Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
  • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
  • G01N15/1492—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties within droplets
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1006—Investigating individual particles for cytology

Definitions

• the present technology relates to a droplet separation system. More specifically, the present invention relates to a droplet separation system, a droplet separation method, and a droplet separation program that perform droplet separation by optically detecting the characteristics of droplets.
• Flow cytometry is a process in which the particles to be analyzed are poured into a fluid in an aligned state, and the particles are irradiated with laser light, etc., and the fluorescence and scattered light emitted from each particle is detected. , is an analytical method for particle analysis and fractionation.
• cells labeled with a fluorescent dye are irradiated with excitation light such as laser light having an appropriate wavelength and intensity. Then, the fluorescence emitted from the fluorescent dye is focused using a lens, etc., light in an appropriate wavelength range is selected using a wavelength selection element such as a filter or dichroic mirror, and the selected light is transferred to a photomultiplier tube (PMT). Detection is performed using a photodetector such as a multiplier tube.
• PMT photomultiplier tube
• Detection is performed using a photodetector such as a multiplier tube.
• Fluorescence detection in flow cytometry involves selecting multiple discontinuous wavelength ranges of light using a wavelength selection element such as a filter and measuring the intensity of light in each wavelength range. Another method is to measure the intensity of light as a fluorescence spectrum.
• fluorescence emitted from particles is separated using a spectroscopic element such as a prism or a grating. Then, the separated fluorescence is detected using a light receiving element array in which a plurality of light receiving elements having different detection wavelength ranges are arranged.
• the light-receiving element array includes a PMT array or photodiode array in which light-receiving elements such as PMTs and photodiodes are arranged in one dimension, or a plurality of independent detection channels such as two-dimensional light-receiving elements such as CCD or CMOS. It is used.
• Particle analysis such as flow cytometry, often uses optical methods that irradiate the particles to be analyzed with light such as a laser and detect the fluorescence and scattered light emitted from the particles. Based on the detected optical information, an analysis computer and software extract a histogram and perform analysis.
• Patent Document 1 discloses an optical mechanism that irradiates each biological particle with light and detects the light from the biological particle, and an optical mechanism that irradiates each biological particle with light and detects the light from the biological particle. , comprising a control unit that detects the movement speed of the biological particles in the liquid flow, and a charging unit that applies an electric charge to the biological particles based on the movement speed of each of the biological particles.
• Devices have been proposed for separating biological particles contained in a liquid flow.
• Patent Document 2 discloses that a droplet sorting device includes a detection unit that detects the state of droplets discharged from an orifice that generates a fluid stream and satellite droplets existing between the droplets, and a detection unit that detects the state of the satellite droplets that exist between the droplets.
• a technology that can stably form droplets is provided by providing a control unit that controls the frequency of the driving voltage supplied to the vibration element that vibrates the orifice based on the position where the droplet is present.
• the main purpose of the present technology is to provide a new technology for stably forming droplets in droplet separation technology.
• the present technology first includes a droplet imaging unit that images the state of a fluid stream including droplets discharged from an orifice that generates the fluid stream; a vibrating element for forming the droplet; a control unit that specifies a control parameter for the vibrating element based on a state of the satellite with respect to a fluid stream image including a droplet fused with a satellite imaged by the droplet imaging unit; provide the system.
• the control parameter may be one or more parameters selected from the frequency, amplitude, and intensity of the drive voltage of the vibration element.
• the droplet separation system according to the present technology can include a processing unit that performs separation processing of the satellite part and the droplet part with respect to the fluid stream image.
• the control unit in the droplet separation system according to the present technology can specify control parameters for the vibrating element based on the fluid stream image after the separation process.
• the separation process can be performed based on droplet width information. In this case, the separation process can be performed at a position that has a specific width relative to the length of the droplet. Further, the separation process can also be performed at a position where the width of the droplet is the minimum, or at a position where the width of the droplet is a specific width with respect to the maximum width.
• the processing unit in the droplet separation system according to the present technology can perform a first determination process of determining whether or not the separation process is necessary based on a preset threshold value.
• the threshold value may be a threshold value related to one or more selected from the width, length, and center of gravity of the droplet.
• the processing unit in the droplet separation system determines whether the separation process is possible based on the droplet state parameter calculated from the fluid stream image captured by the droplet imaging unit.
• a second determination process can be performed.
• the state parameters include the ratio of the width and length of the droplet, the position of the center of gravity with respect to the length of the droplet, and the width of the droplet with respect to the length of the droplet.
• One or more state parameters can be selected from the positions.
• the processing unit in the droplet separation system according to the present technology can scan the fluid stream image from the downstream side and calculate the minimum value of the width of the droplet.
• the second determination process it can be determined that separation is possible when the state parameter is within a predetermined range.
• the fluid stream image is scanned from the upstream side, and if the position where the width of the droplet is minimum is within a predetermined range with respect to the length of the droplet, It can be determined that it is separable.
• the fluid stream image is scanned from the downstream side, and the position where the width of the droplet becomes a specific width with respect to the maximum width is determined within a predetermined range with respect to the length of the droplet. It can be determined that separability is possible if the
• the present technology then includes an imaging step of imaging the state of a fluid stream including droplets discharged from an orifice that generates the fluid stream; a droplet forming step of forming droplets using a vibrating element; a control step of specifying a control parameter for the vibration element based on a state of the satellite with respect to a fluid stream image fused with the satellite imaged in the imaging step; A droplet separation method is provided.
• the present technology further includes forming the droplets based on the state of the satellite with respect to a fluid stream image in which the state of the fluid stream including the droplet fused with the satellite ejected from the orifice that generates the fluid stream is captured.
• a droplet sorting program that allows a computer to implement a control function that specifies control parameters for a vibrating element to achieve this goal.
• FIG. 1 is a schematic conceptual diagram schematically showing a first embodiment of a droplet separation system 1 according to the present technology. It is a schematic conceptual diagram which shows typically 2nd Embodiment of the droplet separation system 1 based on this technique. It is a schematic conceptual diagram which shows typically 3rd Embodiment of the droplet separation system 1 based on this technique.
• 3 is a flowchart illustrating an example of an overview of a processing method performed by the processing unit 103.
• FIG. 4 is a flowchart illustrating an example of an overview of a processing method performed by the processing unit 103, which is different from FIG. 4.
• FIG. 5 is a flowchart illustrating an example of an outline of a processing method performed by the processing unit 103, which is different from FIGS. 4 and 5.
• This is a photograph substituted for a drawing showing an example of a fluid stream image captured by the droplet imaging unit 101 and for explaining a method of calculating the ratio of the center of gravity position to the length of a droplet.
• It is an example of the flowchart of the second determination process S2 when the position where the width of the droplet becomes a specific width with respect to the length of the droplet is used as the droplet state parameter.
• 2 is a drawing-substitute photograph showing an example of a droplet image captured by the droplet imaging unit 101.
• FIG. 10 is a photograph substituted for a drawing showing an example of a droplet image captured by the droplet imaging unit 101, which is different from that shown in FIG. 10.
• FIG. 5 is a flowchart illustrating an example of a series of steps of a processing method performed by the processing unit 103.
• FIG. FIG. 3 is a schematic conceptual diagram showing an installation example of a vibration element V and a charging section 106a.
• Droplet separation system 1 (1) Flow path P (2) Light irradiation section 104 (3) Detection unit 105 (4) Vibration element V (5) Droplet imaging unit 101 (6) Control unit 102 (7) Processing unit 103 (8) Preparation section 106 (9) Storage unit 107 (10) Display section 108 (11) User interface 109 2. Droplet separation method 3. Droplet separation program
• FIG. 1 is a schematic conceptual diagram schematically showing a first embodiment of a droplet separation system 1 according to the present technology.
• FIG. 2 is a schematic conceptual diagram schematically showing a second embodiment of the droplet separation system 1 according to the present technology.
• the droplet separation system 1 according to the present technology includes at least a droplet imaging section 101, a vibration element V, and a control section 102. Further, if necessary, a flow path P (P11 to P13), a light irradiation section 104, a detection section 105, a processing section 103, a sorting section 106, a storage section 107, a display section 108, a user interface 109, etc. may be provided. I can do it. The details of each part will be explained below.
• the droplet separation system 1 may include an information processing device 20 having a processing section 102, a processing section 103, a storage section 107, a display section 108, and a user interface 109.
• control unit 102, the processing unit 103, the storage unit 107, and the display unit 108 can be provided in a cloud environment and connected to the droplet separation system 1 via a network.
• a control unit 102, a processing unit 103, a display unit 108, and a user interface 109 are provided in the information processing device 20, and a storage unit 107 is provided in a cloud environment to perform droplet sorting via a network. It is also possible to connect to the device 10 and the information processing device 20. In this case, it is also possible to store records of various processes in the information processing device 20 in the storage unit 107 on the cloud, and to share the various information stored in the storage unit 107 with multiple users.
• Flow path P In the droplet separation system 1 according to the present technology, particle analysis and separation can be performed by detecting optical information obtained from particles aligned in a line in a flow cell (channel P).
• the flow path P may be provided in the droplet separation system 1 in advance, but it is also possible to install a commercially available flow path P or a disposable chip provided with a flow path P to perform analysis or separation. It is.
• the form of the flow path P is also not particularly limited and can be freely designed.
• a flow path P formed in a two-dimensional or three-dimensional substrate T such as plastic or glass as shown in FIGS. 1 and 3
• conventional flow cytometers can be used as shown in FIG.
• a flow path P such as that used can also be used in the droplet separation system 1.
• the channel width, channel depth, and channel cross-sectional shape of the channel P are not particularly limited as long as they can form laminar flow, and can be freely designed.
• a microchannel with a channel width of 1 mm or less can also be used in the droplet separation system 1.
• a microchannel having a channel width of approximately 10 ⁇ m or more and 1 mm or less can be suitably used in the present technology.
• the method for sending the particles is not particularly limited, and the particles can be passed through the flow path P depending on the form of the flow path P used.
• the sample liquid containing particles is introduced into the sample liquid flow path P11, and the sheath liquid is introduced into the two sheath liquid flow paths P12a and P12b.
• the sample liquid flow path P11 and the sheath liquid flow paths P12a and P12b merge to form a main flow path P13.
• sample liquid laminar flow sent through the sample liquid flow path P11 and the sheath liquid laminar flow sent through the sheath liquid flow paths P12a and P12b merge in the main flow path P13, and the sample liquid laminar flow is A sheath flow sandwiched between sheath liquid laminar flows can be formed.
• Particles flowing through the channel P include a wide range of biologically related microparticles such as cells, microorganisms, and ribosomes, and synthetic particles such as latex particles, gel particles, and industrial particles.
• Biologically related microparticles include chromosomes, ribosomes, mitochondria, organelles (cellular organelles), etc. that make up various cells.
• Cells include animal cells (eg, blood cells, etc.) and plant cells.
• Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast.
• biologically relevant microparticles may also include biologically relevant macromolecules such as nucleic acids, proteins, and complexes thereof.
• the industrial particles may be, for example, organic or inorganic polymeric materials, metals, and the like.
• Organic polymer materials include polystyrene, styrene/divinylbenzene, polymethyl methacrylate, and the like.
• Inorganic polymer materials include glass, silica, magnetic materials, and the like.
• Metals include colloidal gold, aluminum, and the like. Although the shape of these particles is generally spherical, in the present technology, they may be non-spherical, and their size, mass, etc. are not particularly limited.
• the particles flowing through the channel P can be labeled with one or more types of dyes such as fluorescent dyes.
• fluorescent dyes that can be used in this technology include, for example, Cascade Blue, Pacific Blue, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Propidium iodide (PI), Texas red (TR), Peridinin chlorophyll protein (PerCP ), Allophycocyanin (APC), 4',6-Diamidino-2-phenylindole (DAPI), Cy3, Cy5, Cy7, Brilliant Violet (BV421), etc.
• FITC Fluorescein isothiocyanate
• PE Phycoerythrin
• PI Propidium iodide
• TR Texas red
• API Allophycocyanin
• DAPI 4',6-Diamidino-2-phenylindole
• the light irradiation unit 104 irradiates particles contained in the fluid with excitation light.
• the light irradiation unit 104 can also be equipped with a plurality of light sources so that excitation light of different wavelengths can be irradiated. In this case, it is possible to irradiate a plurality of excitation lights with different wavelengths at different positions in the flow direction of the fluid.
• the type of light irradiated from the light irradiation unit 104 is not particularly limited, but in order to reliably generate fluorescence and scattered light from the particles, it is desirable that the light has a constant direction, wavelength, and light intensity.
• Examples include lasers, LEDs, etc.
• the type is not particularly limited, but it may be an argon ion (Ar) laser, a helium-neon (He-Ne) laser, a dye laser, a krypton (Cr) laser, a semiconductor laser, or a semiconductor laser.
• Ar argon ion
• He-Ne helium-neon
• Ce helium-neon
• dye laser a krypton
• semiconductor laser or a semiconductor laser.
• One type or two or more types of solid-state lasers combined with wavelength conversion optical elements can be used in any combination.
• the detection unit 105 detects light from particles contained in the fluid. Specifically, upon irradiation with the excitation light, fluorescence and scattered light emitted from the particles are detected and converted into electrical signals.
• the photodetector that can be used in the detection unit 105 is not particularly limited in its specific photodetection method as long as it can detect light from particles, and any photodetector used in known photodetectors may be used.
• the light detection method can be freely selected and employed. For example, fluorescence measuring instruments, scattered light measuring instruments, transmitted light measuring instruments, reflected light measuring instruments, diffracted light measuring instruments, ultraviolet spectrometers, infrared spectrometers, Raman spectrometers, FRET measuring instruments, FISH measuring instruments, etc.
• the vibrating element V forms droplets containing the particles. Specifically, when fluid containing particles is ejected as a jet flow JF from the orifice P14 of the main flow path P13, a vibration element V that vibrates at a predetermined frequency is used to vibrate all or part of the main flow path P13. By adding this, the horizontal section of the jet flow JF is modulated along the vertical direction in synchronization with the frequency of the vibrating element V, and droplets D are separated and generated at the break-off point BOP.
• the vibration element V used in the present technology is not particularly limited, and any vibration element V that can be used in a droplet separation device such as a general flow cytometer can be freely selected and used.
• An example is a piezo vibrating element.
• the size of the droplet D can be adjusted by adjusting the amount of liquid sent to the sample liquid flow path P11, the sheath liquid flow paths P12a, P12b, and the main flow path P13, the diameter of the discharge port, the vibration frequency of the vibration element V, etc. can be adjusted to generate droplets D each containing a certain amount of particles.
• the position of the vibrating element V is not particularly limited, and can be freely placed as long as it is possible to form droplets containing the particles.
• the vibration element V can be placed near the orifice P14 of the main flow path P13, or as shown in FIG. 4, the vibration element V can be placed upstream of the flow path P. It is also possible to apply vibration to the whole or part of the flow path P or to the sheath flow inside the flow path P.
• Droplet imaging unit 101 images the state of a fluid stream containing droplets (hereinafter also referred to as "the fluid stream"). Further, the droplet imaging section 101 is arranged downstream of the detection section 105.
• the specific configuration of the droplet imaging unit 101 is not limited as long as it can image the state of the fluid stream.
• the configuration is not limited to a configuration including an image pickup device such as a CCD camera or a CMOS sensor, but can also be configured with a so-called line sensor, etc., in which a plurality of sensors capable of detecting light brightness information such as a light amount sensor are lined up.
• the droplet imaging unit 101 is arranged at a position where it can image the state of the fluid stream between the orifice P14 and a counter electrode 106b, which will be described later.
• the fluid stream image obtained by the droplet imaging unit 101 is displayed on a display unit 108 such as a display to be described later, so that the user can check the droplet formation status and particle information (size, shape, spacing, etc.) in the fluid stream. etc.) can also be used to check.
• a strobe S As a light source for imaging the state of the fluid stream in the droplet imaging unit 101, for example, a strobe S can be used.
• the strobe S can also be controlled by a control unit 102, which will be described later.
• the strobe S can be composed of an LED for imaging the fluid stream and a laser (for example, a red laser light source) for imaging the fluid stream, and the control unit 102 controls the light source to be used depending on the purpose of detection. Switching can be done.
• the specific structure of the strobe S is not particularly limited, and one or more known circuits or elements can be selected and freely combined.
• Control unit 102 specifies control parameters for the vibrating element V based on the state of the satellite, with respect to the fluid stream image including the droplet fused with the satellite imaged by the droplet imaging unit 101.
• Patent Document 2 the state of the satellite droplet was detected, and the control parameters of the vibration element V were specified based on this.
• satellite droplets do not necessarily exist, and when satellite droplets do not exist, that is, when satellites are fused into droplets, based on this, It was not possible to specify the control parameters of the vibration element V.
• the state of the satellite can be extracted from the droplet image fused with the satellite, and the control parameters of the vibration element V can be specified based on this.
• the state of the satellite can be extracted, for example, by performing separation processing of the satellite part and the droplet part on the fluid stream image by the processing unit 103, which will be described later. That is, the control unit 102 can specify the control parameters for the vibration element V based on the fluid stream image that has been subjected to separation processing by the processing unit 103, which will be described later. Details of the separation process in the processing unit 103 will be described later.
• the control unit 102 Based on the extracted satellite state, the control unit 102 specifies the control parameters for the vibration element V. Examples of the control parameters of the vibration element V include the frequency, amplitude, and intensity of the drive voltage of the vibration element V, and the control unit 102 specifies one or more control parameters of the vibration element V. be able to.
• the satellite status is, for example, satellite position information. More specifically, by determining whether the satellite is a fast satellite or a slow satellite and executing a programmed control algorithm based on this, the frequency of the drive voltage supplied to the vibrating element V is determined. One or more control parameters selected from , amplitude, and intensity can be automatically controlled.
• control parameters of the vibration element V based on the state of the satellite can be specified using, for example, the method shown in Patent Document 2 mentioned above.
• Processing unit 103 performs separation processing between the satellite portion and the droplet portion on the fluid stream image including the droplet fused with the satellite imaged by the droplet imaging portion 101 .
• the processing unit 103 performs separation processing between the satellite portion and the droplet portion on the fluid stream image including the droplet fused with the satellite imaged by the droplet imaging portion 101 .
• FIG. 4 is a flowchart illustrating an example of an overview of a processing method performed by the processing unit 103.
• the processing unit 103 can perform a first determination process S1, a second determination process S2, and a separation process S3. These processes are performed as necessary. For example, if the first determination process S1 is determined to be "NG”, the process ends without performing the second determination process S2 and separation process S3, and the process proceeds to control by the control unit 102. On the other hand, if it is determined to be "OK” in the first determination process S1 and further determined to be "OK” in the second determination process S2, a separation process S3 is performed and the process in the processing unit 103 ends. The process then proceeds to control by the control unit 102.
• the process ends without performing the separation process S3, and the process proceeds to control by the control unit 102.
• the first determination process S1 is not essential, and may be set in advance according to the specifications of the droplet separation device 10, the size of the orifice P14, etc., or the user himself can perform the same steps as the first determination process S1. It is also possible to make a determination.
• the second determination process S2 is not essential, and when the user himself/herself performs the second determination process S2 and the result is determined to be "OK", the processing unit 103 may perform the separation process S3. After performing the separation process S3, the processing in the processing unit 103 is completed, and control proceeds to the control unit 102.
• the first determination process S1 and the second determination process S2 can be performed multiple times by changing their respective criteria as necessary. For example, as shown in the flowchart shown in FIG. 5, after performing the first determination process S1 at the first time ⁇ the second determination process S2 at the first time, the first determination process S1 at the second time ⁇ the second determination process at the second time. Process S2 can also be performed.
• the second determination process S2 for the first time ⁇ the second determination process S2 for the second time may be performed.
• the number of times of the first determination process S1 and the number of times of the second determination process S2 may not be the same.
• the first determination process S1 may be performed only once, and the second determination process S2 may be performed multiple times using different criteria. Each process will be explained in detail below.
• First determination process S1 In the first determination process S1, it is determined whether or not the separation process S3 is necessary based on a preset threshold value. More specifically, in the first determination process S1, a threshold value preset according to the specifications of the droplet sorting device 10, a threshold value determined at the time of design evaluation of the droplet sorting device 10, or a flow rate to be used is used. Based on a threshold value etc. set in advance according to the size of the orifice P14 of the path P, etc., it is determined whether or not to perform the separation process S3.
• the threshold value can be a threshold value related to one or more selected from the width, length, and center of gravity of the droplet.
• Second determination process S2 In the second determination process S2, it is determined whether the separation process S3 is possible based on the droplet state parameters calculated from the fluid stream image captured by the droplet imaging unit 101. More specifically, in the second determination process S2, it is determined that the droplet can be separated if the state parameter of the droplet calculated from the fluid stream image captured by the droplet imaging unit 101 is within a predetermined range, If it is outside the predetermined range, it is determined that separation is not possible.
• Droplet condition parameters include, for example, the ratio of the width and length of the droplet, the position of the center of gravity relative to the length of the droplet, and the position where the width of the droplet becomes a specific width relative to the length of the droplet. Based on one or more of these state parameters, the processing unit 103 determines whether the separation process S3 is possible.
• FIG. 7 shows an example of a fluid stream image captured by the droplet imaging unit 101, and is a photograph substituted for a drawing for explaining a method of calculating the ratio between the width and length of a droplet.
• Ratio (%) (width / height) x 100
• the ratio (%) value is 1 to 99, so the predetermined range is set to 1 to 99, and the ratio is determined based on the ratio of the width and length of the droplet. If the 2 determination process S2 is performed, it can be determined that the separation process S3 is possible only in the case of vertically long droplets.
• FIG. 8 shows an example of a fluid stream image captured by the droplet imaging unit 101, and is a photograph substituted for a drawing for explaining a method of calculating the ratio of the center of gravity position to the length of a droplet.
• the ratio (%) of the center of gravity position to the length (height) of the droplet can be calculated using the following formula.
• Ratio of center of gravity position (ratio (%)) (distance to center of gravity/length (height)) x 100
• the droplet has its center of gravity at the top (upstream side).
• the ratio (%) is 100%, the droplet has its center of gravity at the bottom (downstream side).
• the ratio (%) is 50%, the droplet has its center of gravity in the middle with respect to its length.
• the ratio (%) of the center of gravity is between 1 and 49. If the second determination process S2 is performed based on the position of the center of gravity relative to the length of the droplet, it can be determined that the separation process S3 is possible only for droplets whose center of gravity is below (downstream) from the center. can.
• FIG. 9 is an example of a flowchart of the second determination process S2 when the position where the width of the droplet becomes a specific width with respect to the length of the droplet is used as the state parameter of the droplet.
• the processing unit 103 first calculates the minimum width of the droplet (Min width).
• the minimum width of the droplet (Min width) can be calculated by scanning the fluid stream image from the downstream side (bottom side).
• the droplet has a part where the width widens again after reaching the minimum width, and the width of that part is XX% or less with respect to the maximum width of the droplet, narrowing is considered. It can be determined that the part is "present".
• the threshold value of the position relative to the length of the droplet is set to XX%, as shown in the example of the droplet image shown in Fig. 10, there is clearly a constricted part in the droplet, and the position of the narrowed part
• the droplet is located more than XX% downstream with respect to the length of the droplet, it can be estimated that the part above the constriction part corresponds to the satellite, and in the separation process S3 described later, Separation is possible at the stenosis. In this case, it is determined that separation is possible in check S233 of the position of the constricted portion of the droplet, and the process proceeds to separation processing S3, which will be described later.
• the width of the droplet is If separation is performed at the minimum position, there is a risk that separation will occur upstream of the position where separation should occur. If the control unit 102 specifies the control parameters for the vibration element V based on the information of the satellite part that has been separated upstream of the position where it should be separated, there is a risk that the control accuracy will be reduced. Therefore, in this case, the process advances to step S234 to check the position where the width of the next droplet becomes a specific width with respect to the maximum width.
• the width of a droplet is determined from a fluid stream image, and a predetermined width is calculated using the following formula.
• Predetermined width droplet width ⁇ (width threshold (%) for maximum droplet width/100)
• the length (height) of the droplet is determined from the fluid stream image, a predetermined position is calculated using the same formula as in checking S233 for the position of the constriction part of the droplet, and the calculated position and the predetermined width are Judgment can be made by comparing the position where (Next min width).
• the droplet length position threshold For example, if you set the droplet length position threshold to XX%, scan the fluid stream image from downstream until the droplet width is a certain width (Next min width) relative to the maximum width. If the position is more than XX% downstream of the length of the droplet, it can be determined that the droplet can be separated.
• the droplet A part that cannot be separated at the position where the droplet width is minimum, but is estimated to correspond to a satellite by separating at a part where the droplet width is a certain width (Next min width) compared to the maximum width. can be separated from the droplet portion.
• it is determined that separation is possible in step S234 of checking the position where the width of the droplet is a specific width with respect to the maximum width, and the process proceeds to separation processing S3, which will be described later.
• the constriction part of the droplet is not clear, the position where the width of the droplet is minimum (Min width) is too high, and the width of the droplet is If the position at which the specified width (Next min width) is also too high compared to the maximum width, if the separation is performed at the Next min width position, there is a risk that the separation will occur upstream of the position where it should be separated. If the control unit 102 specifies the control parameters for the vibration element V based on the information of the satellite part that has been separated upstream of the position where it should be separated, there is a risk that the control accuracy will be reduced.
• separation processing S3 separation processing of the satellite portion and the droplet portion is performed on the fluid stream image.
• the separation process between the satellite part and the droplet part can be performed based on the width information of the droplet. Specifically, the separation process between the satellite part and the droplet part can be performed at a position having a specific width relative to the length of the droplet. More specifically, the separation process of the satellite part and the droplet part is carried out at the position where the width of the droplet is the minimum (Min width) or the position where the width of the droplet is a specific width relative to the maximum width (Min width). Next min width).
• the constricted part is If it is determined that the length is within a predetermined range, in separation process S3, the satellite part and the droplet part are separated at the narrowing part, that is, the position where the width of the droplet is minimum (Min width). can be processed.
• the second determination process S2 in which the droplet has a constricted part, it is determined that there is no constricted part in the droplet, or in the second determination process S2, in which the droplet has a constricted part, it is determined that the droplet has no constricted part.
• check S232 it is determined that the droplet has a constricted part, but in check S233 of the position of the constricted part of the droplet, it is determined that the position of the constricted part is outside the predetermined range.
• next min width a specific width
• separation process S3 the width of the droplet is set to the maximum width. Separation processing of the satellite part and the droplet part can be performed at a position where the width becomes a certain width (Next min width).
• the first determination process S11 is performed for the first time. In the first first determination process S11, it is determined whether the separation process is necessary or not based on a preset threshold value. If it is determined that the separation process is not necessary, the process ends without performing the second determination process S2 and the separation process S3, and the process proceeds to control by the control unit 102. On the other hand, if it is determined in the first first determination process S11 that it is necessary to perform the separation process, the process proceeds to the first second determination process S21.
• the ratio of the width and length of the droplet is calculated (S211). If the calculated width-to-length ratio of the droplet is outside the predetermined range, the process ends without performing the separation process S3, and the process proceeds to control by the control unit 102. On the other hand, if the calculated ratio of the width and length of the droplet is within the predetermined range, the process proceeds to the second first determination process S12.
• the second first determination process S12 it is determined whether the separation process is necessary or not based on a preset threshold value. If it is determined that the separation process is not necessary, the process ends without performing the second determination process S22 and the separation process S3, and the process proceeds to control by the control unit 102. On the other hand, if it is determined in the second first determination process S12 that it is necessary to perform the separation process, the process proceeds to the second second determination process S22.
• the ratio of the center of gravity position to the length of the droplet is calculated (S221). If the calculated ratio of the center of gravity position is outside the predetermined range, the process ends without performing the separation process S3, and the process proceeds to control by the control unit 102. On the other hand, if the calculated ratio of the center of gravity is within the predetermined range, the process proceeds to S231 for calculating the minimum width of the droplet.
• next min width a specific width
• S234 is performed. In checking S234 for the position where the width of the droplet becomes a specific width (Next min width) with respect to the maximum width, if it is determined that the Next min width is within a predetermined range, the process proceeds to separation processing S3, and the droplet Separation processing between the satellite part and the droplet part is performed at a position where the width of the droplet part becomes a specific width (Next min width) with respect to the maximum width.
• Preparation section 106 the droplet D containing the particles formed by the vibrating element V is fractionated. Specifically, the droplet D is charged with a positive or negative charge based on the analysis results of the particle size, shape, internal structure, etc., analyzed from the optical signal detected by the detection unit 105 (sign 106a). Then, the course of the charged droplet D is changed to a desired direction by the counter electrode 106b to which a voltage is applied, and the droplet D is fractionated.
• the position of the charging unit 106a is not particularly limited, and can be freely positioned as long as it is possible to charge the droplet D containing the particles.
• the position of the charging unit 106a is not particularly limited, and can be freely positioned as long as it is possible to charge the droplet D containing the particles.
• a charging unit 105a composed of an electrode or the like and charge the droplet D via the sheath liquid immediately before forming the droplet D containing the target particles.
• the droplet separation system 1 can include a storage unit 107 that stores various data.
• the storage unit 107 stores, for example, image data captured by the droplet imaging unit 101, optical signal data from particles detected by the detection unit 105, control data controlled by the control unit 102, and processing processed by the processing unit 103. It is possible to store all kinds of data related to particle detection and droplet sorting, such as data and sorting data of particles sorted by the sorting section 106.
• the storage unit 107 can be provided in a cloud environment, so each user can share various information recorded in the storage unit 107 on the cloud via a network. It is.
• the storage unit 107 is not essential, and it is also possible to store various data using an external storage device or the like.
• the droplet separation system 1 can include a display section 108 that displays various data.
• the display unit 108 displays, for example, image data captured by the droplet imaging unit 101, optical signal data from particles detected by the detection unit 105, control data controlled by the control unit 102, and processing processed by the processing unit 103. It is possible to display all kinds of data related to particle detection and droplet separation, such as data and separation data of particles separated by the separation unit 106.
• the display unit 108 is not essential, and an external display device may be connected.
• the display unit 108 for example, a display, a printer, etc. can be used.
• the droplet separation system 1 can include a user interface 109 that is a part operated by a user. A user can access each part and each device through the user interface 109 and control each part and each device.
• the user interface 109 is not essential, and an external operating device may be connected.
• an external operating device may be connected.
• the user interface 109 for example, a mouse, a keyboard, etc. can be used.
• Droplet separation method includes at least an imaging step, a droplet formation step, and a control step. Further, a light irradiation step, a detection step, a treatment step, a fractionation step, a storage step, a display step, etc. can be performed as necessary.
• the droplet sorting program according to the present technology is a program for causing a computer to realize at least a control function of specifying control parameters of a vibrating element for forming droplets.
• the droplet separation program according to the present technology may be a program for causing a computer to realize a processing function of separating a satellite part and a droplet part with respect to a fluid stream image.
• the droplet separation program according to the present technology may be stored in a recording medium such as a magnetic disk, optical disk, magneto-optical disk, or flash memory, and may also be distributed via a network.
• a recording medium such as a magnetic disk, optical disk, magneto-optical disk, or flash memory
• each function is the same as the function performed by each part of the droplet separation system 1 according to the present technology described above, so a description thereof will be omitted here.
• the present technology can also take the following configuration.
• a droplet imaging unit that images the state of a fluid stream including droplets discharged from an orifice that generates the fluid stream; a vibrating element for forming the droplet; a control unit that specifies a control parameter for the vibrating element based on a state of the satellite with respect to a fluid stream image including a droplet fused with a satellite imaged by the droplet imaging unit; system.
• the control parameter is one or more parameters selected from the frequency, amplitude, and intensity of the driving voltage of the vibrating element.
• the droplet separation system according to any one of (3) to (7), wherein the processing unit performs a first determination process for determining whether or not the separation process is necessary based on a preset threshold value. .
• the threshold value is a threshold value related to one or more selected from the width, length, and center of gravity of the droplet.
• the processing unit performs a second determination process of determining whether the separation process is possible based on the droplet state parameter calculated from the fluid stream image captured by the droplet imaging unit.
• the droplet separation system according to any one of 3) to (9).
• the condition parameter is one or more selected from the ratio of the width and length of the droplet, the position of the center of gravity relative to the length of the droplet, and the position where the width of the droplet becomes a specific width relative to the length of the droplet.
• the processing unit scans the fluid stream image from the downstream side and calculates the minimum width of the droplet.
• (13) The droplet separation system according to (11), wherein in the second determination process, it is determined that separation is possible when the state parameter is within a predetermined range.
• the fluid stream image is scanned from the upstream side, and if the position where the width of the droplet is minimum is within a predetermined range with respect to the length of the droplet, it is determined that separation is possible.
• the fluid stream image is scanned from the downstream side, and the position where the width of the droplet becomes a specific width with respect to the maximum width is within a predetermined range with respect to the length of the droplet.
• the droplet separation system according to (13) which determines that separation is possible in certain cases.
• a droplet separation method comprising: (17) Based on the fluid stream image in which the state of the fluid stream including the droplet fused with the satellite discharged from the orifice that generates the fluid stream is imaged, the vibration element for forming the droplet is determined based on the state of the satellite.
• a droplet separation program that enables a computer to perform control functions that specify control parameters.
• Droplet separation system 10 Droplet separation device 20 Information processing device P, P11, P12, P13 Channel P14 Orifice 101 Droplet imaging section 102 Control section 103 Processing section 104 Light irradiation section 105 Detection section V Vibration element 106 Taking part 107 Storage part 108 Display part 109 User interface 106a Charging part 106b Counter electrode JF Jet flow BOP Break-off point D Droplet S Strobe

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• 2023
  • 2023-04-27 JP JP2024520393A patent/JPWO2023218986A1/ja active Pending
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