JP6735207B2 - Cell imaging method - Google Patents

Description

The present invention relates to a method of holding a cell in a translucent dish and imaging the cell with an imaging device in order to recognize the shape of the cell.

For example, in medical or biological research applications, single cells, or cell aggregates formed by three-dimensionally aggregating cells (hereinafter, simply referred to as cells in the present specification) are used for observation, confirmation of drug efficacy, examination or For processing operations such as culturing, the microplate having wells arranged in a matrix may be housed in the wells. The cells contained in the wells are sorted on a dish having a recess for holding the cells. Prior to this selection, a cell suspension containing a large amount of cells is dispersed on the dish, and the cells are retained in the recesses. Then, an image of the dish holding the cells is captured, and the usable cells are separated from the unusable cells and contaminants by the image processing technique. Thereafter, usable cells are sucked from the recess by the suction tip, and the sucked cells are discharged into the well of the microplate (for example, refer to Patent Document 1).

WO2015/087371A1

When recognizing the shape of cells and the like from an image of a dish holding cells, it is desirable that the constituent parts of the dish and cells can be clearly distinguished and that the shape of cells can be easily recognized. Furthermore, the angle of view of the optical system for imaging micron-order cells naturally becomes small, and many imaging operations are required to image the entire area of the dish. For this reason, it is desirable to shorten the imaging time by enabling quick focusing on cells. However, at present, there has not been proposed a method for imaging cells that sufficiently satisfies these requirements.

An object of the present invention is to perform cell shape recognition from an image of a dish holding cells, to easily recognize the cells on the image, and to image a cell capable of shortening the imaging time of the dish. To provide a method.

A method for imaging a cell according to one aspect of the present invention includes a main body made of a light-transmissive member and formed of a flat plate having an upper surface and a lower surface, and a plurality of cells extending vertically from the upper surface to the lower surface of the main body to hold cells. a dish having a recess for holding, and the holding recess, the Chi lifting the first inclination selected from a range of 25 ° to 55 ° with respect to the vertical direction, gradually opening area downward The first portion has a smaller tapered surface and the tubular portion has an inner wall surface that is substantially parallel to the vertical direction. A second portion that restricts movement and a second inclination that is selected from the range of 55° to 80° with respect to the vertical direction and that is larger than the first inclination, and the opening area gradually decreases downward. smaller a tapered surface comprises a third portion that do and the ground plane of the cell, and a fourth portion that cells retaining the object consists of a bottom plate having a discharge hole of a size that can not pass through, and to the lower surface from the upper surface Toward, the first step of horizontally arranging the dish, which is configured by sequentially connecting the first portion to the fourth portion, at a predetermined position, and arranging the imaging device on the lower surface side. And a second step of recognizing the height position of a predetermined shape feature of the dish based on the focus information at the time of the image capturing operation, and performing the image capturing operation of the image of the dish. In the state of being immersed in, the cell suspension containing cells is sprinkled from the side of the upper surface, and the cells are guided by gravity into the holding recess through the first portion, and the cells are moved along the second portion. In the third step of allowing the cells to settle and contacting the third portion, and in the imaging device arranged on the lower surface side, the cells were held in the holding recess with the height position of the shape feature as a reference position. It includes a fourth step of performing an image pickup operation of the image of the dish in the state, and a fifth step of specifying the shape of the cell based on the image data of the dish obtained by the image pickup operation.

According to this imaging method, since the taper portion having the relatively small first inclination with respect to the vertical direction is present in the first portion of the holding concave portion provided in the dish, the cell suspension is sprinkled in the third step. Then, the cells are surely guided into the holding recess along the taper portion. Further, since the second portion is a cylindrical portion extending in the vertical direction, the cells can settle as they are without receiving resistance. Further, the third portion where the cells are in contact with the ground has a second inclination that is relatively large with respect to the vertical direction, so that the contact surface becomes closer to horizontal. Therefore, the distance from the ground surface of the cell to the lower surface of the dish can be shortened, the influence of the presence of the dish can be reduced when focusing on the cell in the imaging operation of the fourth step, and the image It also contributes to the improvement of resolution. Furthermore, it becomes possible to guide the cells to the desired position along the second slope. 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. In addition to this, since the second portion of the holding recess is a cylindrical portion having an inner wall surface that is substantially parallel to the vertical direction, the second portion of the holding concave portion is included in the image obtained by imaging from the lower surface side in the fourth step. The inner wall surface is less likely to be reflected, and the recognizability of cells on the image is improved. Further, since the fourth portion of the holding concave portion is provided with the discharge hole, it is possible to allow foreign substances, which are obstacles for imaging, to escape from the discharge port, and cell recognition is enhanced. If the discharge hole is a hole extending in the vertical direction, it is possible to make it difficult for the discharge hole to be reflected in the image acquired by imaging.

Further, according to the above-mentioned image pickup method, the height position of the predetermined shape feature of the dish is recognized in the second step, and the image pickup operation of the dish is performed with the height position of the shape feature being the reference position in the fourth step. Is done. Therefore, by grasping the positional relationship between the shape-characteristic portion and the third portion where the cell is grounded in the holding concave portion, it becomes possible to quickly focus on the cell and shorten the imaging time. Can be planned.

A method for imaging a cell according to another aspect of the present invention is to hold a cell, which is composed of a flat plate formed of a translucent member and has an upper surface and a lower surface, and extends in a vertical direction from the upper surface to the lower surface of the main body. A dish having a plurality of holding recesses, wherein the holding recess is substantially parallel to the vertical direction with a first portion formed of a tapered portion having a first inclination with respect to the vertical direction. The second portion is composed of a cylindrical portion having an inner wall surface, defines a space for accommodating cells, regulates the horizontal movement of the accommodated cells, and more than the first inclination with respect to the vertical direction. From the top surface, a third portion having a large second inclination and formed of a tapered portion that serves as a ground contact surface of cells, and a fourth portion that includes a bottom plate having a discharge hole of a size that does not allow cells to be retained to pass therethrough. A first step of horizontally arranging the dish, which is configured by sequentially connecting the first portion to the fourth portion toward the lower surface, at a predetermined position; And a second step of recognizing the height position of a predetermined shape feature of the dish based on the focus information at the time of the imaging operation. While being immersed in a liquid, a cell suspension containing cells is sprinkled from the side of the upper surface, and the cells are guided by gravity to the holding recess through the first portion, and the cells are placed in the second portion. The third step of causing the cells to settle along the third portion to contact the third portion, and the imaging device arranged on the lower surface side, the cells are stored in the holding recess with the height position of the shape feature as a reference position. A cell including a fourth step of performing an image pickup operation of the image of the dish in a held state, and a fifth step of specifying the shape of the cell based on the image data of the dish obtained by the image pickup operation In the imaging method described above, in the dish, a ridge portion is formed on the upper surface by the top of the first portion of the one holding recess being adjacent to the top of the first portion of the other holding recess. , wherein the shape feature recognized in the second step is, Ru said ridge portion der formed on the upper surface of the dish.

According to this imaging method, since the ridge line portion and the taper portion having a relatively small first inclination with respect to the vertical direction are present in the first portion, when the cell suspension is sprinkled in the third step, The cells are reliably guided to the holding recess without staying on the upper surface of the dish. Since the ridge portion is on the upper surface relatively far from the third portion where the cells are grounded, and is located between the adjacent holding recesses, it is not hidden by the cells and is easily recognized on the image as a shape feature. .. Therefore, the reference position can be obtained easily and accurately.

In the above-mentioned imaging method, the main body of the dish is a rectangular flat plate, and in the second step, the height of the ridge line portion at least near the four corners of the body and the ridge line portion near the intersection of the diagonal lines of the four corners. Position is recognized, and in the fourth step, the height position of the ridge line portion of any holding concave portion to be imaged is calculated based on the height position of the ridge line portion near the four corners and the intersection. It is desirable that the calculated height position is used as the reference position.

Even if the dish is placed at a predetermined position in the first step, it is difficult to make it perfectly horizontal, and the dish may warp. Therefore, it is difficult to obtain the reference positions of all the holding recesses by simply obtaining the height position of one ridge portion, and ideally, it is desirable to obtain the height positions of the ridge portions corresponding to all the holding recesses. .. However, according to the above-mentioned imaging method, without recognizing the height position of the ridge line portion with respect to all the holding recesses provided in the dish, the height position of any ridge line portion of the dish can be set in the vicinity of the four corners and the intersection point. It is possible to calculate based on the height position of the ridge portion. Therefore, the time required for imaging can be shortened.

In the above-mentioned imaging method, as the dish, a dish having the thinnest shape of the fourth portion in the thickness in the vertical direction among the first to fourth portions forming the holding recess may be used. 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 may cause a light amount loss or image distortion when imaging a cell. However, according to the above-mentioned imaging method, the fourth portion is the thinnest, so that the factor of image deterioration can be suppressed to the minimum.

In the above-mentioned imaging method, it is preferable that the discharge hole of the fourth portion has a polygonal shape in a plan view as seen from the lower surface side.

The discharge hole of the fourth portion is inevitably reflected in the image acquired by the imaging in the fourth step. However, according to the above-described imaging method, since the discharge hole has a polygonal shape, cells (cell aggregates) having a generally spherical shape can be easily distinguished from the discharge hole on an image. Become.

According to the present invention, when the shape of a cell is recognized from the image of the dish holding the cell, the cell can be easily recognized on the image, and the imaging time of the dish can be shortened.

FIG. 1A is a schematic diagram showing a part of the configuration of a cell migration 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 migration 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 included in the sorting container. FIG. 4 is a sectional view taken along line IV-IV in FIG. FIG. 5 is a top view showing a plurality of dishes and a shooting range. FIG. 6 is a diagram showing a procedure of the cell imaging method according to the present invention. FIG. 7 is a cross-sectional view for explaining the details of the dish. FIG. 8 is a diagram schematically showing an imaging mode of the ridge line portion of the dish. FIG. 9 is a plan view showing an imaging location in the imaging of the ridge portion. FIG. 10 is a cross-sectional view showing a mode in which cells are accommodated in the holding recess of the dish. FIG. 11 is a cross-sectional view showing a state where the dish is imaged in a state where cells are held in the holding recess. FIG. 12(A) is a diagram showing an image of the holding recesses and cells obtained by the imaging, and FIG. 12(B) is a diagram showing an image of cells after image processing. FIG. 13 is a block diagram showing the electrical configuration of the cell transfer device. FIG. 14 is a flowchart showing the overall flow of a cell imaging operation in the cell moving device. FIG. 15 is a detailed flowchart of the edge line detection processing. FIG. 16 is a detailed flowchart of the cell imaging process.

Hereinafter, an embodiment of a cell imaging method according to the present invention will be described in detail with reference to the drawings. In the present invention, cells to be imaged are cells of biological origin, particularly cell aggregates (spheroids). A cell aggregate derived from a living body is formed by aggregating several to several hundred thousand cells. Therefore, the sizes of cell aggregates vary. The cell aggregates formed by living cells are substantially spherical, but if some of the cells that make up the cell aggregates are altered or become dead cells, the shape of the cell aggregates becomes distorted, or The density may be non-uniform. In the following description, it is simply referred to as a cell C in the sense that it includes the above-mentioned cell aggregate.

The cell imaging method according to the present invention is preferably applied to, for example, a cell migration device. The cell transfer device picks up usable cells from a plurality of cells with various shapes carried on the dish on the selection stage in tests in the fields of biotechnology and medicine, and selects them from the microplate to the microplate. Moving. In the microplate, various kinds of processing such as observation, confirmation of drug efficacy, inspection, and culture are performed on the cells. Imaging of cells is performed, for example, to determine usable cell aggregates on a dish. Hereinafter, an example in which the cell imaging method according to the present invention is applied to such a cell migration device will be mainly described.

[Overall configuration of cell transfer device]
1A and 1B are diagrams schematically showing the overall configuration of the cell transfer device S. Here, a cell moving device S that moves the cells C between the three containers is illustrated. 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. There is. In general, the cell transfer device S sucks a cell suspension LC containing a large number of cells C from a dispensing container 100 and spreads the cell suspension LC on a dish 10 of a sorting container 1 to obtain cells according to the present invention. It is an apparatus for selecting the usable cells C by applying the imaging method of (3), sucking the usable cells C from the dish 10 and discharging the cells to the microplate 4. FIG. 1A shows the dispensing container 100 and the sorting container 1 of the cell transfer device S, and FIG. 1B shows the sorting container 1 and the microplate 4 respectively.

The dispensing container 100 is a container with an upper surface opening that stores a cell suspension LC containing a large number of cells C. The cell suspension LC contains the cells C to be sorted in the sorting container 1 and impurities that are inevitably mixed.

The sorting container 1 is a container from which the cells C are transferred, stores the medium L, and holds the dish 10 for cell sorting immersed in the medium L. The dish 10 is a plate that carries cells C, and has a plurality of holding recesses 3 on the upper surface that can individually accommodate the cells C.

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, serum and the like, as well as glycerol and cell banker (Juji Field Co., Ltd.) added before frozen storage Cell suspension, formalin, reagent for fluorescent staining, antibody, purified water, physiological saline and the like. For example, when BxPC-3 (human pancreatic adenocarcinoma cell) derived from a living body is used as the cell C, RPMI-1640 medium as the medium L was mixed with 10% fetal bovine serum FBS (Fetal Bovine Serum). If necessary, supplements such as antibiotics and sodium pyruvate may be used. Such 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 inputting the cells C and picking up the sorted cells C. The dish 10 is arranged below the upper opening 1H. As the sorting container 1 and the dish 10, those made of a translucent resin material or glass are used. This is because the cells C carried on the dish 10 can be observed by the camera unit 5 arranged below the sorting container 1.

The cell suspension LC is injected into the sorting container 1 from the dispensing tip 60. The dispensing tip 60 is a tubular 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. At the time of suction, 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. At the time of the discharge, the cell suspension LC (cell C) held in the dispensing tip 60 is discharged with the tip opening 60H of the dispensing tip 60 immersed in the medium L of the sorting container 1.

The microplate 4 is a container to which the cells C move, and has a plurality of wells 41 that receive the cells C. One well 41 accommodates the required 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 for making it possible to observe the cells C carried on the microplate 4 by the camera unit 5 arranged below the microplate 4.

The camera unit 5 includes a camera lens 51 and captures an image of the cells C carried on the dish 10 in the sorting container 1 or the cells C held in the wells 41 of 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 pickup device.

The camera unit 5 is arranged below these so that the camera lens 51 faces the respective lower surfaces of the sorting container 1 and the microplate 4. That is, the camera unit 5 captures an image of the cells C carried on the sorting container 1 or the microplate 4 from the lower surface side thereof. The camera unit 5 is horizontally movable along the guide rail 52 between the lower portion of the sorting container 1 and the lower portion of the microplate 4, as indicated by an arrow X2 in the figure.

The tip 6 is a tubular 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 carried in the holding recesses 3 (FIG. 3) of the dish 10 together with the medium L, and sucks them. 4 to the well 41. Although not shown in the drawing, the chip 6 aspirates a reagent solution or the like as necessary and discharges the reagent solution or the like 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, and a head body 62 and a head 63. Equipped with. 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 horizontally along the guide rail 61R from the dispensing container 100 to the arrangement position of the microplate 4 as shown by an arrow X1 in the figure. Is. Although not shown in FIGS. 1A and 1B, the head main body 62 can also move in a direction (front-back direction) orthogonal to the paper surface of FIG.

The head 63 is a hollow rod. The dispensing tip 60 and the tip 6 are attached to the lower end of the head 63. Although one head 63 is illustrated here, a plurality of heads may be used. The heads 62 for the dispensing tip 60 and the tip 6 may be provided in the head main body 62, respectively. Alternatively, the dispensing tip 60 and the tip 6 may be alternately attached to and detached from the single head 63. A piston mechanism is mounted in the hollow portion of the head 63, and suction and discharge forces 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 contains a power unit of the piston mechanism, a lifting mechanism for moving the head 63 in the vertical direction, and a power unit thereof.

The operation of the cell transfer device S will be described. First, the head unit 61 in which the dispensing tip 60 is 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. In this state, a suction force is generated in the head 63, and the cell suspension LC is sucked into the dispensing tip 60. After that, the head 63 is lifted 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 with the tip opening 60H of the dispensing tip 60 immersed in the medium L of the sorting container 1. That is, the cells C are scattered on the dish 10 (see FIG. 1A).

When the cells C are moved from the dish 10 to the microplate 4, the head unit 61 in which the chip 6 is mounted on the head 63 is moved above the sorting container 1. At the previous stage, the imaging of the cells C carried on the dish 10 according to the present embodiment and the selection of usable cells C based on the captured image are executed to obtain the coordinates of the cells C to be extracted. ing. Then, 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. In this state, a suction force is generated in the head 63, and usable cells C are suctioned from the dish 10 into the chip 6. Then, the head 63 is lifted and the head unit 61 is moved to the position above the micro plate 4. The head 63 is lowered again, and the cells C in the chip 6 are discharged into the well 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, and FIG. 4 is a sectional view taken along line IV-IV of FIG. The sorting container 1 includes a bottom plate 11, an outer peripheral wall 12, an inner peripheral wall 13 and a ceiling wall 14. The bottom plate 11 is a cylindrical plate 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 ceiling wall 14 form a lid member that covers the bottom plate 11. The outer peripheral wall 12 is a portion having a diameter larger than that of the side peripheral wall of the bottom plate 11, and the inner peripheral wall 13 is a square tubular 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 1H on the upper surface side of the sorting container 1.

The inner peripheral wall 13 is a wall that defines the upper opening 1H, and is inclined so that the opening area gradually decreases from the upper opening 1H downward. The top wall 14 is provided with a working hole 15 which is a vertical through hole. Through this working hole 15, operations such as injecting the medium L into the cavity of the sorting container 1, injecting chemicals, absorbing the liquid L, or draining the medium L are performed. Further, the ceiling wall 14 is provided with a pipe connection port 16 for adjusting the atmospheric pressure in the cavity of the sorting container 1.

The dish 10 includes a dish main body 2 formed of a translucent member, and a plurality of holding recesses 3 formed in the dish main body 2. The dish main body 2 is made of a flat plate-shaped 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 for holding the cells C to be moved. The dish 10 is held at the lower end of the inner peripheral wall 13 in a state where the lower surface 22 is 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 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 medium L.

Each of the holding concave portions 3 includes an opening portion 31, a bottom portion 32, a cylindrical wall surface 33, a hole portion 34 (discharge hole), and a ridge line portion 35. In the present embodiment, an example is shown in which the holding recesses 3 that are square in a top view are arranged in a matrix. 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 portion 32 is located inside the dish main body 2 and near the lower surface 22. The bottom portion 32 is an inclined surface that gently inclines downward toward the center (the center of the square). The cylindrical wall surface 33 is a wall surface that extends vertically downward from the opening portion 31 toward the bottom portion 32. The hole portion 34 is a through hole that vertically penetrates between the center of the bottom portion 32 and the lower surface 22. The shape of the hole 34 is square in a top view and is concentric with the opening 31. The ridge line portion 35 is a portion which is located on the upper surface 21 and serves as an opening edge of each holding concave portion 3 and which divides the holding concave portions 3 from each other. The shape of the holding recess 3 in a top view may be a round shape, a triangular shape, a pentagonal shape, a hexagonal shape, etc., and these may be arranged in the dish main body 2 in a honeycomb shape, a linear shape, or randomly. The mode of the holding recess 3 will be described in more detail later with reference to FIG.

The bottom 32 and the cylindrical wall surface 33 of each holding recess 3 define a storage space 3H that stores cells C therein. It is generally intended that one cell C is 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 portion 34 is provided to allow small cells or foreign substances other than a desired size to escape from the accommodation space 3H. Therefore, the size of the hole 34 is selected so that the cells C having a desired size cannot pass through but small cells and contaminants other than the desired size can pass through. As a result, the cells C to be sorted are trapped in the holding recesses 3, while the impurities and the like drop from the holes 34 to the bottom plate 11 of the sorting container 1.

FIG. 5 is a diagram showing an arrangement example of the actual dishes 10 in the sorting container 1. Here, the dish 10 in which four small dishes 10A, 10B, 10C, and 10D having a square shape are arranged to form one large square is illustrated. The holding recess 3 of the dish 10 for holding the micron-order cells C has a very small size, so that a thin plate is naturally used as the dish main body 2. In this case, if the plate size is increased, the flatness of the plate is difficult to be obtained, so that the small-sized small dishes 10A to 10D are often assembled to form the dish 10 of a required size. 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 for imaging the micron-order cells C naturally becomes small. Generally, a camera with an angle of view of about 2 mm is used. Therefore, it is not possible to cover the entire area of the dish 10 with one shot of the camera unit 5. Therefore, a method of sequentially capturing a part of the dish 10 with the camera unit 5 is adopted. In FIG. 5, the shooting range AV that covers about 1/4 of the small dish 10A is drawn, but in reality, by covering the entire area of one small dish 10A, the imaging is performed several tens to 100 times. Is required. Therefore, in the example of FIG. 5, four small dishes 10A to 10D are used, and thus four times as many imaging operations are required.

[Imaging method of cells on dish]
FIG. 6 is a diagram showing a procedure of the method of imaging the cell C according to the present embodiment. The imaging method of the cells C is carried out sequentially, the first step #1 of preparing the dish 10 and arranging the dish 10 at a predetermined position, recognizing the height position of the ridge line portion 35 (shape characteristic portion) of the dish 10. 2 step #2, 3rd step #3 of accommodating the cell C in the holding recess 3 of the dish 10, 4th step #4 of imaging the dish 10 holding the cell C with the camera unit 5, and It includes a fifth step #5 of identifying (evaluating) the shape of the cell C based on the obtained image. Each step will be described below. Note that the third step #3 may be executed before the second step #2.

<First step>
In the first step #1, a dish 10 having a holding recess 3 suitable for sorting and imaging cells C is prepared. FIG. 7 is a cross-sectional view for explaining the details of the dish 10 used in this embodiment. As described above, the dish 10 includes the dish main body 2 having the upper surface 21 and the lower surface 22, and the holding concave portion 3 having the opening 31 in the upper surface 21 and extending from the upper surface 21 toward the lower surface 22. In FIG. 7, a vertical line V orthogonal to the upper surface 21 and the lower surface 22 is drawn at the core position of the holding recess 3. The holding recess 3 extends in the vertical direction in which the vertical line V extends.

The translucent material forming the dish body 2 is not particularly limited, but it is preferable to employ, for example, a thermoplastic resin, a thermosetting resin, a photocurable resin, or the like. More specifically, as the translucent material, polyethylene resin, polyethylene naphthalate resin, polypropylene resin, polyimide resin, polyvinyl chloride resin, cycloolefin polymer (COP), cycloolefin copolymer (CPC), norbornene-containing resin, poly Ether sulfone resin, polyethylene naphthalate resin, cellophane, aromatic polyamide resin, (meth)acrylic resin such as poly(meth)acrylate, styrene resin such as polystyrene or styrene-acrylonitrile copolymer, polycarbonate resin, polyester resin, Examples thereof include phenoxy resin, butyral resin, polyvinyl alcohol, cellulose resin such as ethyl cellulose, cellulose acetate and cellulose acetate butyrate, epoxy resin, phenol resin, silicone resin and polylactic acid.

Among the above, it is desirable to use heat resistant COP. Since the COP is excellent in transparency, it is advantageous when the cells C carried on the dish 10 are imaged from the lower surface 22 side, and by having heat resistance, it can endure heat sterilization treatment. Further, COP has a property that proteins are unlikely to adhere to it, and thus cells C can be prevented from adhering 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. In FIG. 7, the holding recess 3 is divided into four characteristic portions, that is, the first portion P1, the second portion P2, the third portion P3, and the fourth portion P4, with the joint between these portions as a boundary. That is, the holding concave portion 3 is configured by sequentially arranging the first portion P1 to the fourth portion P4 from the upper surface 21 toward the lower surface 22.

The first portion P1 is a portion having a ridge line portion 35 flush with the upper surface 21 and an opening taper portion 31T (taper portion having a first inclination). The opening taper portion 31T is formed by a taper surface whose opening area gradually decreases downward. The inclination angle θ1 (first inclination) of the tapered surface with respect to the vertical line V is set to be relatively small. A preferable range of the tilt angle θ1 is about 25° to 55°.

In the present embodiment, the plurality of holding recesses 3 are arranged in a matrix, and the apexes of the opening tapered portions 31T of one holding recess 3 and the apexes of the opening tapered portions 31T of the other holding recesses 3 are adjacent to each other, A ridge line portion 35 is formed on the upper surface 21. That is, a sharp ridge is formed on the upper surface 21 by the adjacent opening tapered portions 31T, and the remaining portion of the upper surface 21 is the opening portion 31. Therefore, even when the cell suspension LC is sprinkled on the upper surface 21, the cells C do not stay on the upper surface 21 and are guided to the accommodation space 3H by the opening tapered portion 31T. The preferable apex angle θA of the above-mentioned sharp peak is about 50° to 110°.

The second portion P2 is an area 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 composed of a cylindrical portion having an inner wall surface (vertical wall surface) that is substantially parallel to the vertical line V, and defines most of the housing space 3H for housing the cells C. .. When the cells C are accommodated in the holding recess 3, the cylindrical wall surface 33 guides the cells C settled by gravity downward. At this time, since the cylindrical wall surface 33 is a vertical wall surface, the cells C can settle by their own weight without receiving resistance. Furthermore, the cylindrical wall surface 33 also serves to regulate the horizontal movement of the contained cells C. Further, as will be described later in detail, since 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 is reflected in the image. You can prevent it from getting crowded.

The third portion P3 is an area where the bottom portion 32 exists. The bottom portion 32 is formed of 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 becomes the ground contact surface of the cell C. The inclination angle θ2 (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 θ1. A preferable range of the inclination angle θ2 is about 55° to 80°. With such an inclination angle θ2, the cell C can be stably grounded to the bottom portion 32, and the contaminants 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. As shown in FIG. 3, in a plan view seen from the upper surface 21 side, the bottom portion 32 is a surface having four trapezoidal pieces and a truncated cone shape.

The fourth portion P4 is a portion including a bottom plate having a hole 34 having a size that does not allow the cells C to be held to pass through. The hole portion 34 is a hole extending in the vertical direction, which is arranged at the position of the hole core (corresponding to the vertical line V in FIG. 7) of the holding concave portion 3. As shown in FIG. 3, the hole portion 34 has a square shape when seen in a plan view from the lower surface 22 side. As a result, the cells C (cell aggregates) having a generally spherical shape and the holes 34 can be easily distinguished on the image. The hole 34 may be a polygon such as a triangle or a pentagon. The circular hole 34 may be used if it can be easily distinguished from the cell C.

The fourth portion P4 is the thinnest portion in the thickness in the direction of the vertical line V among the first to fourth portions P1 to P4 forming the holding recess 3. The fourth portion P4 is located between the cell C grounded on the bottom 32 of the third portion P3 and the camera unit 5. For this reason, the presence of the fourth portion P4 in addition to the third portion P3 may cause a loss of light amount or image distortion when the cell C is imaged. However, the fourth portion P4 is the thinnest, and the bottom portion 32 is also a tapered surface having a relatively large inclination angle θ2 with respect to the vertical line V to thin the third portion P3. It can be minimized.

The dish 10 having the characteristics of the first to fourth portions P1 to P4 described above is prepared, and is horizontally arranged at a predetermined position. In the present embodiment, the dish 10 faces the upper opening 1H of the sorting container 1 and is arranged so as to be immersed in the medium L.

<Second step>
In the second step #2, the height position of the dish 10 installed at the predetermined position in the first step #1 is calibrated. Specifically, a process of capturing an image of the dish 10 with the camera unit 5 and recognizing the height position of a predetermined shape feature of the dish 10 is performed. In the present embodiment, an example is shown in which the recognized shape feature is the 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 characteristic portion may be another portion of the dish 10, for example, the lower end edge of the hole portion 34.

FIG. 8 is a diagram schematically showing an imaging mode of the ridge line portion 35 in the second step #2. In FIG. 8, the sorting container 1 is schematically illustrated. 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 the state. The camera unit 5 is arranged on the lower surface 22 side and executes an image capturing operation of an 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 medium L is injected.

The camera lens 51 focuses on the ridge line portion 35. Since the sorting container 1, the dish 10, and the medium L have translucency, the ridge line portion 35 can be imaged from the lower surface 22 side. For this focusing, for example, a contrast detection method can be adopted. Specifically, the camera unit 5 causes the image of the dish 10 to be captured while shifting the focus position upward by a unit 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 image capturing end point is a predetermined position that can be determined to be above the ridgeline portion 35. That is, the imaging end point is a position where it can be confirmed that the contrast value gradually increases due to the upward shift of the focus position, and after the highest value (focus position with respect to the ridge line portion 35), the contrast value gradually decreases.

In the obtained images, the focus position at which 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 calculated based on the focus distance. The position is required. In this way, the height position of the ridge line portion 35 as the shape characteristic portion is recognized based on the focus information at the time of the image pickup operation. The shape feature may be other than the ridge line portion 35, but since the ridge line portion 35 is located on the upper surface 21 relatively far from the third portion P3 where the cell C is grounded, and is located between the adjacent holding concave portions 3, the cell C Since it is not hidden by and is a simple linear shape, it has an advantage that it can be easily recognized on the image as a geometrical feature.

Even if the dish 10 is placed at a predetermined position in the first step #1, it is difficult to make the dish 10 perfectly horizontal, and the dish 10 may warp. For this reason, it is difficult to obtain the reference positions of all the holding recesses 3 by simply obtaining the height positions of one ridge line portion 35, and ideally, the height positions of the ridge line parts 35 corresponding to all the holding recess portions 3 are determined. It is desirable to ask. 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 images are required to image the entire area of one dish, the ridge line portions 35 corresponding to all the holding concave portions 3 are imaged. Doing so would take too much time. Therefore, a method of imaging only the main part of the dish 10 and calculating the height position of the ridge part 35 at an arbitrary position on the upper surface 21 of the dish 10 based on the obtained height position of the ridge part 35 of each main part is used. It is desirable to collect.

FIG. 9 is a plan view showing an example of an imaged portion of the ridge portion 35. The dish body 2 is a rectangular flat plate. In this case, the holding recesses 3A, 3B, 3C and 3D located at the four corners of the dish body 2 (holding recesses in the vicinity thereof) and the holding recess 3E at the center of the dish body 2 are selected as the main parts. The height positions of the ridge portions 35 corresponding to these are recognized by the above-described contrast detection method, for example. The holding recess 3E is a holding recess that exists near the intersections of the diagonal lines of the four corners of the dish body 2. The dish body 2 may have a shape other than a quadrangle, such as a polygon, a circle, and an ellipse. Further, what is actually imaged is the holding concave portion 3 included in the range of the photographing range AV as described in FIG. 5, and the photographing range AV is set so as to include the selected holding concave portions 3A to 3E.

In this way, by recognizing the height positions of the ridgeline portions 35 of the upper surface 21 of the dish main body 2 which are separated from each other, the height position of the ridgeline portion 35 at an arbitrary position of the upper surface 21 can be calculated. Is possible. Therefore, in the subsequent fourth step #4, when obtaining the height position information of the ridge line portion 35 of the arbitrary holding concave portion 3 to be imaged of the cell C, the ridge line portions 35 of all the holding concave portions 3 included in the dish 10 are obtained. It is possible to calculate it based on the information of the height position of the ridge line portion 35 near the four corners and near the intersection without recognizing the height position of the. Therefore, the time required for imaging can be shortened. Of course, you may make it recognize the height position of the ridgeline part 35 about all the holding recessed parts 3. Further, the height position of each ridge line portion 35 of all the holding concave portions 3 included in the range of the photographing range AV when photographing the holding concave portions 3A to 3E may be obtained. Then, these average values may be calculated, and the average values may be treated as the height position information of each of the four corners and the center of the dish main body 2.

<Third step>
The third step #3 is a step of accommodating the cells C in the holding concave portion 3 of the dish 10. As described based on FIG. 1A, the cell suspension LC containing the cells C is spread from the upper surface 21 side in a state where the dish 10 is immersed in the medium L (liquid) in the selection container 1. The cell C is guided by gravity into the holding recess 3 through the first portion P1 (opening 31). Furthermore, the cell C is settled along the second portion P2 (cylindrical wall surface 33) and is grounded to the third portion P3 (bottom portion 32).

FIG. 10 is a cross-sectional view showing an aspect in which the cells C are accommodated in the holding recess 3 of the dish 10. The cell suspension LC contains cells C of various sizes and shapes (simple circles are drawn in FIG. 10) and impurities Cx that are inevitably mixed. These sink in the medium L due to gravity and reach the upper surface 21 of the dish body 2. The cells C settled on the opening 31 of the holding recess 3 enter the accommodation space 3H as they are. On the other hand, the cells C settled on the walls between the holding recesses 3 collide with the ridges 35. However, in the first portion P1, the ridgeline portion 35 is formed by a sharp ridge, and the opening taper portion 31T following the ridgeline portion 35 has a relatively steep inclination. Therefore, the cells C are guided by the opening tapered portion 31T and smoothly enter the second portion P2.

In 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 this sedimentation, the cells C do not receive resistance from the substantially cylindrical wall surface 33. As the sedimentation proceeds, the cells C eventually reach the third portion P3 and come into contact with the tapered surface of the bottom portion 32. A cell C (a cell C indicated by a dotted line in FIG. 10) that is in contact with a position deviated from the hole core (the position of the hole 34) of the holding recess 3 is guided by the tapered surface of the bottom 32 and is 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 does not settle further. On the other hand, the foreign matter Cx also contacts the bottom portion 32 and is guided toward the hole portion 34. Since the size of the contaminant Cx is smaller than the opening size of the hole 34, the contaminant Cx passes through the hole 34 (fourth portion P4). Thereafter, the foreign matters Cx are received by the bottom plate 11 (FIG. 2) of the sorting container 1. Therefore, only the cells C are held in the holding recess 3. In addition, when a plurality of cells C enter one holding recessed portion 3, a jet flow directed upward through the hole 34 may be generated to disperse the cells C.

<Fourth step>
In the fourth step #4, the camera unit 5 performs the image capturing operation of the image of the dish 10 in which the cells C are held in the holding recesses 3. FIG. 11 is a cross-sectional view showing a state in which the dish 10 is imaged. The camera unit 5 includes an image pickup element 53 that photoelectrically converts an optical image, a camera lens 51 as an image pickup optical system, and a diaphragm 54. While the dish 10 is illuminated by an illumination system (not shown), the camera unit 5 captures an image of the dish 10 holding the cells C1 and C2 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 size. Therefore, the optical images M1 and M2 of the cells C1 and C2 are imaged on the image sensor 53 as an object having a corresponding horizontal width. Therefore, the contours of the cells C1 and C2 can be captured as an image. On the other hand, since the cylindrical wall surface 33 of the holding recess 3 is a vertical wall, it has almost no width in the horizontal direction even if it is displaced from the optical axis. Therefore, the optical image M3 of the cylindrical wall surface 33 is hardly reflected in the captured image (to the extent of being reflected as a narrow line). Further, since the third portion P3 and the fourth portion P4 of the holding concave portion 3 are thinned as described above, loss of light amount and image distortion are small. These contribute to the clarification of the images of the captured cells C1 and C2.

In the above imaging operation, the information on the height position of the ridge portion 35 obtained in the second step #2 is used. Specifically, the height position of the ridge 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 in consideration of the depth of the holding recess 3 and the like according to the distance from the ridge portion 35 to a position corresponding to the vicinity of the lower surface of the cell C carried in the holding recess 3. For example, in the vertical direction, the total length of the first portion P1 and the second portion P2 (the distance from the ridge portion 35 to the lower end of the cylindrical wall surface 33) is 293 μm, and the first portion P1 to the third portion P3 If the combined length is 360 μm and the combined length of the first portion P1 to the fourth portion P4 (distance between the upper surface 21 to the lower surface 22) is 390 μm, the predetermined distance may be 360 μm, for example. ..

After the image of the dish 10 is captured by the camera unit 5 at the image capturing start point, subsequently, the camera unit 5 is caused to capture the image of the dish 10 a plurality of times while shifting the focus position upward by several tens of microns. The pitch for shifting upward is, for example, 20 μm to 40 μm. The image capturing end point is an appropriate position below the ridgeline portion 35. Of the images acquired by these imaging, for example, the image in which the line estimated to be the contour of the cell C appears with the highest contrast is selected, and the image data relating to the image is selected in the fifth step #5 of the next stage. It can be used for image processing. Contrary to the above, the imaging start point may be set to a position above the dish 10 and the focus position may be gradually shifted downward.

<Fifth step>
In the fifth step #5, the shape and color tone of the cell C are specified based on the image data of the dish 10 obtained by the image capturing operation. For example, the image data obtained in the fourth step #4 is subjected to image processing, and the process of recognizing the presence of the cell C on the image, the process of recognizing the shape of the recognized cell C, and the like are executed. Further, based on the specified shape and shade, it is evaluated whether or not the cell C is a healthy cell that can be used for experiments and inspections.

The shape of the dish 10 (holding recess 3) is inevitably reflected in the image obtained in the fourth step #4. Therefore, in the above-mentioned image processing, it is desirable to filter the shape portion of the dish 10 from the image. FIG. 12A is an example of an image of the holding recess 3 and the cells C obtained by the imaging operation of the fourth step #4. In the image, the line corresponding to the ridge line portion 35, the contour lines of the cylindrical wall surface 33 and the hole portion 34, and the quadrangular truncated pyramid line of the bottom portion 32 are reflected. All of these lines are straight lines, and can be easily detected by general-purpose straight line edge detection processing or the like. In particular, the hole 34 is reflected in the image in such a manner that it completely overlaps the cell C, but the hole 34 of the present embodiment is square, so that it is easy to detect on the image. FIG. 12B is a diagram showing an image of the cell C after the image processing for erasing the detected straight line is performed. A clear image of the cells C can be obtained by the image processing.

[Electrical configuration of cell transfer device]
FIG. 23 is a block diagram showing the 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 elevation of the head 63, the suction and discharge operation of the cells C by the head 63, and the movement and imaging operation of the camera unit 5. A section 7 is provided. Further, the cell moving device S includes a camera shaft drive unit 55 as a mechanism for horizontally moving the camera unit 5, a head unit shaft drive unit 64 as a mechanism for horizontally moving the head unit 61, a mechanism for moving the head 63 up and down, and suction and discharge operations. A head drive 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. In a preferred aspect, a ball screw is laid along the guide rail 52, the camera unit 5 is attached to a nut member screwed to the ball screw, and the drive motor rotates the ball screw forward or backward. This is a mode in which 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 mode is a mode in which a ball screw and a nut member are provided, like the camera shaft drive unit 55, and the drive motor rotates the ball screw forward or backward. When the head main body 62 is moved in two XY directions, the movement is attached to the first ball screw (X direction) along the guide rail 61R and the first nut member screwed to the first ball screw. The second ball screw (Y direction) mounted on the plate is used. In this case, the head body 62 is attached to the second nut member screwed to the second ball screw.

The head drive unit 65 corresponds to a power unit for a lifting mechanism for moving 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. .. As described above, the elevating 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 housed. The power unit of the piston mechanism raises and lowers the piston member arranged in the head 63, and thereby the suction force and the discharge force are applied to the tip opening 6H of the tip 6 mounted on the head 63 or the tip opening 60H of the dispensing tip 60. Generate.

The display unit 66 includes a liquid crystal display or the like, and displays an image captured by the camera unit 5, an image processed by the control unit 7, and the like.

The control unit 7 includes a microcomputer and 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 when a predetermined program is executed. Works like. The image pickup control unit 71 controls the movement operation and the image pickup operation of the camera unit 5, particularly the image pickup operation of the dish 10 described in the above-mentioned second step #2 and fourth step #4, and functionally, the camera. 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 camera axis drive unit 55 to control the operation of moving the camera unit 5 along the guide rail 52. In addition, the camera movement control unit 711 slightly moves the camera unit 5 when capturing an image of 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 drive 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 portion 35 described in the second step #2. The ridge line height calculation unit 713 calculates the ridge line portions of the arbitrary holding recesses 3 based on the height positions of the ridge line portions 35 of some holding recesses 3 (for example, the four corners and the center of the dish 10) detected by the ridge line detecting unit 712. 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 provided in the microcomputer, an external storage, and the like, and temporarily stores the 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, based on the image of the dish 10 after the cells C are dispensed, a process of recognizing the presence of the cells C on the dish 10 on the image, as described in the fifth step #5, The process of recognizing the distribution of the cells C, the process of recognizing the recognized shape of the cells C, and the like are executed using an image processing technique. Further, the image processing unit 73 executes a process of filtering a line (ridge line portion 35) corresponding to the holding concave portion 3 from the acquired image.

The axis controller 74 controls the operation of the head unit axis driver 64. That is, the axis controller 74 controls the head unit axis driver 64 to move the head unit 61 to a predetermined horizontal target position. The movement of the head 63 (chip 6 or dispensing tip 60) between the dispensing container 100 and the sorting container 1, the positioning of the holding recess 3 of the dish 10 to be sucked in the vertical sky, and the micro to be discharged. Positioning of the well 41 of the plate 4 in the vertical sky is realized by the control of the head unit axis drive section 64 by the axis control section 74.

The head control unit 75 controls the head drive unit 65. The head control unit 75 controls the power unit for the elevating mechanism of the head drive unit 65 to raise and lower the head 63 to be controlled toward a predetermined target position. Further, the head control unit 75 controls the power unit of the piston mechanism for the head 63 to be controlled, so that the tip opening of the tip 6 or the dispensing tip 60 mounted on 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 setting values and data in the cell transfer device S. In addition, the storage unit 76 also stores the imaging sequence of the dish 10 by the camera unit 5, the height position data of the ridge line portion 35 detected by the ridge line detection unit 712, and the like.

[Flow of imaging operation]
Subsequently, a flow of an image pickup operation of the cell C by the cell transfer device S of the present embodiment will be described. FIG. 14 is a flowchart showing the overall flow of the imaging operation, FIG. 15 is a detailed flowchart of ridge line detection processing, and FIG. 16 is a detailed flowchart of cell imaging processing. Here, as illustrated in FIG. 5, an imaging operation example of a case in which four dishes 10 (dish numbers N=1 to 4) are arranged in the sorting container 1 is shown. The flow of FIG. 14 is started after the preparation of the dish 10 and the placement in the sorting container 1 in the first step #1 shown in FIG. 6 are completed.

Referring to FIG. 14, when the imaging operation is started, control unit 7 reads the arrangement data of dish 10 (step S1). The arrangement data is XY coordinate data that specifies the positions of the four dishes 10 in the sorting container 1 arranged on a predetermined work stage. Although an example in which four dishes 10 are used is shown here, five or more dishes 10 may be used, or three or less dishes 10 may be used.

Next, a process 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 processing corresponds to the recognition of the ridge line portion 35 described in the second step #2 of FIG. In the flowchart of FIG. 15, as shown in FIG. 9, a process of recognizing the height positions of the five ridge line portions 35 on the upper surface 21 of the dish body 2 which are separated from each other is illustrated.

After that, the axis control unit 74 and the head control unit 75 operate the head unit 61 via the head unit axis drive unit 64 and the head drive unit 65, and select from the dispensing tip 60 as illustrated in FIG. 1A. The cell suspension LC is discharged into the container 1. By this operation, the cells C are scattered on the dish 10 (step S3). A waiting time is set for a while until the cells C are 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.

Thereafter, the process of imaging the cells 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. In the flowchart of FIG. 16, as shown in FIG. 5, processing is performed for each of the four dishes 10A to 10D in the case where imaging is required a plurality of times to image the entire dish.

<Ridge detection processing>
Details of the ridge line detection processing will be described with reference to FIG. 15. First, the control unit 7 sets the dish number N=1 and designates the first dish 10 to be the target of ridge line detection processing (step S11). In the example of FIG. 5, for example, the small dish 10A is designated as the processing target. In response to this, the camera movement control unit 711 controls the camera axis drive unit 55 to move the camera unit 5 to immediately below the Nth dish 10 (step S12).

More specifically, the camera unit 5 is moved so that the optical axis of the camera is aligned with the holding concave portion 3 that is set to take an image first among the holding concave portions 3 where the ridge line portion 35 is imaged. .. For example, it is immediately below the holding recess 3A at the upper left corner shown in FIG. It should be noted that although it may be specified that the central holding concave portion 3E is imaged first, since the central portion is likely to bend, the height position tends to be different from the other four corner portions. It is preferable to image any one of the corners first and use this as a reference when imaging the remaining four places. In the following description of the flow, the ridge line portions 3 of the holding recesses 3A, 3B, 3C, and 3D at 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 center ridge line portion 3.

When the camera unit 5 is moved to directly below the holding concave portion 3A of the first corner portion, the ridge line detection unit 712 causes the camera unit 5 to perform an image capturing operation for detecting the ridge line portion height Z1 of the first corner portion. (Step S13). Specifically, the ridge line detection unit 712 sets a predetermined position, which is clearly lower than the arrangement position of the dish 10 (ridge line portion 35), 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 sufficient number of times that the focus position can be estimated to reach above the ridge line portion 35 by the upward shift of the predetermined pitch.

The ridge line detection unit 712 detects the ridge line portion 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 portion 35 has the highest contrast among the images obtained by the above-described 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 focus position, and detects the ridge line portion height Z1 based on the focus distance.

Then, it is confirmed whether or not the ridge line height Z1 can be detected (step S14). For example, if the dish 10 having the dish number N=1 is not installed at a predetermined position due to the forgetting of arrangement or the like, the ridge line portion height Z1 cannot be detected even if the detection operation of step S13 is performed, and another ridge line portion is not detected. 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 is skipped to step S20.

When the ridge line height Z1 can be detected (YES in step S14), the camera unit 5 is moved to immediately below the second corner holding recess 3B to detect the ridge line height Z2 of the second corner portion. The imaging operation of is executed (step S15). Specifically, the camera movement control unit 711 controls the camera shaft drive unit 55 to move the camera unit 5 to directly below the holding recess 3B at the second corner. Then, the ridge line detection unit 712 causes the camera unit 5 to perform the imaging operation of the region including the holding recess 3B, 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 the predetermined image capturing start point.

At this time, the information on the ridge line portion height Z1 obtained in step S13 is used. Specifically, the image pickup start point is set at a position below the ridge line portion height Z1 as a reference position by a predetermined distance. That is, since the ridge line height Z1 is known, the range in which the ridge line height Z2 exists can be estimated to some extent. As a result, the number of imaging shifts can be reduced and the work time can be shortened. For example, if the reference position is unknown in the detection of the ridge line height Z1, and it is necessary to shift the focus position to the information at a predetermined pitch about 30 times, the shift is detected in the detection of the ridge line height Z2. It is possible to reduce the number of times of making to about 10 times.

Similarly, the camera unit 5 is moved to directly below the holding concave portion 3C at the third corner portion, and the image capturing operation for detecting the ridge line height Z3 of the third corner portion is executed (step S16). Subsequently, the camera unit 5 is moved to immediately below the holding recess 3D at the fourth corner, and the image capturing operation for detecting the ridge line height Z4 at the fourth corner is executed (step S17). Then, the camera unit 5 is moved to immediately below the holding recess 3E near the central portion, and the image capturing operation for detecting the ridgeline portion height Z5 of the central portion is executed (step S18).

The ridge line detection unit 712 stores the data of the ridge line portion heights Z1 to Z5 detected by the above processing in the storage unit 76 (step S19). This makes it possible to calculate the height position of the ridgeline portion 35 at any position on the upper surface 21 based on the data of the ridgeline portion heights Z1 to Z5. Therefore, it is possible to save the trouble of acquiring the ridge line portion height data for all the holding concave portions 3.

Next, the control unit 7 confirms whether or not the dish number N is Max (N=4 in this embodiment) (step S20). If the dish number N is not Max (NO in step S20), the dish number N is incremented (step S21), the process returns to step S12, and the same process is performed for the next dish 10. On the other hand, when the dish number N is Max (YES in step S20), the control unit 7 ends the process.

<Cell imaging processing>
Details of the cell imaging process will be described with reference to FIG. 16. First, the control unit 7 sets the dish number N=1 and designates the first dish 10 to be the target of ridge line detection processing (step S31). In response to this, the camera movement control unit 711 controls the camera axis drive unit 55 to move the camera unit 5 to directly below the Nth dish 10 (step S32).

Subsequently, the control unit 7 sets the cell imaging point M=1 for the Nth dish 10 (step S33). The number of cell imaging points M for one dish 10 is determined by the angle of view of the camera unit 5. For example, if the imaging of the entire area of one dish 10 requires 80 times, Max of the cell imaging point M is M=80. In response to this, the camera movement control unit 711 controls the camera axis drive unit 55 to slightly move the camera unit 5 directly below the M-th cell imaging point (step S34).

After that, the ridge line height calculation unit 713 uses the coordinates of the cell imaging point M and the data of the ridge line portion heights Z1 to Z5 stored in the storage unit 76 to hold the cell imaging point M at the position. The height position Zm of the ridgeline portion 35 in the recess 3 is calculated (step S35). If the dish 10 has no warp and is arranged completely horizontally, Z1 to Z5=Zm. On the other hand, if there are variations in Z1 to Z5, Zm can be calculated from the XY coordinates and inclinations of the height positions of three points surrounding Zm and the XY coordinates of Zm.

After that, the cell detection unit 714 performs focus adjustment with the calculated height position Zm as a reference position and 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 is caused to image the cell C carried in the holding concave portion 3 existing at the position of the cell imaging point M. Subsequently, the cell detection unit 714 causes the camera unit 5 to capture the image of the dish 10 multiple times while shifting the focus position upward by a unit of several tens of microns. Of the images acquired by these imaging, for example, the image in which the line estimated to be the contour of the cell C appears with the highest contrast is selected as the image of the cell C (step S36).

Next, the control unit 7 confirms whether or not the cell imaging point M is Max (step S37). When the cell imaging point M is not Max (NO in step S37), the cell imaging point M is incremented (step S38), the process returns to step S34, and the same process is executed for the next cell imaging point M. On the other hand, when the cell imaging point M is Max (YES in step S37), the control unit 7 confirms whether the dish number N is Max (N=4 in this embodiment) (step S39). If the dish number N is not Max (NO in step S39), the dish number N is incremented (step S40), the process returns to step S32, and the similar cell imaging process is executed for the next dish 10. On the other hand, when the dish number N is Max (YES in step S39), the control unit 7 ends the process.

[Main effects]
According to the cell transfer device S according to the present embodiment described above, the ridge line portion 35 and the opening taper portion 31T having a relatively small inclination with respect to the vertical direction are provided in the first portion P1 of the holding concave portion 3 included in the dish 10. Since the cells C are present, when the cell suspension LC is sprinkled 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. In addition, since the second portion P2 is configured by the cylindrical wall surface 33 formed of a cylindrical portion extending in the vertical direction, the cells C can be settled as they are by their own weight without receiving resistance.

Further, the bottom portion 32 of the third portion P3 on which the cell C contacts the ground has a relatively large inclination with respect to the vertical direction, so that the ground surface is closer to the horizontal. Therefore, the distance from the ground 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 can be reduced when focusing on the cell C in the imaging operation of the fourth step #4. This also contributes to the improvement of image resolution. Furthermore, the inclination of the taper portion of the third portion P3 enables the cells C to be collected in the core of the holding recess 3. In addition to this, since the second portion P2 of the holding recess 3 is the cylindrical wall surface 33 having the inner wall surface substantially parallel to the vertical direction, the imaging from the lower surface 22 side in the fourth step #4. It becomes difficult for the inner wall surface to be reflected in the image acquired by, and the recognizability of the cells C on the image is improved. Further, since the fourth portion P4 of the holding concave portion 3 is provided with the hole portion 34, it is possible to allow foreign matters and the like which are obstacles for imaging to escape from the hole portion 34, and the recognizability of the cell C is enhanced. .. Since the hole portion 34 is a hole extending in the vertical direction, it is possible to make it difficult for the hole portion 34 to be reflected in the image acquired by imaging.

Further, in the second step #2, the height position of the ridge line portion 35 of the dish 10 is recognized, and in the fourth step #4, the imaging operation of the dish 10 is performed with the height position of the ridge line portion 35 as the reference position. Therefore, by grasping the positional relationship between the ridge line portion 35 and the third portion P3 where the cell C is grounded in the holding recessed portion 3, focusing on the cell C can be performed quickly, and the imaging time is shortened. Can be promoted. In particular, the ridge portion 35 is located on the upper surface 21 relatively far from the third portion P3 where the cells C are grounded, and is located between the adjacent holding recesses 3, so that it is not hidden by the cells C and has a shape characteristic. Easy to recognize on the image. Therefore, the reference position can be obtained easily and accurately.

C cell LC cell suspension S cell transfer device P1 first part P2 second part P3 third part P4 fourth part 1 sorting container 10, 10A to 10D dish 2 dish body 21 upper surface 22 lower surface 3 holding recess 31 opening 31T Opening taper portion 32 Bottom portion 33 Cylindrical wall surface 34 Hole portion (discharge hole)
35 Ridge part (shape feature part)
5 Camera unit (imaging device)

Claims (5)

A dish having a main body made of a flat plate having a top surface and a bottom surface formed of a translucent member, and a plurality of holding recesses extending vertically from the top surface of the main body toward the bottom surface to hold cells, the dish comprising: The holding recess is
The Chi lifting the first inclination selected from a range of 25 ° to 55 ° with respect to the vertical direction, a first portion comprising a tapered surface opening area gradually decreases downward,
A second part consisting of a cylindrical part having an inner wall surface substantially parallel to the vertical direction, defining a space for accommodating cells, and restricting the horizontal movement of the accommodated cells;
The cell is a tapered surface selected from the range of 55° to 80° with respect to the vertical direction and having a second inclination larger than the first inclination, and having an opening area that gradually decreases toward the lower side. and a third part that Do and the ground plane of,
A fourth portion including a bottom plate having a discharge hole of a size that does not allow cells to be retained to pass through,
A first step of horizontally arranging the dish configured by sequentially connecting the first portion to the fourth portion from the upper surface to the lower surface at a predetermined position;
An image pickup device is arranged on the lower surface side to perform an image pickup operation of the image of the dish, and the height position of a predetermined shape feature of the dish is recognized based on focus information at the time of the image pickup operation. The second step,
While the dish is immersed in a liquid, a cell suspension containing cells is sprinkled from the upper surface side, and the cells are guided by gravity into the holding recess through the first portion, and the cells are placed in the first section. A third step of settling along the second portion and contacting the third portion with the ground;
A fourth step of performing an image capturing operation of an image of the dish in a state in which the cells are held in the holding recess with the height position of the shape characteristic portion as a reference position by the image pickup device arranged on the lower surface side. When,
And a fifth step of identifying the shape of the cell based on the image data of the dish obtained by the image capturing operation. A dish having a main body made of a flat plate having a top surface and a bottom surface formed of a translucent member, and a plurality of holding recesses extending vertically from the top surface of the main body toward the bottom surface to hold cells, the dish comprising: The holding recess is
A first portion including a tapered portion having a first inclination with respect to the vertical direction;
A second part consisting of a cylindrical part having an inner wall surface substantially parallel to the vertical direction, defining a space for accommodating cells, and restricting the horizontal movement of the accommodated cells;
A third portion having a second inclination larger than the first inclination with respect to the vertical direction, the third portion including a tapered portion serving as a ground contact surface of a cell;
A fourth portion including a bottom plate having a discharge hole of a size that does not allow cells to be retained to pass through,
A first step of horizontally arranging the dish configured by sequentially connecting the first portion to the fourth portion from the upper surface to the lower surface at a predetermined position;
An image pickup device is arranged on the lower surface side to perform an image pickup operation of the image of the dish, and the height position of a predetermined shape feature of the dish is recognized based on focus information at the time of the image pickup operation. The second step,
While the dish is immersed in a liquid, a cell suspension containing cells is sprinkled from the upper surface side, and the cells are guided by gravity into the holding recess through the first portion, and the cells are placed in the first section. A third step of settling along the second portion and contacting the third portion with the ground;
A fourth step of performing an image capturing operation of an image of the dish in a state in which the cells are held in the holding recess with the height position of the shape characteristic portion as a reference position by the image pickup device arranged on the lower surface side. When,
A fifth step of identifying the shape of the cell based on the image data of the dish obtained by the image capturing operation ;
In the dish, the top of the first portion of the one holding recess is adjacent to the top of the first portion of the other holding recess, thereby forming a ridge portion on the upper surface,
The method for imaging cells, wherein the shape feature recognized in the second step is the ridge portion formed on the upper surface of the dish. The cell imaging method according to claim 2, wherein
The body of the dish is a rectangular flat plate,
In the second step, the height position of the ridge line portion at least near the four corners of the main body and the ridge line portion near the intersection of the diagonal lines of the four corners is recognized,
In the fourth step, the height position of the ridge line portion of any of the holding recesses to be imaged is calculated based on the height position of the ridge line portion near the four corners and the intersection, and the calculated height is calculated. A method for imaging a cell, wherein a position is used as the reference position. The method for imaging a cell according to any one of claims 1 to 3,
A method for imaging cells, wherein, as the dish, a dish in which the fourth portion has the thinnest shape in the vertical thickness among the first to fourth portions forming the holding recess is used. The method for imaging a cell according to any one of claims 1 to 4,
As the dish, a cell imaging method is used in which the discharge hole of the fourth portion has a polygonal shape in a plan view seen from the lower surface side.

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