 53. If the gas-liquid interface operating unit 101 does not have a flow path exchange unit 53, a nozzle 49 with an extremely narrow flow path 51 may be attached to the nozzle actuator 40 by the operator.

[0256] To give another example of how 910 is specifically performed, as shown in 702 of Figure 13D, 912 involves filling the channel 51 in the nozzle 49 with a filler material (e.g., beads 291) to block a portion of the space inside the channel 51, thereby reducing the volume of gas continuous with the bubble and stably controlling the bubble. The beads 291 may be made of metal, plastic, or rubber, but are not limited to these. The size of the beads 291 may be such that they fit within the channel 51, and the shape of the beads 291 may be spherical, but is not limited to this; beads 291 of any size and shape can be used.

[0257] The total volume of the filler material in relation to the total volume of the flow path 51 may be 95% or less. The total volume of the filler material in relation to the total volume of the flow path 51 may be 30% or more, 60% or more, and even 90% or more. Here, the total volume of the flow path 51 may be the volume of the space sealed between the end 254 of the nozzle 49 and the plunger of the syringe pump connected to the nozzle 49.

[0258] The method of 912 can be performed by pre-installing the nozzle 49 filled with beads 291 onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle 49 in S180. The step of attaching the nozzle 49 may be performed in the same manner as in S180.

[0259] Here, the nozzle 49 filled with beads 291 may be stored in the nozzle storage section of the flow path exchange section 53 after the beads 291 have been pre-filled into the flow path 51 inside the nozzle 49. Alternatively, a nozzle 49 without beads 291 may be stored in the nozzle storage section, and just before attaching it to the nozzle actuator 40, the operator may manually fill the flow path 51 inside the nozzle 49 with beads 291, and then attach the nozzle 49 to the nozzle actuator 40 by the operator.

[0260] To give another example of how 910 is specifically performed, in 703 of Figure 13D, liquid 261 is drawn into the channel 51 in the nozzle 49, and the drawn-in liquid 261 partitions the gas inside the channel 51. This reduces the volume of the channel involved in bubble formation, thereby reducing the volume of gas continuous with the bubble, which helps to maintain the bubble stably and makes it easier to control. The liquid 261 drawn into the channel 51 may be the liquid that fills the container 25, or the liquid stored in the liquid storage section of the liquid storage unit 54, but is not limited to these, and any liquid can be used.

[0261] The amount (volume) of the liquid 261 taken in may be 1% or more of the total volume of the flow path 51. The amount (volume) of the liquid 261 taken in may be 90% or less, 50% or less, or even 20% or less of the total volume of the flow path 51. Here, the total volume of the flow path 51 may be the volume of the space sealed between the end 254 of the nozzle 49 and the plunger of the syringe pump connected to the nozzle 49.

[0262] The method of 913 can be performed by pre-attaching the nozzle 49, which has already taken in the liquid 261, to the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle in S180. Alternatively, the liquid 261 may be taken into the flow path 51 inside the nozzle 49 after the nozzle 49 has been attached to the nozzle actuator 40.

[0263] The intake of liquid 261 into the flow path 51 within the nozzle 49 may be carried out in the same manner as in step S510. For example, the intake of liquid 261 may be carried out by the bubble forming unit 200 controlling the pressure generating unit 47 to perform a suction operation on the flow path 51, thereby drawing in liquid 261 from the tip of the flow path 51. After the intake of liquid 261 into the flow path 51 within the nozzle 49, gas may be further introduced into the flow path 51 within the nozzle 49. The intake of gas may be carried out in the same manner as in step S436. For example, the bubble forming unit 200 may control the pressure generating unit 47 and the nozzle actuator 40 to draw in gas in order to form new bubbles at the end 254 of the nozzle 49. The bubble forming unit 200 may instruct the nozzle actuator 40 to remove the nozzle 49 from the liquid 261. After the nozzle actuator 40 removes the nozzle 49 from the liquid 261, the pressure generating unit 47 may pull the plunger of the syringe pump to draw in the required amount of gas. The amount of air drawn in can be used to adjust the amount of gas partitioned within the flow path 51.

[0264] Other examples of how 910 is specifically performed are given. For example, in 704a, 704b, and 704c of Figure 13E, 914 is performed by providing a partitioning member (e.g., a diaphragm 292) in the flow path 51 within the nozzle 49 to partition the space inside the flow path 51, thereby reducing the volume of gas continuous with the bubble, maintaining the bubble stably, and making control easier. The diaphragm 292 may be made movable within the flow path 51 by controlling the pressure generation unit 47 to perform an intake or suction operation on the flow path 51. The diaphragm 292 may be made movable within the flow path 51 by being pushed or pulled by a press 294 by an arbitrary distance. Based on the control of the bubble forming unit 200, the press 20 may move the diaphragm 292 within the flow path 51 by pushing or pulling the diaphragm 292 by a preset distance using a nozzle actuator 40 or a press actuator (not shown). The diaphragm 292 may be made of metal, plastic, or rubber, but is not limited to these. In this case, the method of 914 can be carried out by pre-installing the nozzle 49, which is equipped with the diaphragm 292, onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle 49 in S180. The step of attaching the nozzle 49 may be carried out in the same manner as in S180.

[0265] Here, the volume of the space separated by the dividing member (for example, in the case of 704a, the volume of the space below the diaphragm 292) relative to the total volume of the flow path 51 may be 1% or more. The volume of the space separated by the dividing member relative to the total volume of the flow path 51 may be 50% or less, 25% or less, and even 10% or less. Here, the total volume of the flow path 51 may be the volume of the space sealed between the end 254 of the nozzle 49 and the plunger of the syringe pump connected to the nozzle 49.

[0266] A nozzle 49 equipped with a diaphragm 292 may be stored in the nozzle storage section of the flow path exchange section 53 after the diaphragm 292 and press 294 have been installed in the flow path 51 inside the nozzle 49. Alternatively, a nozzle 49 without a diaphragm 292 and press 294 may be stored in the nozzle storage section, and the diaphragm 292 and press 294 may be installed in the flow path 51 inside the nozzle 49 by the operator, and then the nozzle 49 may be attached to the nozzle actuator 40 by the operator.

[0267] Let's look at another example of how 910 is specifically implemented. As one example, 915, as shown in 705 of Figure 13E, provides a structure in the flow path 51 within the nozzle 49 that includes a portion with a wider flow path diameter and a portion with a narrower flow path diameter. For example, the structure in the flow path 51 that includes a portion with a wider flow path diameter and a portion with a narrower flow path diameter may be an orifice structure in which a part of the flow path 51 is narrowed. In addition, the flow path 51 may have one or more portions with a wider flow path diameter and portions with a narrower flow path diameter. In 705 of Figure 13E, the nozzle 49 has a portion with a narrower flow path diameter 297, a portion with a wider flow path diameter 295, and a portion with a wider flow path diameter 298, but the number of portions with a narrower flow path diameter and portions with a wider flow path diameter provided in the nozzle 49 is not limited to these.

[0268] In this way, by providing a narrow channel diameter section 297, when gas is drawn into the bubble 256 via the channel 51, the volume of gas present in the channel 51 involved in bubble formation can be considered to be only the volume of 295 over time. This reduces the volume of gas continuous with the bubble, allowing the bubble to be maintained in a stable state, and thus making control easier.

[0269] The diameter of the narrow portion of the channel may be one-tenth or less of the diameter of the wide portion, one-twentieth or less, or even one-twenty-fifth or less, but is not limited to these values. Having the diameter of the narrow portion of the channel be one-tenth or less of the diameter of the wide portion of the channel provides sufficient effectiveness in maintaining and controlling the stability of bubbles.

[0270] In this case, the method of 915 can be performed by pre-installing a nozzle 49 having a wider flow path portion and a narrower flow path portion on the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the procedure may start again from the nozzle installation step of S180. The nozzle installation step may be performed in the same manner as in S180. The nozzle 49 having a wider flow path portion and a narrower flow path portion may be stored in advance in the nozzle storage section of the flow path exchange section 53. If the gas-liquid interface operating section 101 does not have a flow path exchange section 53, the nozzle 49 having a wider flow path portion and a narrower flow path portion may be installed on the nozzle actuator 40 by the operator.

[0271] Next, at 920, the bubbles can also be stabilized by increasing the right-hand side of the above stabilization formula (Equation 2). In other words, the Laplace pressure P of the bubbles can be increased in response to small volume changes. m The bubbles can be maintained stably by increasing the variation in the flow path 51 of the nozzle 49.

[0272] Figure 13F is a graph showing the relationship between dimensionless volume and dimensionless curvature when the contact angle between the flow channel member forming the end 254 of the nozzle 49 and the liquid 261 is 90 degrees. Based on this, 921 will be explained as an example of specifically performing 920. The horizontal axis represents the dimensionless volume of the bubble, and the vertical axis represents the dimensionless curvature of the bubble. Curve 298a (dotted line) shows the case where the cross-sectional shape of the flow channel 51 of the nozzle 49 is square, curve 298b (solid line) shows the case where the cross-sectional shape is cross-shaped, and curve 298c (dashed line) shows the case where the cross-sectional shape is circular. The cross-sectional areas of these cross-sections are all equal; only the shape of the cross-section differs.

[0273] Here, the cross-sectional shape of the flow path 51 of the nozzle 49 affects the stability of the bubbles by increasing the change in Laplace pressure in response to minute volume changes of 920. Specifically, it is shown that the effect of increasing the change in Laplace pressure is greatest when the cross-section is cross-shaped.

[0274] In the curve of the graph in Figure 13F, the dimensionless curvature represents the curvature of the bubble. The greater the curvature, the higher the internal pressure, and the smaller the curvature, the lower the internal pressure. An increase in dimensionless curvature in relation to a dimensionless volume change indicates that the internal pressure of the bubble is increasing with respect to the volume change, and that the Laplace pressure, which is the pressure difference with the liquid phase of liquid 261, is increasing. In other words, the Laplace pressure is increasing with respect to small volume changes, and the right-hand side of the stabilization equation (Equation 2) above is increasing. On the other hand, a decrease in dimensionless curvature in relation to a dimensionless volume change indicates that the Laplace pressure is decreasing with respect to small volume changes, and the right-hand side of the stabilization equation (Equation 2) above is decreasing. From the above, when the dimensionless curvature changes from increasing to decreasing, the right-hand side of the stabilization equation (Equation 2) changes from positive to negative, and the bubble becomes unstable. Therefore, the larger the value of the dimensionless volume that gives the maximum value of the curve, the wider the region of volume in which the bubble can be stably maintained and controlled, and the easier it is to stably maintain and control the bubble. In the graph in Figure 13F, curve 298b for the case of a cross-shaped cross section gives a maximum value of dimensionless curvature at larger dimensionless volumes than curve 298a for the case of a square cross section and curve 298c for the case of a circular cross section. Therefore, it is considered that a cross-shaped cross section can contribute to the stability of the bubble.

[0275] Figure 13F shows the case where the cross-section is cross-shaped, but the shape that contributes to stabilization is not limited to a cross shape. For example, if the cross-sectional shape has a projection that faces inward, that is, a shape with an interior angle, the same effect as a cross shape can be obtained. For example, the shape with a projection that faces inward may be an asterisk shape, but is not limited to these.

[0276] Furthermore, in 921, the angle formed at the end 254 of the nozzle 49 between the inner surface of the flow path 51 and the end surface of the flow path member forming the flow path 51 increases the change in Laplace pressure in response to minute volume changes in 920, thereby influencing the stable maintenance of the bubbles.

[0277] Figure 13G is a graph showing the relationship between the rate of change in Laplace pressure and the volume of bubbles when the chamfer angle (θc of 298u) of the corner formed by the inner surface of the channel 51 of the nozzle 49 and the end surface of the channel member forming the channel 51 is changed. The horizontal axis represents the volume of the bubbles, and the vertical axis represents the change in the Laplace pressure of the bubbles. Curve 298v (solid line) shows the case when the chamfer angle is 15 degrees, curve 298w (dotted line) shows the case when the chamfer angle is 30 degrees, curve 298x (dashed line) shows the case when the chamfer angle is 45 degrees, curve 298y (dotted line) shows the case when the chamfer angle is 60 degrees, and curve 298z (double-dotted line) shows the case when the chamfer angle is 75 degrees. From the graph, it can be seen that as the chamfer angle increases, the change in Laplace pressure decreases in relation to the volume increase associated with bubble formation, especially in the initial volume increase stage, making it difficult to stably maintain and control the bubbles. In the absence of chamfering, the angle formed by the inner surface of the channel 51 and the end surface of the channel member forming the channel 51 is 90 degrees. As shown in Figure 13C, there is a region where the change in Laplace pressure increases with bubble formation, and there is a region where bubbles can be stably maintained and controlled. In other words, at the end 254 of the nozzle 49, if the angle formed by the inner surface of the channel 51 and the end surface of the channel member forming the channel 51, that is, the angle of the cross-section at the end of the nozzle 49, is within the range of 90 ± 5 degrees, bubbles can be stably maintained and controlled.

[0278] The angle at which the bubble is formed may be in the range of 90 ± 5 degrees, 90 ± 3 degrees, or even approximately 90 degrees. Having the forming angle near a right angle increases the right-hand side of the stabilization equation (Equation 2), contributing to the stability of the bubble.

[0279] The method of 921 can be carried out by pre-installing a nozzle 49 having the cross-sectional shape and / or angle described above onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the nozzle installation step of S180. The nozzle installation step may be carried out in the same manner as in S180. A nozzle 49 having the cross-sectional shape and / or angle described above may be stored in advance in the nozzle storage section of the flow path exchange section 53. If the gas-liquid interface operating section 101 does not have a flow path exchange section 53, the nozzle 49 may be installed on the nozzle actuator 40 by the operator.

[0280] Furthermore, the contact angle between the flow channel member forming the flow path 51 of the nozzle 49 and the liquid increases the change in Laplace pressure for a minute volume change of 920, thereby influencing the stable maintenance of the bubbles. Specifically, the bubbles can be stabilized when the contact angle between the flow channel member and the liquid is 90 degrees or less.

[0281] Figure 13H is a graph showing the relationship between dimensionless volume and dimensionless curvature when the contact angle between the flow channel member forming the end 254 of the flow channel 51 of the nozzle 49 and the liquid 261 is changed. Based on this, 922 will be explained as another example of specifically performing 910. The horizontal axis represents the dimensionless volume of the bubble, and the vertical axis represents the dimensionless curvature of the bubble. Curve 299a (solid line) shows the case when the contact angle between the nozzle 49 and the liquid is 30 degrees, curve 299b (dotted line) shows the case when the contact angle is 60 degrees, curve 299c (dashed line) shows the case when the contact angle is 90 degrees, curve 299d (single-dotted line) shows the case when the contact angle is 120 degrees, and curve 299e (double-dotted line) shows the case when the contact angle is 150 degrees.

[0282] Figure 13F shows that when the contact angle exceeds 90 degrees, at 120 degrees and 150 degrees (curves 299d and 299e, respectively), the value of the dimensionless volume that gives the maximum value shifts significantly to the left. In other words, when the contact angle exceeds 90 degrees, the region in which bubbles can be stably maintained narrows, making it difficult to stably maintain and control bubbles. Therefore, in order to contribute to the stable maintenance and control of bubbles, the contact angle between the flow channel member forming the end 254 of the flow channel 51 of the nozzle 49 and the liquid 261 should be 90 degrees or less. The contact angle between the flow channel member forming the end 254 of the flow channel 51 of the nozzle 49 and the liquid 261 should be 60 degrees or less.

[0283] The contact angle between the flow channel member and the liquid 261 can be measured by a known method. For example, the contact angle may be measured by the droplet method. The droplet method involves bringing a droplet of liquid into contact with the flow channel member and measuring the static contact angle between the liquid and the flow channel member using a contact angle meter. A commercially available contact angle meter can be used.

[0284] In this case, the method of 922 can be performed by pre-installing the nozzle 49 having the above-mentioned contact angle onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle in S180. The step of attaching the nozzle 49 may be performed in the same manner as in S180. The nozzle 49 having the above-mentioned contact angle may be stored in advance in the nozzle storage section of the flow path exchange section 53. If the gas-liquid interface operating section 101 does not have a flow path exchange section 53, the nozzle 49 having the above-mentioned contact angle may be attached to the nozzle actuator 40 by the operator.

[0285] Furthermore, let's give another example of how 920 can be specifically implemented. As one example, in 923, providing a step 307 in the flow path 51 within the nozzle 49 can also contribute to stabilizing the bubbles.

[0286] Figure 13I shows a step 307 provided at the connection point between a tip portion 305 located at the end of the flow path 51 within the nozzle 49 and an inner portion 306 connected to the tip portion 305. Here, the step may be a structure in which the inner diameter of the flow path 51 changes in the axial direction of the cylindrical portion 253 of the nozzle 49. The step may also be a structure in which a portion with a narrow flow path diameter enters a portion with a wider flow path diameter within the flow path 51. For example, the tip portion 305 and the inner portion 306 may have different flow path diameters. For example, the flow path diameter of the tip portion 305 may be smaller than the flow path diameter of the inner portion 306. As an example, the flow path diameter of the tip portion 305 may be 1 / 10 or less, 1 / 20 or less, or even 1 / 25 or less of the flow path diameter of the inner portion 306, but is not limited to these.

[0287] Examples of steps include cases where a wide channel diameter and a narrow channel diameter are continuously connected, as shown in 710a, and cases where a narrow channel diameter extends into and connects to a wider channel diameter, as shown in 710b. In the case of 710b, a very small amount of liquid can be measured by adjusting the length over which the narrow channel diameter extends into the wider channel diameter.

[0288] At this time, by introducing liquid 261 into the flow path 51, a gas-liquid interface 255 is formed at the step 307 of the connection portion. In this way, by providing a step 307 in the flow path 51, the above-mentioned stabilization formula (Equation 2) holds true at the formed gas-liquid interface 255. In other words, the gas-liquid interface 255 can be easily maintained and controlled at the position of the step 307. By utilizing this, it is possible to measure a very small amount of liquid only at the tip portion 305, where the flow path diameter is small.

[0289] In this case, the method of 923 can be performed by pre-installing the nozzle 49 with the stepped portion 307 onto the nozzle actuator 40. If it is necessary to attach the nozzle 49 to the nozzle actuator 40, the process may start again from the step of attaching the nozzle in S180. The step of attaching the nozzle 49 may be performed in the same way as in S180. The nozzle 49 with the stepped portion 307 may be stored in advance in the nozzle storage section of the flow path exchange section 53. If the gas-liquid interface operating section 101 does not have a flow path exchange section 53, the nozzle 49 with the stepped portion 307 may be attached to the nozzle actuator 40 by the operator.

[0290] Figure 13J shows a nozzle 49 with a step 307 in the flow path 51. The nozzle 49 shown in Figure 13H has a flow path diameter (diameter) of 0.1 mm at the tip portion 305, a height of 1 mm at the tip portion 305, a flow path diameter (radius) of 1.0 mm at the inner portion 306, and a total length of 20 mm. This nozzle 49 was able to maintain a stable gas-liquid interface 255 at the step 307, and it was possible to measure the liquid volume (approximately 7.5 nL) from the tip portion alone.

[0291] Furthermore, at 930, the bubbles can also be stabilized by reducing the surface tension of the liquid between it and the gas (interfacial free energy at the gas-liquid interface 255). Reducing the surface tension allows the bubbles to expand easily with small changes in internal pressure, thus reducing the change in internal pressure of the bubbles in response to minute volume changes. In other words, the left side of the stabilization equation (equation 2) decreases, making it possible to maintain and control the bubbles more stably.

[0292] Let's give an example of how 930 can be specifically performed. As an example, in 931, the surface tension of the liquid can be reduced by adding a solute that reduces the surface tension of the liquid to the liquid. For example, the solute may be a polar organic compound or a surfactant. A polar organic compound may be an organic compound having highly polar functional groups such as an amino group, a carboxyl group, or a hydroxyl group. For example, polar organic compounds may be alcohols, fatty acids, amino acids, peptides, proteins, sugars, etc. Because polar organic compounds have hydrophobic and hydrophilic functional groups, they interact with the molecules of the gas and liquid 261 that form the gas-liquid interface 255 and adhere to the gas-liquid interface 255. In other words, by adding a polar organic compound to the solution, the gas-liquid interface 255 is covered with molecules of the polar organic compound, resulting in a decrease in the number of water molecules on the surface that come into contact with the gas phase, and thus a decrease in surface tension.

[0293] The surfactant may be an amphiphilic compound having both a hydrophobic and a hydrophilic portion within its molecule. For example, the surfactant may be a cationic surfactant (such as a reverse soap), anionic surfactant (such as sodium fatty acid), amphoteric surfactant (such as a betaine-based surfactant), or nonionic surfactant (such as an octyl glycoside). Like polar organic compounds, surfactants interact with the molecules of the gas and liquid 261 that form the gas-liquid interface 255, but the surface tension decreases at a much lower concentration than in the case of polar organic compounds. Therefore, adding a surfactant to a liquid significantly reduces the surface tension. This step of adding a solute may be performed, for example, by adding a solute-containing solution and performing the same operation as in step S620.

[0294] Let's look at another example of how 930 is specifically carried out. As an example, in 932, the surface tension of the liquid between the liquid and the gas can be reduced by removing a solute that increases the surface tension of the liquid from the liquid. For example, the solute may be an inorganic salt. An inorganic salt may be a compound that dissociates into cations and anions in a liquid such as an aqueous solution. For example, an inorganic salt may be a metal salt such as sodium chloride, potassium chloride, phosphate, or alum. In an inorganic salt, it dissociates in solution to become a cation and anion, and these ions are stabilized by being surrounded by water molecules and hydrated. Water molecules are more stable interacting with cations and anions that have Coulomb forces than interacting with other water molecules to form hydrogen bonds. Therefore, it is not possible for cations and anions originating from the inorganic salt to adhere to the gas-liquid interface 255. As a result, inside the bubble, a stable and stronger interaction occurs between the cations and anions originating from the inorganic salt and water molecules due to Coulomb forces. On the other hand, the gas-liquid interface 255 with the bubbles becomes closer to pure water, with only hydrogen bonds forming. As a result, an energy difference is created between the liquid inside the liquid and the liquid on the surface of the gas-liquid interface 255, and consequently, the interfacial free energy of the gas-liquid interface 255 increases. In other words, adding an inorganic salt to the liquid increases the surface tension. Therefore, removing the inorganic salt from the liquid can reduce the surface tension. The removal of the inorganic salt can be done by adding a chelating agent such as EDTA to the liquid or by performing column treatment with an ion exchange resin on the liquid.

[0295] Other specific examples of how 930 is carried out can be given. For example, in 933, the surface tension of the liquid between the gas and the liquid may be reduced by adding a liquid with a relatively lower surface tension to the liquid. Alternatively, the surface tension of the liquid between the gas and the liquid may be reduced by substituting the liquid with a liquid with a relatively lower surface tension. For example, the surface tension can be reduced and bubbles can be stably maintained and controlled by adding a liquid with a higher concentration of amphiphilic substances, such as polar organic compounds or surfactants, to the liquid filling container 25, or by substituting it with a liquid with a higher concentration of amphiphilic substances. For example, the surface tension can be reduced and bubbles can be stably maintained and controlled by adding a liquid with a lower concentration of inorganic salts to the liquid filling container 25, or by substituting it with a liquid with a lower concentration of inorganic salts.

[0296] Such steps of adding or replacing liquids can be carried out in the same manner as the steps and substeps of S600. For example, all or at least part of the liquid in the basic medium or buffer filling container 25 may be replaced with complete medium, or an appropriate amount of complete medium may be added to the liquid. By replacing or adding in this manner, surface tension can be reduced and bubbles can be stabilized. The basic medium may contain only a small amount of proteins and amino acids. As an example, the basic medium may be DMEM (Dulbecc's modified Eagle medium) or Ham's F-12 (Ham's F-12 medium). The complete medium may be the basic medium to which proteins such as serum or cell growth factors, or amino acids such as L-glutamine, have been added. The buffer may be a solution adjusted to a salt concentration, pH, or osmotic pressure suitable for cells. As an example, the buffer may be PBS (phosphate-buffered saline), HANKS buffer, or HEPES (hydroxyethylpiperazine ethanesulfonic acid) buffer.

[0297] Furthermore, as explained in Figure 13B, originally, θ m If the angle exceeds 90 degrees, the bubbles can become unstable, but θ mEven when the temperature exceeds 90 degrees, bubbles can be maintained stably under certain conditions. Let V0 be the maximum volume of gas involved in bubble formation (gas continuous with the bubble) at which the bubble is stably maintained. By keeping the volume of gas continuous with the bubble below V0, the bubble can be maintained stably. Since the formula for V0 is complex, experiments were conducted by setting the parameter values ​​based on actual usage conditions, and the following was confirmed.

[0298] Figure 13K shows the above experiment. In 720a, a tiny gas space was formed in the flow path 51 of the nozzle 49, with liquid in between. Water was used as the liquid, the contact angle between the nozzle 49 and the liquid was 90 degrees, and air was used as the gas. The radius of the flow path 51 of the nozzle 49 was 50 μm, and the maximum volume V0 of gas continuous with the bubble that the bubble was stable was calculated to be 1.17 μL. In 720a, the volume of the tiny gas space formed in the flow path 51 of the nozzle 49 was approximately 0.9 μL, and θ m Even if the temperature exceeds 90 degrees, the bubbles will remain stable. Such a minute gas space was formed as follows. First, the tip of the channel 51 of the nozzle 49 was placed in water, and a syringe pump was used to draw in approximately 3.5 μL of water into the channel 51. Next, the nozzle 49 was removed from the water, and a syringe pump was used to draw in approximately 0.9 μL of air into the channel 51. In this way, the amount of gas partitioned inside the channel 51 can be adjusted. After forming such a minute gas space, the stability of bubble formation was confirmed as follows. The tip of the channel 51 of the nozzle 49, which had formed the minute gas space, was placed in water, and pressurization was started using a syringe pump from state 720b (720c). At this time, θ was already mAlthough the temperature exceeded 90 degrees, the bubbles remained stable and were easy to control. Pressurization was continued (720d), and even when the bubbles were made larger, they remained stable and were easy to control. Subsequently, pressurization was stopped, and gas suction was started (720e). Gas suction was continued (720f), and even during the process of gradually reducing the size of the bubbles, they remained stable and were easy to control. Gas suction was completed (720g), and the bubbles were taken into the flow path 51 of the nozzle 49.

[0299] First, V0 is maximized when the contact angle between the flow channel member forming the end 254 of the flow channel 51 of the nozzle 49 and the liquid 261 is 90 degrees. Also, the larger the specific heat ratio κ of the gas, the larger V0 becomes. For example, the specific heat ratio κ is 5 / 3 for monatomic molecules such as helium, 7 / 5 for diatomic molecules such as nitrogen, and 8 / 6 for polyatomic molecules such as carbon dioxide. Furthermore, the smaller the surface tension γ, the larger V0 becomes. For example, the surface tension γ at a temperature of 20°C is 72.8 mN / m for water, about 50 mN / m for a culture solution containing protein, and about 5-15 mN / m for an aqueous solution containing a surfactant.

[0300] Therefore, we considered the case where the contact angle between nozzle 49 and the liquid is 90 degrees, and the gas is a monatomic molecule (specific heat ratio κ is 5 / 3) or air (since nitrogen and oxygen, which mainly make up air, are diatomic molecules, we considered air as a diatomic molecule and the specific heat ratio κ is 7 / 5). In this case, V o and the radius r of the end 254 of the flow path 51 h The relationship is V o and r h When plotted on a log-log graph, it is shown as a straight line.

[0301] Figure 13L shows r on the horizontal axis. h V on the vertical axis o The log-log graph when V is taken is shown. The conditions for the bubbles to be stably maintained and controlled are given by V. o This can be defined by: As shown in Figure 13L, V o and r h Since the relationship can be represented by a straight line, the slope of the line can be calculated. By calculating the slope of the line, V oand r h This can be approximated by the following relationship. [Equation 7] V o = a × r h 4 + b×r h 3 Here, a and b are coefficients. Then, in the graph of Figure 13L, if the region is below this line, the volume of the bubble is V o Therefore, bubbles can be stably maintained and controlled.

[0302] For example, in Figure 13L, if the contact angle between the end 254 of the nozzle 49 and the liquid 261 is 90 degrees, the gas is air (diatomic molecules, specific heat ratio κ is 7 / 5), and the liquid is water (in this case, the surface tension γ is 72.8 mN / m at a temperature of 20°C), then a = 1.82 × 10 8 Therefore, b = 2.59 × 10 2 That is the case.

[0303] Therefore, the volume V(m³) of gas occupied by the gas continuous from the gas-liquid interface 255 to the interior of the flow path 51 is 3 ) is the radius r of the end 254 of the flow path 51 h For (m), the following equation may be satisfied. [Formula 8] V ≤ a × r h 4 + b×r h 3 However, a = 1.82 × 10 8 Therefore, b = 2.59 × 10 2 V is r h If the above equation 8 is satisfied, the bubbles are stably maintained and controlled.

[0304] For example, in Figure 13L, if the contact angle between the end 254 of the nozzle 49 and the liquid 261 is 90 degrees, the gas is a monatomic molecule, and the liquid is an aqueous solution containing a surfactant (in this case, the surface tension γ is 5 mN / m at a temperature of 20°C), then the coefficients a and b can be calculated as a = 3.21 × 10⁻⁶. 9 Therefore, b = 3.15 × 102 That is the case.

[0305] Furthermore, in Figure 13L, if the contact angle between the end 254 of the nozzle 49 and the liquid 261 is 90 degrees, the gas is air, and the liquid is a culture solution containing protein (in this case, the surface tension γ is 50 mN / m at a temperature of 20°C), then a = 2.65 × 10 8 Therefore, b = 2.59 × 10 2 Therefore, when these values ​​are substituted for coefficients a and b, the bubbles are stably maintained and controlled within the range that satisfies equation 8. In the example shown in Figure 13K, the contact angle between the end 254 of the nozzle 49 and the liquid 261 is 90 degrees, the gas is air, and the liquid is water, and r h Since it is 50 μm, V o The volume is calculated to be 1.17 μL. In Figure 13K, the volume of the gas is approximately 0.9 μL, so the experiment confirmed that the bubbles are stably maintained and controlled. Note that the above relationship is an example where the contact angle is 90 degrees, but the contact angle is not limited to 90 degrees. For example, by setting a predetermined coefficient, the above relationship will be satisfied even if the contact angle is 80 degrees or 120 degrees.

[0306] Figure 14 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.

[0307] The host controller 2082 connects RAM 2020 to the CPU 2000 and graphics controller 2075, which access RAM 2020 at a high transfer rate. The CPU 2000 operates based on programs stored in ROM 2010 and RAM 2020, and controls each component. The graphics controller 2075 acquires image data generated by the CPU 2000 etc. on a frame buffer provided in RAM 2020 and displays it on the display device 2080. Alternatively, the graphics controller 2075 may include an internal frame buffer for storing image data generated by the CPU 2000 etc. The display device 2080 can display various information generated internally by the information processing device 170 (e.g., images, position information of the target device 35, etc.).

[0308] The I / O controller 2084 connects the host controller 2082 to the relatively high-speed I / O devices: the communication interface 2030, the hard disk drive 2040, and the CD-ROM drive 2060. The communication interface 2030 communicates with other devices via a network, either wired or wirelessly. The communication interface also functions as hardware for communication. The hard disk drive 2040 stores programs and data used by the CPU 2000 in the computer 1900. The CD-ROM drive 2060 reads programs or data from the CD-ROM 2095 and provides them to the hard disk drive 2040 via the RAM 2020.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] (Note) [Item 1] A gas-liquid interface formation step involves immersing the end of a channel in a liquid in which a living organism is submerged, thereby forming a gas-liquid interface within or at the end of the channel, where a restoring force acts against minute interfacial movement. The operational stage involves manipulating living organisms using a gas-liquid interface, A method for manipulating a living organism that possesses the following features. [Item 2] The gas-liquid interface formation step includes a step of releasing or drawing gas from the end. The operating procedure described in item 1. [Item 3] The gas-liquid interface formation step involves releasing gas from the end and maintaining a gas-liquid interface at the end where a restoring force acts against minute volume changes. The operating procedure described in item 1. [Item 4] In the gas-liquid interface formation stage, the gas volume V(m³) occupied by the gas that extends from the gas-liquid interface to the interior of the channel is 3 ) is the radius r at the end of the channel. h For (m), the following equation is satisfied: V ≤ a × r h 4 + b×r h 3 (However, a = 2.65 × 10 8 , and b = 2.59 × 10 2 (Satisfies the condition) The operating procedure described in item 2 or 3. [Item 5] Prior to the gas-liquid interface formation step, a volume reduction step is further included in which the volume of gas occupied by the gas continuous from the end to the interior of the flow path is reduced. The operating procedure described in any one of items 2 through 4. [Item 6] The volume reduction step involves introducing liquid into the channel and using the introduced liquid to partition the gas inside the channel. Instructions for use as described in item 5. [Item 7] The volume reduction step includes filling the channel with a filler material that blocks some of the internal space of the channel. Instructions for use as described in item 5. [Item 8] The flow path includes the space formed inside the nozzle and the pump connected to the nozzle. The volume reduction stage includes reducing the pump capacity to 10% or less of its maximum capacity. Instructions for use as described in item 5. [Item 9] The flow path is provided with a partitioning member that divides the space inside the flow path. Instructions for use as described in item 5. [Item 10] The cross-sectional shape of the end of the channel has a protruding portion facing inward. The operating procedure described in any one of items 1 through 9. [Item 11] At the end of the flow path, an angle is formed between the inner surface of the flow path and the end surface of the flow path member forming the flow path, and the angle of this angle is in the range of 90 ± 5 degrees. The operating procedure described in any one of items 1 through 10. [Item 12] The contact angle between the flow channel member forming the channel and the liquid using the droplet method is 90 degrees or less. The operating procedure described in any one of items 1 through 11. [Item 13] The flow path is The tip portion provided on the end side, An inner portion connected to the tip and having a different flow path diameter from the tip portion, It has, At the connection point between the tip and the inner part, the flow path forms a step. The operating procedure described in any one of items 1 through 12. [Item 14] The flow path is It has a wide section and a narrow section in the flow path diameter, The diameter of the narrowest part of the channel is less than one-tenth of the diameter of the wider part. The operating procedure described in any one of items 1 through 12. [Item 15] Prior to the gas-liquid interface formation step, the process further includes a surface tension reduction step in which the surface tension of the liquid between the gas and the liquid is reduced. The operating procedures described in items 2 through 9 and any one of items 10 through 14 that are indirectly dependent on item 2 or 3. [Item 16] Before the gas-liquid interface formation stage, The process further includes a movement step that moves the flow path and the organism closer together. The operating procedure described in any one of items 1 through 15. [Item 17] An organism manipulating apparatus for manipulating an organism, comprising: a flow channel having an end portion immersed in a liquid in which an organism is immersed; a gas-liquid interface operating section that forms a gas-liquid interface, on which a restoring force acts in response to minute interface movement, inside the flow channel or at the end portion of the flow channel, and manipulates the organism by means of the gas-liquid interface; the organism manipulating apparatus comprising the foregoing. [Item 18] the gas-liquid interface operating section discharges gas from the end portion into the liquid, and maintains the gas-liquid interface, on which a restoring force acts in response to minute volume changes, at the end portion, The organism manipulating apparatus according to Item 17. [Item 19] A gas volume V (m 3 ) occupied by gas continuous from the gas-liquid interface to the interior of the flow channel satisfies the following formula with respect to a radius r h (m) of the end portion of the flow channel, V≦a×r h 4 + b×r h 3 , (where a=2.65×10 8 and b=2.59×10 2 are satisfied) The organism manipulating apparatus according to Item 18. [Item 20] a filler that blocks a part of the space inside the flow channel is filled in the flow channel, The organism manipulating apparatus according to any one of Items 17 to 19. [Item 21] a partitioning member that partitions the space inside the flow channel is provided in the flow channel, The organism manipulating apparatus according to any one of Items 17 to 20. [Item 22] a cross-sectional shape of the end portion of the flow channel is a shape having a protruding portion directed inward, The organism manipulating apparatus according to any one of Items 17 to 21. [Item 23] at the end portion of the flow channel, a corner is formed by an inner side surface of the flow channel and an end-side surface of a flow channel member that forms the flow channel, and an angle formed by the corner is in a range of 90±5 degrees, A biological manipulation device as described in any one of items 17 to 22. [Item 24] The contact angle between the flow channel member forming the channel and the liquid using the droplet method is 90 degrees or less. A biological manipulation device as described in any one of items 17 to 23. [Item 25] The flow path is The tip portion provided on the end side, An inner portion connected to the tip and having a different flow path diameter from the tip portion, It has, At the connection point between the tip and the inner part, the flow path forms a step. A biological manipulation device as described in any one of items 17 to 24. [Item 26] The flow path is It has a wide section and a narrow section in the flow path diameter, The diameter of the narrowest part of the channel is less than one-tenth of the diameter of the wider part. A biological manipulation device as described in any one of items 17 to 25. [Explanation of Symbols]

[0324] 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 31 Normal Ski Prism 32. Analyzer (Polarizing Plate) 35. Target of Operation 37. Polarizer (polarizing plate) 38 Normal Ski Prism 39 Ring aperture 40 Nozzle Actuators 41 Sample Actuator 42 Camera for fluid imaging 45 Light source 46 Light source 47 Pressure generation unit 48 Sensor section 49 nozzles 50 Microscope Section 51 Flow channels 51a First channel 51b Second channel 53 Flow channel replacement section 54 Liquid storage section 58 Sample Lid 59 Sample lid storage section 60 Cameras 70 Cameras 100 Biological body manipulation device 101 Air-liquid interface 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 Second pump 253 Cylindrical part 253a Outer cylinder 253b Inner cylinder 254 End 255 Air-liquid interface 256 bubbles 260 Liquid Control Unit 261 Liquid 291 beads 292 Diaphragm 294 Press 295 Wider part of the channel diameter 297 Narrow section of the channel diameter 298 Wider part of the channel diameter 300 Image Processing Unit 305 Tip part 306 Inner part 307 steps 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. A gas-liquid interface formation step involves immersing the end of a channel in a liquid in which a living organism is submerged, and forming a gas-liquid interface at the end where a restoring force acts against minute interfacial movement, A step of manipulating the living organism using the aforementioned gas-liquid interface, Equipped with, The gas-liquid interface formation step includes releasing gas from the end and maintaining the gas-liquid interface at the end, in which a restoring force acts against minute volume changes. The aforementioned operational step is, The steps include: moving the gas-liquid interface while the gas-liquid interface is in contact with the biological body, thereby detaching the biological body from the solid phase and causing it to adhere to the gas-liquid interface; The step includes recovering the biological material adhering to the gas-liquid interface into the flow path by sucking in the gas present in the flow path, The organism includes cells, Methods for manipulating living organisms.

2. The gas-liquid interface formation step includes a step of drawing gas from the end, The operating method according to claim 1.

3. In the gas-liquid interface formation step, the gas volume V (m³) occupied by the gas that extends from the gas-liquid interface to the space formed inside the cylindrical portion of the flow path from the end of the flow path. ３ ) is the radius r at the end of the flow path. ｈ For (m), the following equation is satisfied: V≦a×r ｈ ４ + b×r ｈ ３ (However, a = 2.65 × 10 ８ , and b = 2.59 × 10 ２ (Satisfies the condition) The operating method according to claim 1 or 2.

4. Prior to the gas-liquid interface formation step, the method further includes a volume reduction step that reduces the volume of gas occupied by the gas that extends from the end of the flow path to the interior. The operating method according to any one of claims 1 to 3.

5. The volume reduction step includes taking the liquid into the flow path and partitioning the gas inside the flow path with the taken-in liquid. The operating method according to claim 4.

6. The volume reduction step includes filling the channel with a filler material that blocks a portion of the space inside the channel. The operating method according to claim 4.

7. The aforementioned flow path includes a space formed inside the nozzle and the pump connected to the nozzle. The volume reduction step includes reducing the capacity of the pump to 10% or less of its maximum capacity. The operating method according to claim 4.

8. The flow path is provided with a partitioning member that divides the space inside the flow path. The operating method according to claim 4.

9. The cross-sectional shape of the end of the aforementioned flow channel has a shape with a protrusion facing inward. The operating method according to any one of claims 1 to 8.

10. At the end of the flow path, an angle is formed between the inner surface of the flow path and the end surface of the flow path member forming the flow path, and the angle of this angle is in the range of 90 ± 5 degrees. The operating method according to any one of claims 1 to 9.

11. The contact angle between the flow channel member forming the flow channel and the liquid by the droplet method is 90 degrees or less. The operating method according to any one of claims 1 to 10.

12. The aforementioned flow path is The tip portion provided on the end side, An inner portion connected to the tip portion and having a flow path diameter different from the flow path diameter of the tip portion, It has, At the connection point between the tip portion and the inner portion, the flow path forms a step. The operating method according to any one of claims 1 to 11.

13. The aforementioned flow path is It has a wide section and a narrow section in the flow path diameter, The diameter of the narrow portion of the aforementioned flow path is one-tenth or less of the diameter of the wide portion of the aforementioned flow path. The operating method according to any one of claims 1 to 11.

14. Prior to the gas-liquid interface formation step, the method further includes a surface tension reduction step in which the surface tension of the liquid between the gas and the liquid is reduced. The operating method according to any one of claims 1 to 13.

15. The system further includes a detection step for detecting a first pressure applied to the gas-liquid interface, The detection step involves detecting the change in the first pressure when gas is continuously released into the flow path, and determining a second pressure at which the gas-liquid interface can be formed based on the changed value of the first pressure. The operating method according to any one of claims 1 to 14.

16. The system further includes a detection step for detecting a first pressure applied to the gas-liquid interface, Based on the change in the first pressure, it is detected that the end of the flow path has come into contact with the liquid, or that the gas-liquid interface formed at the end of the flow path has come into contact with the bottom of the container in which the organism is cultured. The operating method according to any one of claims 1 to 15.

17. Before the gas-liquid interface formation step, The process further includes a movement step in which the flow path and the organism are moved to bring them closer together. The operating method according to any one of claims 1 to 16.

18. A biological manipulation device for manipulating living organisms, A channel whose end is immersed in the liquid in which the organism is immersed, A gas-liquid interface is formed at the end of the aforementioned flow path, where a restoring force acts against minute interfacial movement, and a gas-liquid interface operating section is provided to manipulate a living organism using the gas-liquid interface. Equipped with, The gas-liquid interface operating unit releases gas from its end into the liquid, maintains the gas-liquid interface at the end where a restoring force acts against minute volume changes, moves the gas-liquid interface while it is in contact with the biological material, thereby detaching the biological material from the solid phase and causing it to adhere to the gas-liquid interface, and recovers the biological material attached to the gas-liquid interface into the flow path by sucking in the gas in the flow path. The organism includes cells, Biological body manipulation device.

19. A gas volume V (m ３ ) occupied by the gas that is continuous from the gas-liquid interface to the space formed inside the cylindrical portion of the flow channel from an end of the flow channel satisfies the following formula with respect to a radius r ｈ (m) of the end of the flow channel: V≦a×r ｈ ４ + b×r ｈ ３ 、 (However, a = 2.65 × 10 ８ , and b = 2.59 × 10 ２ (Satisfies the condition) The biological manipulation apparatus according to claim 18.

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