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WO2018070153A1 - Procédé d'imagerie d'une cellule - Google Patents

Procédé d'imagerie d'une cellule Download PDF

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Publication number
WO2018070153A1
WO2018070153A1 PCT/JP2017/032470 JP2017032470W WO2018070153A1 WO 2018070153 A1 WO2018070153 A1 WO 2018070153A1 JP 2017032470 W JP2017032470 W JP 2017032470W WO 2018070153 A1 WO2018070153 A1 WO 2018070153A1
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WO
WIPO (PCT)
Prior art keywords
dish
cell
cells
imaging
ridge line
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2017/032470
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English (en)
Japanese (ja)
Inventor
伊藤　三郎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yamaha Motor Co Ltd
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Yamaha Motor Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yamaha Motor Co Ltd filed Critical Yamaha Motor Co Ltd
Publication of WO2018070153A1 publication Critical patent/WO2018070153A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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

Definitions

  • the present invention relates to a method of holding a cell in a light-transmitting dish for recognizing the shape of the cell and imaging the cell with an imaging device.
  • a microplate having wells arranged in a matrix may be accommodated in the wells.
  • Cells accommodated in the well are sorted on a dish having a recess for holding the cells.
  • a cell suspension containing a large amount of cells is dispersed on the dish, and the cells are held in the recesses.
  • an image of the dish holding the cells is picked up, and the cells that can be used and the unusable cells and contaminants are classified by the image processing technique. Thereafter, usable cells are sucked from the recess by the suction tip, and the sucked cells are discharged to the well of the microplate (for example, see Patent Document 1).
  • the present invention provides a cell imaging method capable of easily recognizing cells on an image and shortening the imaging time of a dish when performing cell shape recognition from an image of a dish holding cells. It is to provide.
  • a cell imaging method includes a main body formed of a light-transmitting member and including a flat plate having an upper surface and a lower surface, and a plurality of cells that extend vertically from the upper surface to the lower surface of the main body and hold cells.
  • a holding recess, wherein the holding recess has a first portion formed of a tapered portion having a first inclination with respect to the vertical direction and an inner portion substantially parallel to the vertical direction.
  • the imaging method includes a first step of horizontally arranging at a predetermined position, an imaging device arranged on the lower surface side, performing an imaging operation of the image of the dish, and based on focus information at the time of the imaging operation.
  • FIG. 1A is a schematic diagram showing a part of the configuration of a cell transfer device for carrying out the cell imaging method according to the present invention.
  • FIG. 1B is a schematic view showing another part of the configuration of the cell transfer device.
  • FIG. 2 is a perspective view of a sorting container used in the cell transfer device.
  • FIG. 3 is a top view of a dish provided in the sorting container.
  • 4 is a cross-sectional view taken along line IV-IV in FIG.
  • FIG. 5 is a top view showing a plurality of dishes and photographing ranges.
  • FIG. 6 is a diagram showing the procedure of the cell imaging method according to the present invention.
  • FIG. 7 is a cross-sectional view for explaining details of the dish.
  • FIG. 1A is a schematic diagram showing a part of the configuration of a cell transfer device for carrying out the cell imaging method according to the present invention.
  • FIG. 1B is a schematic view showing another part of the configuration of the cell transfer device.
  • FIG. 2 is
  • FIG. 8 is a diagram schematically illustrating an imaging mode of the ridge portion of the dish.
  • FIG. 9 is a plan view showing an imaging location in imaging the ridge line portion.
  • FIG. 10 is a cross-sectional view showing a mode in which cells are accommodated in the holding recesses of the dish.
  • FIG. 11 is a cross-sectional view illustrating a state where the dish is imaged in a state where cells are held in the holding recess.
  • FIG. 12A is a diagram showing an image of a holding recess and a cell obtained by the imaging
  • FIG. 12B is a diagram showing an image of a cell after image processing.
  • FIG. 13 is a block diagram showing an electrical configuration of the cell transfer device.
  • FIG. 14 is a flowchart showing an overall flow of a cell imaging operation in the cell transfer device.
  • FIG. 15 is a detailed flowchart of the ridge line detection process.
  • FIG. 16 is a detailed flowchart of the cell imaging process.
  • a subject to be imaged is a cell derived from a living body, particularly a cell aggregate (spheroid).
  • a cell aggregate derived from a living body is formed by aggregation of several to several hundred thousand cells. Therefore, the size of the cell aggregate is various.
  • Cell aggregates formed by living cells are almost spherical, but if some of the cells that make up the cell aggregates are altered or become dead cells, the shape of the cell aggregate is distorted, or The density may be non-uniform.
  • the term “cell C” is simply used to include the cell aggregates as described above.
  • the cell imaging method according to the present invention is preferably applied to, for example, a cell moving device.
  • the cell transfer device picks usable cells from a plurality of cells having various shapes carried on a dish on a sorting stage in tests in the fields of biotechnology and medicine, and uses them to microplates. Moving. In the microplate, various processes such as observation, drug confirmation, inspection, and culture are performed on the cells. Cell imaging is performed, for example, to determine usable cell clumps on a dish.
  • a cell moving device picks usable cells from a plurality of cells having various shapes carried on a dish on a sorting stage in tests in the fields of biotechnology and medicine, and uses them to microplates. Moving. In the microplate, various processes such as observation, drug confirmation, inspection, and culture are performed on the cells. Cell imaging is performed, for example, to determine usable cell clumps on a dish.
  • an example in which the cell imaging method according to the present invention is applied to such a cell moving device will be mainly described.
  • FIG. 1A and 1B are diagrams schematically showing the overall configuration of the cell transfer device S.
  • the cell transfer device S includes a dispensing container 100, a sorting container 1 having a dish 10, a microplate 4, a camera unit 5 (imaging device), and a head unit 61 on which a dispensing chip 60 or a chip 6 is mounted. Yes.
  • the cell transfer device S aspirates a cell suspension LC containing a large number of cells C from the dispensing container 100, spreads the cell suspension LC on the dish 10 of the sorting container 1, and the cells according to the present invention.
  • FIG. 1A illustrates a portion of the dispensing container 100 and the sorting container 1 of the cell transfer device S
  • FIG. 1B illustrates a portion of the sorting container 1 and the microplate 4.
  • the dispensing container 100 is a container having an upper surface opening for storing a cell suspension LC containing a large number of cells C.
  • This cell suspension LC contains the cells C to be sorted in the sorting container 1 and inevitably mixed impurities.
  • the sorting container 1 is a container from which the cells C move, stores the medium L, and holds the cell sorting dish 10 in a state of being immersed in the medium L.
  • the dish 10 is a plate that carries the cells C, and has a plurality of holding recesses 3 on the upper surface that can accommodate the cells C individually.
  • the medium L is not particularly limited as long as it does not deteriorate the properties of the cells C, and can be appropriately selected depending on the type of the cells C.
  • Examples of the medium L include basal medium, synthetic medium, eagle medium, RPMI medium, Fischer medium, ham medium, MCDB medium, and serum medium, as well as glycerol and cell banker (Toji Field Co., Ltd.) that are added before frozen storage.
  • Cell frozen solution such as manufactured), formalin, reagents for fluorescent staining, antibodies, purified water, physiological saline and the like.
  • BxPC-3 human pancreatic adenocarcinoma cell
  • RPMI-1640 medium 10% fetal bovine serum FBS (Fetal Bovine Serum)
  • What added supplements, such as antibiotics and sodium pyruvate as needed, can be used.
  • a medium L can also be used as a base liquid for the cell suspension LC.
  • the sorting container 1 has a cylindrical shape, and has a rectangular upper opening 1H on the upper surface side.
  • the upper opening 1H is an opening for loading the cells C and picking up the sorted cells C.
  • the dish 10 is disposed below the upper opening 1H.
  • the sorting container 1 and the dish 10 are made of a translucent resin material or glass. This is because the cells C carried on the dish 10 can be observed by the camera unit 5 disposed below the sorting container 1.
  • the cell suspension LC is injected from the dispensing tip 60 into the sorting container 1.
  • the dispensing tip 60 is a tube-shaped member having a tip opening 60H, and sucks the cell suspension LC from the dispensing container 100 and discharges the cell suspension LC held therein to the sorting container 1. And do.
  • the tip opening 60H of the dispensing tip 60 is immersed in the cell suspension LC of the dispensing container 100, and the suction operation is executed.
  • the cell suspension LC (cell C) held in the dispensing tip 60 is discharged in a state where the tip opening 60H of the dispensing tip 60 is immersed in the culture medium L of the sorting container 1.
  • the microplate 4 is a container to which the cells C are moved, and has a plurality of wells 41 for receiving the cells C.
  • a single well 41 accommodates a necessary number (usually one) of cells C together with the medium L.
  • the microplate 4 is also made of a translucent resin material or glass. This is because the cells C carried on the microplate 4 can be observed by the camera unit 5 disposed below the microplate 4.
  • the camera unit 5 includes a camera lens 51 and captures an image of the cell C held on the dish 10 in the sorting container 1 or the cell C held in the well 41 on the microplate 4.
  • the camera unit 5 includes an image sensor such as a CCD image sensor.
  • the camera lens 51 forms an optical image of the cell C on the light receiving surface of the image sensor.
  • the camera unit 5 is arranged below these so that the camera lens 51 faces the lower surfaces of the sorting container 1 and the microplate 4. That is, the camera unit 5 captures an image of the cell C carried on the sorting container 1 or the microplate 4 from the lower surface side.
  • the camera unit 5 is movable in the horizontal direction between the lower side of the sorting container 1 and the lower side of the microplate 4 along the guide rail 52 as indicated by an arrow X2 in the drawing.
  • the chip 6 is a tube-shaped member having a tip opening 6H, and sucks and discharges the medium L containing the cells C. Specifically, the chip 6 sucks the cells C from the dish 10 of the sorting container 1, more specifically the cells C held in the holding recess 3 (FIG. 3) of the dish 10 together with the medium L, and these are microplates. 4 wells 41 are discharged. Although not shown, the chip 6 aspirates a reagent solution or the like as necessary, and discharges it into the well 41 carrying the cells C.
  • the head unit 61 is provided to move the cell suspension LC from the dispensing container 100 to the dish 10 of the sorting container 1 and to move the cells C from the dish 10 to the microplate 4.
  • the head main body 62 holds the head 63 so as to be able to advance and retreat in the vertical direction, and can move in the horizontal direction from the dispensing container 100 to the arrangement position of the microplate 4 as indicated by an arrow X1 in the drawing along the guide rail 61R. It is.
  • the head main body 62 can also move in a direction (front-rear direction) orthogonal to the paper surface of FIG.
  • the head 63 is made of a hollow rod.
  • the dispensing tip 60 and the tip 6 are attached to the lower end of the head 63.
  • one head 63 is illustrated, but a plurality of heads 63 may be used.
  • the head 63 for the dispensing tip 60 and the head 63 for the tip 6 may be provided in the head main body 62, respectively.
  • the dispensing tip 60 and the tip 6 may be alternately attached to and detached from one head 63.
  • a piston mechanism is mounted in the hollow portion of the head 63, and suction force and discharge force are applied to the tip opening 60H of the dispensing tip 60 or the tip opening 6H of the tip 6 by the operation of the piston mechanism.
  • the head main body 62 incorporates a power unit of the piston mechanism, an elevating mechanism for moving the head 63 in the vertical direction, and a power unit thereof.
  • the head unit 61 with the dispensing tip 60 mounted on the head 63 is moved above the dispensing container 100.
  • the head 63 is lowered, and the tip opening 60H of the dispensing tip 60 is immersed in the cell suspension LC of the dispensing container 100.
  • a suction force is generated in the head 63 and the cell suspension LC is sucked into the dispensing tip 60.
  • the head 63 is raised, and the head unit 61 is moved to the upper position of the sorting container 1.
  • the head 63 is lowered again, and the cell suspension LC held in the dispensing tip 60 is discharged while the tip opening 60H of the dispensing tip 60 is immersed in the culture medium L of the sorting container 1. That is, the cell C is seeded on the dish 10 (see FIG. 1A).
  • the head unit 61 in which the chip 6 is mounted on the head 63 is moved above the sorting container 1.
  • imaging according to the present embodiment of the cells C carried on the dish 10 and selection of usable cells C based on the captured images are performed, and the coordinates of the cells C to be extracted are obtained.
  • the head 63 is lowered, and the tip opening 6H of the chip 6 accesses the upper surface of the dish 10 through the upper opening 1H.
  • a suction force is generated in the head 63, and usable cells C are sucked into the chip 6 from the dish 10.
  • the head 63 is raised, and the head unit 61 is moved to a position above the microplate 4.
  • the head 63 is lowered again, and the cells C in the chip 6 are discharged into the wells 41 of the microplate 4.
  • [Dish configuration] 2 is a perspective view of the sorting container 1
  • FIG. 3 is a top view of the dish 10
  • FIG. 4 is a sectional view taken along line IV-IV in FIG.
  • the sorting container 1 includes a bottom plate 11, an outer peripheral wall 12, an inner peripheral wall 13, and a top wall 14.
  • the bottom dish 11 is a cylindrical dish member having an upper surface opening that constitutes the bottom of the sorting container 1.
  • the outer peripheral wall 12, the inner peripheral wall 13, and the top wall 14 constitute a lid member that covers the bottom plate 11.
  • the outer peripheral wall 12 is a portion larger in diameter than the side peripheral wall of the bottom plate 11, and the inner peripheral wall 13 is a rectangular tube-shaped portion disposed inside the outer peripheral wall 12.
  • the top wall 14 is a plate member that covers a region other than the upper opening 1 ⁇ / b> H on the upper surface side of the sorting container 1.
  • the inner peripheral wall 13 is a wall that partitions the upper opening 1H, and is inclined so that the opening area gradually decreases from the upper opening 1H downward.
  • the top wall 14 has a work hole 15 formed of a through hole in the vertical direction. Through this working hole 15, operations such as injection of the medium L into the cavity of the sorting container 1, injection of chemicals, liquid absorption or waste liquid of the medium L, and the like are performed. Further, a pipe connection port 16 for adjusting the atmospheric pressure in the cavity of the sorting container 1 is installed on the top wall 14.
  • the dish 10 includes a dish body 2 formed of a translucent member, and a plurality of holding recesses 3 formed in the dish body 2.
  • the dish body 2 is made of a flat plate member having a predetermined thickness and has an upper surface 21 and a lower surface 22.
  • the upper surface 21 is provided with a plurality of holding recesses 3 that hold cells C to be moved.
  • the dish 10 is held at the lower end of the inner peripheral wall 13 with the lower surface 22 spaced from the bottom plate 11 of the sorting container 1.
  • the dish 10 is immersed in the medium L in the sorting container 1. That is, the culture medium L is poured into the sorting container 1 so that the upper surface 21 of the dish 10 is located below the liquid surface of the culture medium L.
  • Each holding recess 3 includes an opening 31, a bottom 32, a cylindrical wall surface 33, a hole 34 (discharge hole) and a ridge line part 35.
  • the opening 31 is a square opening provided on the upper surface 21 and has a size that allows the tip opening 6H of the sorting chip 6 to enter.
  • the bottom 32 is located inside the dish body 2 and near the lower surface 22.
  • the bottom 32 is an inclined surface that is gently inclined downward toward the center (the center of the square).
  • the cylindrical wall surface 33 is a wall surface extending vertically downward from the opening 31 toward the bottom 32.
  • the hole 34 is a through hole that vertically penetrates between the center of the bottom 32 and the lower surface 22.
  • the shape of the hole 34 is square when viewed from above, and is concentric with the opening 31.
  • the ridge line portion 35 is located on the upper surface 21 and serves as an opening edge of each holding recess 3 and is a ridge line that partitions the holding recesses 3.
  • the holding recess 3 may have a round shape, a triangular shape, a pentagonal shape, a hexagonal shape, or the like, and these may be arranged in the dish body 2 in a honeycomb shape, a linear shape, or at random. The mode of the holding recess 3 will be described in detail later with reference to FIG.
  • each holding recess 3 defines an accommodation space 3H that accommodates the cells C. It is contemplated that one cell C is generally accommodated in the accommodation space 3H. Therefore, the holding recess 3 is set according to the size of the target cell C. However, in the operation of dispensing a cell culture solution containing a large number of cells C into the sorting container 1, a plurality of cells C may enter one holding recess 3.
  • the hole 34 is provided in order to let small cells and impurities other than the desired size escape from the accommodation space 3H. Therefore, the size of the hole 34 is selected so that cells C having a desired size cannot pass through and small cells or impurities other than the desired size can pass therethrough. As a result, the cells C to be sorted are trapped in the holding recesses 3, while impurities and the like fall from the holes 34 to the bottom dish 11 of the sorting container 1.
  • FIG. 5 is a diagram showing an arrangement example of the actual dish 10 in the sorting container 1.
  • the dish 10 in which four square small dishes 10A, 10B, 10C, and 10D are arranged so as to form one large square is illustrated.
  • the holding recess 3 of the dish 10 holding micron-order cells C has a very small size, and a thin plate is naturally used as the dish body 2.
  • the dish 10 having a required size is often formed by gathering the small dishes 10A to 10D. The same applies to the microplate 4.
  • FIG. 5 also shows an example of the shooting range AV of the dish 10 by the camera unit 5.
  • the angle of view of the optical system that images the micron-order cell C is naturally reduced.
  • a shooting range AV that covers about 1 ⁇ 4 of the small dish 10A is depicted, but in practice, the entire range of one small dish 10A is covered, and imaging is performed several tens to 100 times. Is required. Accordingly, since the four small dishes 10A to 10D are used in the example of FIG. 5, an image capturing operation four times as large is necessary.
  • FIG. 6 is a diagram illustrating a procedure of an imaging method for the cell C according to the present embodiment.
  • the imaging method of the cell C is executed sequentially, the first step # 1 in which the dish 10 is prepared and placed at a predetermined position, and the height position of the ridge line part 35 (shape feature part) of the dish 10 is recognized.
  • 2 step # 2 third step # 3 for accommodating the cell C in the holding recess 3 of the dish 10, fourth step # 4 for imaging the dish 10 holding the cell C with the camera unit 5, and the imaging
  • a fifth step # 5 for specifying (evaluating) the shape of the cell C based on the obtained image is included.
  • each step will be described.
  • FIG. 7 is a cross-sectional view for explaining details of the dish 10 used in the present embodiment.
  • the dish 10 includes the dish body 2 having the upper surface 21 and the lower surface 22, and the holding recess 3 having the opening 31 on the upper surface 21 and extending from the upper surface 21 toward the lower surface 22.
  • a vertical line V perpendicular to the upper surface 21 and the lower surface 22 is drawn at the hole core position of the holding recess 3.
  • the direction in which the holding recess 3 extends is the vertical direction in which the vertical line V extends.
  • a translucent material which comprises the dish main body 2 Although it does not specifically limit as a translucent material which comprises the dish main body 2, for example, it is preferable to employ
  • COP heat-resistant COP. Since COP is excellent in transparency, it is advantageous when imaging the cells C carried on the dish 10 from the lower surface 22 side, and can withstand heat sterilization by providing heat resistance. Furthermore, COP has the property that protein is hard to adhere, and can prevent the cell C from attaching to the dish 10.
  • the holding recess 3 includes a ridge line portion 35, an opening taper portion 31T, a cylindrical wall surface 33, a bottom portion 32, and a hole portion 34 from the upper surface 21 to the lower surface 22.
  • the holding recess 3 is divided into four characteristic parts, that is, a first part P1, a second part P2, a third part P3, and a fourth part P4, with the joints of these parts as boundaries. That is, the holding recess 3 is configured by sequentially connecting the first portion P1 to the fourth portion P4 from the upper surface 21 to the lower surface 22.
  • 1st part P1 is a part which has the upper surface 21, the same ridgeline part 35, and the opening taper part 31T (taper part with 1st inclination).
  • the opening taper portion 31T is formed of a tapered surface whose opening area gradually decreases downward.
  • the inclination angle ⁇ 1 (first inclination) with respect to the vertical line V of the tapered surface is set to be relatively small.
  • a preferable range of the inclination angle ⁇ 1 is about 25 ° to 55 °.
  • the plurality of holding recesses 3 are arranged in a matrix, and the apex of the opening taper portion 31T of one holding recess 3 and the apex of the opening taper portion 31T of the other holding recess 3 are adjacent to each other, A ridge line portion 35 is formed on the upper surface 21. That is, a sharp peak is formed on the upper surface 21 by the adjacent opening tapered portion 31 ⁇ / b> T, and the remaining portion of the upper surface 21 is the opening 31. Therefore, even when the cell suspension LC is spread on the upper surface 21 side, the cells C do not stay on the upper surface 21 and are guided to the accommodation space 3H by the opening taper portion 31T.
  • a preferable apex angle ⁇ A of the sharp peak is about 50 ° to 110 °.
  • the second portion P2 is a region where the cylindrical wall surface 33 exists, and is the portion having the longest vertical length among the first to fourth portions P1 to P4.
  • the cylindrical wall surface 33 is formed of a cylindrical portion having an inner wall surface (vertical wall surface) substantially parallel to the vertical line V, and divides most of the accommodation space 3H for accommodating the cells C. .
  • the cylindrical wall surface 33 guides the cells C that are sedimented by gravity downward.
  • the cylindrical wall surface 33 is a vertical wall surface, the cell C can settle by its own weight without receiving resistance.
  • the cylindrical wall surface 33 also serves to regulate the movement of the accommodated cells C in the horizontal direction.
  • the cylindrical wall surface 33 is a vertical wall surface, when the camera 10 captures an image of the dish 10 from the lower surface 22 side of the dish body 2, the cylindrical wall surface 33 appears in the image. You can avoid it.
  • the third portion P3 is a region where the bottom portion 32 exists.
  • the bottom portion 32 has a tapered surface whose opening area gradually decreases downward, and extends from the lower end of the cylindrical wall surface 33 to the upper end of the hole portion 34.
  • the taper surface serves as a contact surface of the cell C.
  • the inclination angle ⁇ 2 (second inclination) with respect to the vertical line V of the tapered surface of the bottom portion 32 is set to be larger than the inclination angle ⁇ 1.
  • a preferable range of the inclination angle ⁇ 2 is about 55 ° to 80 °.
  • the cell C can be stably grounded to the bottom portion 32, and impurities can be guided to the hole portion 34 along the tapered surface of the bottom portion 32. Furthermore, the thickness of the third portion P3 can be reduced.
  • the bottom portion 32 is a surface having four trapezoidal pieces and having a frustum shape.
  • the 4th part P4 is a part which consists of a baseplate which has the hole 34 of the size which the cell C of a holding
  • the hole 34 is a hole extending in the vertical direction and disposed at the position of the hole core (corresponding to the vertical line V in FIG. 7) of the holding recess 3. As shown in FIG. 3, the hole 34 has a square shape in a plan view viewed from the lower surface 22 side. As a result, the cell C (cell aggregate) having a generally spherical shape and the hole 34 can be easily distinguished on the image.
  • the hole 34 may be a polygon such as a triangle or a pentagon. Further, if it can be easily distinguished from the cell C, the circular hole 34 may be used.
  • the fourth part P4 is the thinnest part in the thickness in the direction of the vertical line V among the first to fourth parts P1 to P4 constituting the holding recess 3.
  • the fourth part P4 is located between the camera unit 5 and the cell C grounded to the bottom 32 of the third part P3. For this reason, the presence of the fourth portion P4 in addition to the third portion P3 can cause light loss and image distortion when the cell C is imaged.
  • the fourth portion P4 is the thinnest and the bottom portion 32 is also a tapered surface having a relatively large inclination angle ⁇ 2 with respect to the vertical line V, the third portion P3 is thinned. It can be minimized.
  • the dish 10 having the characteristics of the first to fourth portions P1 to P4 is prepared, and this is arranged horizontally at a predetermined position.
  • the dish 10 faces the upper opening 1H of the sorting container 1 and is disposed in a state of being immersed in the medium L.
  • ⁇ Second step> In the second step # 2, calibration of the height position of the dish 10 installed at the predetermined position in the first step # 1 is performed. Specifically, processing is performed in which the dish 10 is imaged by the camera unit 5 and the height position of a predetermined shape feature of the dish 10 is determined in advance.
  • the recognized shape feature portion is a ridge line portion 35 formed on the upper surface 21 of the dish 10.
  • the ridge line portion 35 is an example, and the shape feature portion may be another portion of the dish 10, and may be, for example, the lower edge of the hole portion 34.
  • FIG. 8 is a diagram schematically illustrating an imaging mode of the ridge line portion 35 in the second step # 2.
  • the sorting container 1 is simply drawn.
  • the dish 10 is installed in the sorting container 1 in a horizontal state, the upper surface 21 is below the liquid level of the medium L, and the lower surface 22 is separated from the bottom surface of the container. It is immersed in the medium L in a state.
  • the camera unit 5 is disposed on the lower surface 22 side, and performs an image capturing operation of the image of the dish 10 from below. The imaging operation may be performed after the dish 10 is installed in the sorting container 1 and before the culture medium L is injected.
  • the ridge line portion 35 is focused by the camera lens 51. Since the sorting container 1, the dish 10, and the medium L have translucency, it is possible to image the ridge line portion 35 from the lower surface 22 side.
  • a contrast detection method can be adopted. Specifically, the camera unit 5 is caused to capture the image of the dish 10 while shifting the focus position upward in units of several tens of microns with a predetermined position that can be determined to be below the ridge line portion 35 as an imaging start point.
  • the imaging end point is a predetermined position that can be determined to be above the ridge line portion 35. That is, the imaging end point is a position where it can be confirmed that the contrast value gradually decreases after the contrast value gradually increases due to the upward shift of the focus position and becomes the highest (focus position with respect to the ridge line portion 35).
  • the focus position where the image in which the line estimated to be the ridge line portion 35 is captured with the highest contrast is treated as the focus position, and the height of the ridge line portion 35 is determined based on the focus distance. A position is required.
  • the height position of the ridge line portion 35 as the shape feature portion is recognized based on the focus information at the time of the imaging operation.
  • the shape feature portion may be other than the ridge line portion 35
  • the ridge line portion 35 is located on the upper surface 21 relatively far from the third portion P3 to which the cell C is grounded, and is located between the adjacent holding recesses 3 so that the cell C Since it has a simple linear shape, it has an advantage that it can be easily recognized on the image as a shape feature.
  • the dish 10 Even if the dish 10 is arranged at a predetermined position in the first step # 1, it is difficult to create a complete horizontal state, and the dish 10 may be warped. For this reason, it is difficult to obtain the reference position of all the holding recesses 3 only by obtaining the height position of one ridge line portion 35. Ideally, the height positions of the ridgeline portions 35 corresponding to all the holding recesses 3 are determined. Desirable. However, as described above with reference to FIG. 5, since the angle of view of the camera unit 5 is small and a large number of imaging operations are required to capture the entire area of one dish, the ridge line portions 35 corresponding to all the holding recesses 3 are imaged. It takes too much time.
  • FIG. 9 is a plan view showing an example of an imaging location of the ridge line portion 35.
  • the dish body 2 is a rectangular flat plate.
  • holding recesses 3A, 3B, 3C, and 3D may be holding recesses in the vicinity thereof located at the four corners of the dish body 2 and a holding recess 3E at the center of the dish body 2 are selected as the main parts.
  • the height position of each ridge line portion 35 corresponding to these is recognized, for example, by the contrast detection method described above.
  • the holding recess 3 ⁇ / b> E is a holding recess that exists near the intersection of the diagonal lines of the four corners of the dish body 2.
  • the dish body 2 may have a shape such as a polygon other than a quadrangle, a circle, or an ellipse. Further, what is actually imaged is the holding recess 3 included in the range of the shooting range AV as described in FIG. 5, and the shooting range AV is set so as to include the selected holding recesses 3A to 3E.
  • the height position of the ridge line portion 35 at an arbitrary position on the upper surface 21 is calculated. Is possible. Accordingly, in the fourth step # 4 in the subsequent stage, when obtaining the height position information of the ridge line portion 35 of any holding recess 3 to be imaged of the cell C, the ridge line portion 35 for all the holding recesses 3 included in the dish 10 is obtained. Without recognizing the height position, it is possible to calculate based on the information on the height positions of the ridge portions 35 near the four corners and the intersection. Therefore, the time required for imaging can be shortened.
  • the height positions of the ridge lines 35 of all the holding recesses 3 included in the imaging range AV when shooting the holding recesses 3A to 3E may be obtained. Then, these average values may be calculated, and the average values may be handled as height position information at each of the four corners and the center of the dish body 2.
  • the third step # 3 is a step of accommodating the cell C in the holding recess 3 of the dish 10.
  • the cell suspension LC containing the cells C is spread from the upper surface 21 side while the dish 10 is immersed in the medium L (liquid) in the sorting container 1.
  • the cell C is guided by gravity into the holding recess 3 through the first portion P1 (opening 31). Further, the cell C settles along the second portion P2 (cylindrical wall surface 33) and contacts the third portion P3 (bottom portion 32).
  • FIG. 10 is a cross-sectional view showing a mode in which the cells C are accommodated in the holding recesses 3 of the dish 10.
  • the cell suspension LC includes cells C of various sizes and shapes (simply drawn as circles in FIG. 10) and impurities Cx that are inevitably mixed. These settle in the medium L by gravity and reach the upper surface 21 of the dish body 2.
  • the cell C settled on the opening 31 of the holding recess 3 enters the accommodation space 3H as it is.
  • the cell C settled on the wall portion between the holding recesses 3 collides with the ridge line portion 35.
  • the ridge line portion 35 is formed by a sharp peak in the first portion P1, and the opening taper portion 31T continuing below the ridge line portion 35 has a relatively steep inclination. For this reason, the cell C is guided to the opening taper portion 31T and smoothly enters the second portion P2.
  • the cells C settle by their own weight while being restricted from moving in the horizontal direction by the cylindrical wall surface 33 extending in the vertical direction. During the sedimentation, the cell C does not receive resistance from the substantially cylindrical wall surface 33. As sedimentation proceeds, the cell C eventually reaches the third portion P3 and contacts the tapered surface of the bottom portion 32. A cell C (cell C indicated by a dotted line in FIG. 10) that is in contact with a position deviated from the hole core (position of the hole 34) of the holding recess 3 is guided by the tapered surface of the bottom 32 and above the hole 34. Settle in position. Since the size of the cell C is larger than the opening size of the hole 34, the cell C will not settle any further.
  • the foreign matter Cx also contacts the bottom 32 and is guided toward the hole 34. Since the size of the contaminant Cx is smaller than the opening size of the hole 34, it passes through the hole 34 (fourth portion P4). Thereafter, the contaminant Cx is received by the bottom plate 11 (FIG. 2) of the sorting container 1. Accordingly, only the cell C is held in the holding recess 3. In addition, when a plurality of cells C enter one holding recess 3, an upward jet through the hole 34 may be generated to disperse the cells C.
  • FIG. 11 is a cross-sectional view showing a state in which the dish 10 is imaged.
  • the camera unit 5 includes an imaging element 53 that photoelectrically converts a light image, a camera lens 51 and an aperture 54 as an imaging optical system. While the dish 10 is illuminated by an unillustrated illumination system, an image of the dish 10 holding the cells C1 and C2 is captured by the camera unit 5 from the lower surface 22 side of the dish body 2.
  • the cells C1 and C2 have a width in the horizontal direction according to their sizes. Therefore, the light images M1 and M2 of the cells C1 and C2 are formed on the image sensor 53 as an object having a corresponding horizontal width. For this reason, the outline of the cells C1 and C2 can be captured as an image.
  • the cylindrical wall surface 33 of the holding recess 3 is a vertical wall, it has little width in the horizontal direction even if it is in a position shifted from the optical axis. Therefore, the optical image M3 of the cylindrical wall surface 33 hardly appears in the captured image (to the extent that it appears as a narrow line). Further, since the third portion P3 and the fourth portion P4 of the holding recess 3 are thinned as described above, there is little light amount loss and image distortion. These things contribute to the clarification of the images of the captured cells C1 and C2.
  • the information on the height position of the ridge line portion 35 obtained in the second step # 2 is used.
  • the height position of the ridge line portion 35 is set as a reference position, and a position below the reference position by a predetermined distance is set as an imaging start point.
  • This predetermined distance can be set according to the distance from the ridge line portion 35 to a position corresponding to the vicinity of the lower surface of the cell C carried on the holding recess 3 in consideration of the depth of the holding recess 3 and the like.
  • the predetermined distance can be set to 360 ⁇ m, for example.
  • the camera unit 5 When the image of the dish 10 is taken by the camera unit 5 at the above imaging start point, the camera unit 5 is caused to take the image of the dish 10 a plurality of times while shifting the focus position upward in units of several tens of microns.
  • the pitch shifted upward is, for example, 20 ⁇ m to 40 ⁇ m.
  • the imaging end point is an appropriate position below the ridge line portion 35.
  • the imaging start point may be set to the upper position of the dish 10 and the focus position may be gradually shifted downward.
  • the shape and hue of the cell C are specified based on the image data of the dish 10 obtained by the imaging operation. For example, image processing is performed on the image data obtained in the fourth step # 4, and processing for recognizing the presence of the cell C on the image, processing for recognizing the shape of the recognized cell C, and the like are executed. Furthermore, based on the specified shape and color, it is evaluated whether or not the cell C is a healthy cell that can be used for experiments and examinations.
  • FIG. 12A is an example of an image of the holding recess 3 and the cell C obtained by the imaging operation in the fourth step # 4.
  • a line corresponding to the ridge line part 35, a contour line of the cylindrical wall surface 33 and the hole part 34, and a line of a quadrangular pyramid on the bottom part 32 are reflected.
  • These lines are all straight lines and can be easily detected by a general-purpose straight edge detection process or the like.
  • the hole portion 34 is reflected in the image so as to completely overlap the cell C.
  • FIG. 12B is a diagram illustrating an image of the cell C after performing image processing for deleting the detected straight line. A clear image of the cell C can be obtained by the image processing.
  • FIG. 23 is a block diagram showing an electrical configuration of the cell transfer device S.
  • the cell transfer device S controls the movement of the head unit 61 (FIGS. 1A and 1B), the positioning and raising / lowering of the head 63, the suction and discharge operations of the cells C by the head 63, and the movement and imaging operations of the camera unit 5.
  • the unit 7 is provided.
  • the cell moving device S includes a camera shaft driving unit 55 as a mechanism for moving the camera unit 5 horizontally, a head unit shaft driving unit 64 as a mechanism for moving the head unit 61 horizontally, a mechanism for moving the head 63 up and down, and suction and discharge operations.
  • a head driving unit 65 and a display unit 66 are provided as a mechanism for performing the above.
  • the camera shaft drive unit 55 includes a drive motor that moves the camera unit 5 along the guide rail 52.
  • a ball screw is laid along the guide rail 52, the camera unit 5 is attached to a nut member screwed into the ball screw, and the drive motor rotates the ball screw forward or backward. In this mode, the camera unit 5 is moved to the target position.
  • the head unit shaft drive unit 64 includes a drive motor that moves the head unit 61 (head body 62) along the guide rail 61R.
  • a preferred embodiment is a mode in which a ball screw and a nut member are provided as in the camera shaft driving section 55, and the drive motor rotates the ball screw forward or backward.
  • the head main body 62 is moved in two directions XY, the first ball screw (X direction) along the guide rail 61R and the movement attached to the first nut member screwed to the first ball screw are mounted.
  • a second ball screw (Y direction) mounted on the plate is used. In this case, the head main body 62 is attached to a second nut member screwed into the second ball screw.
  • the head driving unit 65 corresponds to a power unit for an elevating mechanism that moves the head 63 in the vertical direction, and a power unit (for example, a motor) for driving a piston mechanism assembled in the hollow portion of the head 63 formed of a hollow rod. .
  • the lifting mechanism moves the head 63 up and down between the lowered position where the head 63 extends downward from the head body 62 and the raised position where most of the head body 62 is accommodated.
  • the power unit of the piston mechanism raises and lowers the piston member arranged in the head 63, so that the suction force and the discharge force are applied to the tip opening 6 ⁇ / b> H of the tip 6 attached to the head 63 or the tip opening 60 ⁇ / b> H of the dispensing tip 60. Is generated.
  • the display unit 66 is composed of a liquid crystal display or the like, and displays an image taken by the camera unit 5, an image subjected to image processing by the control unit 7, and the like.
  • the control unit 7 includes a microcomputer or the like, and includes an imaging control unit 71, an image memory 72, an image processing unit 73, an axis control unit 74, a head control unit 75, and a storage unit 76 by executing a predetermined program.
  • the imaging control unit 71 controls the moving operation and imaging operation of the camera unit 5, particularly the imaging operation of the dish 10 described in the second step # 2 and the fourth step # 4 described above.
  • a movement control unit 711, a ridge line detection unit 712, a ridge line height calculation unit 713, and a cell detection unit 714 are provided.
  • the camera movement control unit 711 controls the operation of moving the camera unit 5 along the guide rail 52 by controlling the camera axis driving unit 55. Further, the camera movement control unit 711 moves the camera unit 5 minutely when imaging the dish 10. As described above, since the angle of view of the camera unit 5 is considerably smaller than the size of the dish 10, the camera movement control unit 711 controls the camera axis driving unit 55 to slightly move the camera unit 5 in the XY directions. Meanwhile, the imaging operation of the dish 10 is executed.
  • the ridge line detection unit 712 controls the imaging operation for detecting the ridge line part 35 described in the second step # 2.
  • the ridge line height calculation unit 713 is based on the height positions of the ridge line portions 35 of several holding recesses 3 (for example, the four corners and the center of the dish 10) detected by the ridge line detection unit 712, and the ridge line portions of the arbitrary holding recesses 3 A process of calculating the height position of 35 is performed.
  • the cell detection unit 714 controls the imaging operation for detecting the cell C described in the fourth step # 4.
  • the image memory 72 includes a storage area and an external storage provided in the microcomputer, and temporarily stores image data acquired by the camera unit 5.
  • the image processing unit 73 performs image processing on the image data captured by the camera unit 5 and stored in the image memory 72.
  • the image processing unit 73 recognizes the presence of the cell C on the dish 10 as described in the fifth step # 5 based on the image of the dish 10 after the cells C are dispensed, Processing for recognizing the distribution of the cells C, processing for recognizing the shape of the recognized cells C, and the like are executed using an image processing technique.
  • the image processing unit 73 executes processing for filtering a line (ridge line portion 35) corresponding to the holding recess 3 from the acquired image.
  • the axis control unit 74 controls the operation of the head unit axis driving unit 64. That is, the axis control unit 74 controls the head unit axis driving unit 64 to move the head unit 61 to a predetermined target position in the horizontal direction. Movement of the head 63 (chip 6 or dispensing tip 60) between the dispensing container 100 and the sorting container 1, positioning of the holding recess 3 of the dish 10 to be aspirated in the vertical direction, and micro to be ejected The positioning of the well 41 of the plate 4 in the vertical direction is realized by the control of the head unit shaft drive unit 64 by the shaft control unit 74.
  • the head control unit 75 controls the head driving unit 65.
  • the head control unit 75 controls the power unit for the lifting mechanism of the head driving unit 65 to raise and lower the head 63 to be controlled toward a predetermined target position.
  • the head controller 75 controls the power unit of the piston mechanism for the head 63 to be controlled, thereby opening the tip of the tip 6 or the dispensing tip 60 attached to the head 63 at a predetermined timing.
  • a suction force or a discharge force is generated at 6H and 60H.
  • the storage unit 76 stores various set values and data in the cell transfer device S. In addition, the storage unit 76 also stores an imaging sequence of the dish 10 by the camera unit 5, height position data of the ridge line part 35 detected by the ridge line detection part 712, and the like.
  • FIG. 14 is a flowchart showing the overall flow of the imaging operation
  • FIG. 15 is a detailed flowchart of the ridge line detection process
  • FIG. 16 is a detailed flowchart of the cell imaging process.
  • the flow of FIG. 14 is started after the preparation of the dish 10 and the arrangement in the sorting container 1 in the first step # 1 shown in FIG. 6 are completed.
  • the control unit 7 reads the arrangement data of the dish 10 (step S1).
  • the arrangement data is XY coordinate data for specifying the positions of the four dishes 10 in the sorting container 1 arranged on a predetermined work stage.
  • an example in which four dishes 10 are used is shown, but five or more dishes 10 may be used, or three or less dishes 10 may be used.
  • step S2 processing for detecting the height position of the ridge line portion 35 of the dish 10 is executed mainly by the ridge line detection unit 712 of the imaging control unit 71 (step S2).
  • This ridge line detection process corresponds to the recognition of the ridge line portion 35 described in the second step # 2 of FIG.
  • FIG. 9 a process in the case of recognizing the height positions of the five ridge line portions 35 on the upper surface 21 of the dish body 2 that are separated from each other is illustrated.
  • step S3 The waiting time is set for a while until the cell C is accommodated in the holding recess 3, that is, until the third step # 3 in FIG. 6 is completed, and then the next step S4 is started. Note that step S3 may be executed prior to step S2.
  • step S4 a process of imaging the cell C carried on the dish 10 is executed mainly by the cell detection unit 714 of the imaging control unit 71 (step S4).
  • This cell imaging process corresponds to the imaging of the dish 10 described in the fourth step # 4 of FIG.
  • FIG. 16 As shown in FIG. 5, for each of the four dishes 10A to 10D, a process is illustrated when imaging for the entire area of the dish requires multiple imaging.
  • the small dish 10A is designated as a processing target.
  • the camera movement control unit 711 controls the camera axis driving unit 55 to move the camera unit 5 directly below the Nth dish 10 (step S12).
  • the camera unit 5 is moved so that the camera optical axis is aligned with the holding recesses 3 that are set to perform imaging first among the five holding recesses 3 that perform imaging of the ridge line portion 35. .
  • the central holding recess 3E is imaged first, since the central portion tends to bend, the height position tends to be different from the corners of the other four corners.
  • any one of the corners is imaged first, and this is used as a reference for imaging the remaining four locations.
  • the ridge line portions 3 of the holding recesses 3A, 3B, 3C, and 3D at the corners of the four corners of FIG. 5 are referred to as the ridge line portions 3 of the first, second, third, and fourth corners.
  • the ridge line portion 3 of the central holding recess 3E is referred to as the central ridge line portion 3.
  • the ridge line detection unit 712 causes the camera unit 5 to perform an imaging operation for detecting the ridge line height Z1 of the first corner.
  • Step S13 the ridge line detection unit 712 uses a predetermined position clearly below the position where the dish 10 (ridge line part 35) is arranged as an imaging start point, and sequentially shifts the focus position upward at a predetermined pitch while the image of the dish 10 is displayed.
  • the camera unit 5 is caused to perform a predetermined number of imaging operations by a method of performing imaging.
  • the predetermined pitch is, for example, several tens of microns.
  • the predetermined number of times is a number of times sufficient to estimate that the focus position reaches above the ridge line portion 35 by shifting upward by a predetermined pitch.
  • the ridge line detection unit 712 detects the ridge line part height Z1 by a contrast detection method. That is, the ridge line detection unit 712 selects an image in which the line estimated to be the ridge line part 35 is captured with the highest contrast among the images obtained by the predetermined number of times of imaging. Then, the ridge line detection unit 712 treats the focus position where the image with the highest contrast is captured as the in-focus position, and detects the ridge line part height Z1 based on the focus distance.
  • step S14 it is confirmed whether or not the ridge line height Z1 has been detected.
  • the ridge line part height Z1 cannot be detected even if the detection operation of step S13 is performed, and other ridge line parts Of course, the height cannot be detected. Therefore, when the ridge line portion height Z1 cannot be detected (NO in step S14), the ridge line detection process in the dish 10 is stopped, and the process skips to step S20.
  • the camera unit 5 is moved directly below the holding concave part 3B of the second corner part to detect the ridge line part height Z2 of the second corner part.
  • the imaging operation is executed (step S15). Specifically, the camera movement control unit 711 controls the camera axis driving unit 55 to move the camera unit 5 directly below the holding recess 3B at the second corner. Then, the ridge line detection unit 712 causes the camera unit 5 to perform an imaging operation of the region including the holding recess 3B in the same manner as in step S13. That is, the image of the dish 10 is captured while the focus position is sequentially shifted upward at a predetermined pitch from a predetermined imaging start point.
  • the imaging start point is set at a position below a predetermined distance with the ridge line height Z1 as a reference position. That is, since the ridge line part height Z1 is known, the range in which the ridge line part height Z2 exists can be estimated to some extent. Thereby, the frequency
  • the camera unit 5 is moved directly below the holding recess 3C at the third corner, and an imaging operation for detecting the ridge line height Z3 of the third corner is executed (step S16). Subsequently, the camera unit 5 is moved directly below the holding recess 3D at the fourth corner, and an imaging operation for detecting the ridge line height Z4 of the fourth corner is executed (step S17). In addition, the camera unit 5 is moved directly below the holding recess 3E in the vicinity of the central portion, and an imaging operation for detecting the ridge line portion height Z5 in the central portion is executed (step S18).
  • the ridge line detection unit 712 stores the data of the ridge line part heights Z1 to Z5 detected by the above processing in the storage unit 76 (step S19). Accordingly, the height position of the ridge line portion 35 at an arbitrary position on the upper surface 21 can be calculated based on the data of the ridge line portion heights Z1 to Z5. Therefore, it is possible to save the trouble of acquiring the ridge line height data for all the holding recesses 3.
  • the camera movement control unit 711 controls the camera axis driving unit 55 to move the camera unit 5 directly below the Nth dish 10 (step S32).
  • the camera movement control unit 711 controls the camera axis driving unit 55 to slightly move the camera unit 5 directly below the Mth cell imaging point (step S34).
  • the ridge line height calculation unit 713 uses the coordinates of the cell imaging point M and the data of the ridge line part heights Z1 to Z5 stored in the storage unit 76 to hold at the position of the cell imaging point M.
  • the cell detection unit 714 uses the calculated height position Zm as a reference position, and performs focus adjustment using a position below the reference position by a predetermined distance as an imaging start point. Then, at the imaging start point, the camera unit 5 causes the cell C held in the holding recess 3 located at the cell imaging point M to be imaged. Subsequently, the cell detection unit 714 causes the camera unit 5 to capture the image of the dish 10 a plurality of times while shifting the focus position upward in units of several tens of microns. Of the images acquired by these imaging operations, for example, an image in which a line estimated to be the outline of the cell C is captured with the highest contrast is selected as the image of the cell C (step S36).
  • the ridge line portion 35 and the opening taper portion 31T having a relatively small inclination with respect to the vertical direction are formed in the first portion P1 of the holding recess 3 included in the dish 10. Therefore, when the cell suspension LC is sown in the third step # 3, the cells C are reliably guided into the holding recess 3 without staying on the upper surface 21 of the dish 10. Moreover, since the 2nd part P2 is comprised with the cylindrical wall surface 33 which consists of a cylindrical part extended in a perpendicular direction, the cell C can settle as it is with its own weight, without receiving resistance.
  • the bottom portion 32 of the third portion P3 where the cell C contacts the ground has a relatively large inclination with respect to the vertical direction, and thus becomes a ground contact surface closer to the horizontal. For this reason, the distance from the grounding surface of the cell C to the lower surface 22 of the dish 10 can be shortened, and the influence of the presence of the dish 10 is reduced when focusing on the cell C in the imaging operation of the fourth step # 4. This also contributes to an improvement in image resolution. Further, the cells C can be collected in the hole core of the holding recess 3 by the inclination of the tapered portion of the third portion P3.
  • the second portion P2 of the holding recess 3 is a cylindrical wall surface 33 having an inner wall surface substantially parallel to the vertical direction, imaging from the lower surface 22 side in the fourth step # 4. This makes it difficult for the inner wall surface to be reflected in the image obtained by the above-described method, and the recognition of the cell C on the image is improved.
  • the hole 34 is provided in the fourth portion P4 of the holding recess 3, it is possible to escape foreign matter from the hole 34 as an imaging obstacle, and the recognition of the cells C is enhanced. .
  • the hole 34 is a hole extending in the vertical direction, the hole 34 can be hardly reflected in an image acquired by imaging.
  • the height position of the ridge line portion 35 of the dish 10 is recognized in the second step # 2, and the imaging operation of the dish 10 is performed using the height position of the ridge line portion 35 as a reference position in the fourth step # 4.
  • the ridge 35 is located on the upper surface 21 that is relatively far from the third portion P3 to which the cell C contacts the ground, and is located between the adjacent holding recesses 3 so that it is not hidden by the cell C. Easy to recognize on the image. Therefore, the reference position can be obtained easily and accurately.
  • a cell imaging method includes a main body formed of a light-transmitting member and including a flat plate having an upper surface and a lower surface, and a plurality of cells that extend vertically from the upper surface to the lower surface of the main body and hold cells.
  • a holding recess, wherein the holding recess has a first portion formed of a tapered portion having a first inclination with respect to the vertical direction and an inner portion substantially parallel to the vertical direction.
  • Towards the bottom A first step in which the dish configured by sequentially connecting the first part to the fourth part is horizontally arranged at a predetermined position; and an image of the dish by arranging an imaging device on the lower surface side.
  • a second step of recognizing a height position of a predetermined shape feature of the dish based on focus information at the time of the imaging operation, and a state in which the dish is immersed in a liquid Then, the cell suspension containing the cells is spread from the upper surface side, and the cells are guided through the first portion into the holding recess by gravity, and the cells are allowed to settle along the second portion.
  • the third step of grounding to three portions and the imaging device arranged on the lower surface side the cell is held in the holding recess with the height position of the shape feature as a reference position
  • a fourth step for executing the imaging operation of the dish of the image based on the image data of the dish obtained by the imaging operation and a fifth step for specifying the shape of the cells, a.
  • the cell suspension is spread in the third step. Then, the cells are surely guided into the holding recess along the tapered portion. Further, since the second portion is a cylindrical portion extending in the vertical direction, the cells can settle as they are without being subjected to resistance. Furthermore, since the third portion where the cell contacts the ground has a relatively large second inclination with respect to the vertical direction, the third portion becomes a contact surface closer to the horizontal.
  • the distance from the ground contact surface of the cell to the lower surface of the dish can be shortened, and the influence of the presence of the dish can be reduced when focusing on the cell in the imaging operation of the fourth step. Contributes to improved resolution. Furthermore, it becomes possible to guide the cell to the intended position along the second inclination. For example, when the tapered portion of the third portion is a tapered portion that descends toward the hole core (hole center) of the holding recess, cells can be collected at the hole core position.
  • the second portion of the holding recess is a cylindrical portion having an inner wall surface substantially parallel to the vertical direction, the image acquired by imaging from the lower surface side in the fourth step The inner wall surface becomes difficult to be reflected, and the recognizability of cells on the image is improved.
  • the fourth portion of the holding recess is provided with a discharge hole, foreign matter that becomes an obstacle to imaging can be released from the discharge port, and the recognition of cells is enhanced. If the discharge hole is a hole extending in the vertical direction, the discharge hole can be made difficult to be reflected in an image acquired by imaging.
  • the height position of the predetermined shape feature of the dish is recognized in the second step, and the dish imaging operation is performed with the height position of the shape feature as the reference position in the fourth step. Is done. Therefore, by grasping the positional relationship between the third portion where the cell contacts the ground in the holding recess and the shape feature, the focusing on the cell can be performed quickly, and the imaging time can be shortened. Can be planned.
  • the dish has a ridge line portion formed on the upper surface when the top of the first portion of one holding recess is adjacent to the top of the first portion of another holding recess.
  • the shape feature recognized in the second step is preferably the ridge line formed on the upper surface of the dish.
  • the imaging method since there is a ridge portion in the first portion and a tapered portion having a relatively small first inclination with respect to the vertical direction, when the cell suspension is sown in the third step, The cells are surely guided into the holding recess without staying on the upper surface of the dish.
  • the ridge line portion is on the upper surface relatively far from the third portion where the cell contacts the ground, and is located between adjacent holding recesses so that it is not hidden by the cell, and is easily recognized on the image as a shape feature. . Therefore, the reference position can be obtained easily and accurately.
  • the main body of the dish is a rectangular flat plate, and in the second step, at least a height of the ridge line portion near the four corners of the main body and the ridge line portion near the intersection of the diagonal lines of the four corners.
  • the height position is recognized, and in the fourth step, the height position of the ridge line portion of any holding recess to be imaged is calculated based on the height position of the ridge line portion near the four corners and the intersection. It is preferable that the calculated height position is used as the reference position.
  • the dish Even if the dish is placed at a predetermined position in the first step, it is difficult to create a complete horizontal state, and dish warping may occur. For this reason, it is difficult to obtain the reference position of all the holding recesses only by obtaining the height position of one ridge line portion, and ideally it is desirable to obtain the height positions of the ridge line portions corresponding to all the holding recesses. .
  • the height positions of the arbitrary ridge line portions of the dish can be set near the four corners and the intersections without recognizing the height positions of the ridge line portions of all the holding recesses included in the dish. It is possible to calculate based on the height position of the ridge line portion. Therefore, the time required for imaging can be shortened.
  • the dish having the thinnest shape in the vertical direction among the first to fourth parts constituting the holding recess may be used as the dish. desirable.
  • the fourth portion of the holding recess is located between the cell grounded on the third portion and the imaging device. For this reason, the presence of the fourth portion in addition to the third portion can cause light loss and image distortion during cell imaging. However, according to the imaging method described above, since the fourth portion is the thinnest, the cause of image degradation can be minimized.
  • the dish has a polygonal shape in plan view when the discharge hole of the fourth portion is viewed from the lower surface side.
  • the discharge hole in the fourth part is inevitably reflected in the image acquired by the imaging in the fourth step.
  • the discharge hole has a polygonal shape, it is possible to easily distinguish between cells having a generally spherical shape (cell aggregate) and the discharge hole on the image. Become.
  • the cell in the case of recognizing the shape of a cell from an image of a dish holding cells, the cell can be easily recognized on the image, and the imaging time of the dish can be shortened. .

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Abstract

La présente invention concerne une coupelle possédant une partie de retenue en creux pourvue : d'une première partie comprenant une partie effilée à proximité d'une ouverture; d'une deuxième partie comprenant une partie tubulaire possédant une surface de paroi interne verticale; d'une troisième partie comprenant une partie effilée servant de surface de sol pour des cellules; et une quatrième partie comprenant une plaque inférieure possédant un trou de décharge. Le procédé d'imagerie comporte : une première étape dans laquelle la coupelle est placée horizontalement à une position prédéterminée; une deuxième étape dans laquelle la position en hauteur d'une partie caractéristique de forme prédéterminée de la coupelle est reconnue sur la base d'informations de mise au point au moment où la coupelle est imagée; une troisième étape dans laquelle des cellules sont pulvérisées à partir du côté de surface supérieure de la coupelle, précipitées le long de la première partie et de la deuxième partie, et mises à la terre par rapport à la troisième partie; une quatrième étape dans laquelle la coupelle est imagée dans un état dans lequel des cellules sont retenues dans la partie de retenue en creux; et une cinquième étape dans laquelle les formes des cellules sont identifiées sur la base des données d'image sur la coupelle obtenues par l'imagerie.
PCT/JP2017/032470 2016-10-13 2017-09-08 Procédé d'imagerie d'une cellule Ceased WO2018070153A1 (fr)

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JP2016201943A JP6735207B2 (ja) 2016-10-13 2016-10-13 細胞の撮像方法

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JP2010256077A (ja) * 2009-04-22 2010-11-11 Olympus Corp 細胞画像取得装置及び細胞画像取得方法
WO2014080932A1 (fr) * 2012-11-26 2014-05-30 国立大学法人大阪大学 Microscope optique et dispositif de mise au point automatique pour microscope optique
WO2015093344A1 (fr) * 2013-12-19 2015-06-25 株式会社Ｓｃｒｅｅｎホールディングス Appareil de capture d'image et procédé de capture d'image
WO2016020988A1 (fr) * 2014-08-05 2016-02-11 ヤマハ発動機株式会社 Dispositif de maintien d'objet
WO2017110005A1 (fr) * 2015-12-25 2017-06-29 ヤマハ発動機株式会社 Procédé de prélèvement d'objet cible

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Publication number Priority date Publication date Assignee Title
JP2009204451A (ja) * 2008-02-28 2009-09-10 Yokogawa Electric Corp ウェルプレートとそれを用いた蛍光イメージングシステム
JP2010256077A (ja) * 2009-04-22 2010-11-11 Olympus Corp 細胞画像取得装置及び細胞画像取得方法
WO2014080932A1 (fr) * 2012-11-26 2014-05-30 国立大学法人大阪大学 Microscope optique et dispositif de mise au point automatique pour microscope optique
WO2015093344A1 (fr) * 2013-12-19 2015-06-25 株式会社Ｓｃｒｅｅｎホールディングス Appareil de capture d'image et procédé de capture d'image
WO2016020988A1 (fr) * 2014-08-05 2016-02-11 ヤマハ発動機株式会社 Dispositif de maintien d'objet
WO2017110005A1 (fr) * 2015-12-25 2017-06-29 ヤマハ発動機株式会社 Procédé de prélèvement d'objet cible

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