WO2022230672A1 - 細胞生死判定方法、細胞生死判定装置及び細胞生死判定システム - Google Patents

細胞生死判定方法、細胞生死判定装置及び細胞生死判定システム Download PDF

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WO2022230672A1
WO2022230672A1 PCT/JP2022/017730 JP2022017730W WO2022230672A1 WO 2022230672 A1 WO2022230672 A1 WO 2022230672A1 JP 2022017730 W JP2022017730 W JP 2022017730W WO 2022230672 A1 WO2022230672 A1 WO 2022230672A1
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cell
image
life
death
cells
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English (en)
French (fr)
Japanese (ja)
Inventor
翔太 亀井
隆史 守本
知之 下田
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Fujifilm Corp
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Fujifilm Corp
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Priority to AU2022263866A priority Critical patent/AU2022263866B2/en
Priority to EP22795586.1A priority patent/EP4317970A4/en
Priority to CN202280030722.4A priority patent/CN117203525A/zh
Priority to JP2023517433A priority patent/JPWO2022230672A1/ja
Priority to KR1020237036082A priority patent/KR20230159871A/ko
Publication of WO2022230672A1 publication Critical patent/WO2022230672A1/ja
Priority to US18/491,962 priority patent/US12399103B2/en
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Definitions

  • the present disclosure relates to a method for judging cell life and death, a device for judging cell life and death, and a system for judging cell life and death.
  • the present disclosure has been made in view of such circumstances, and a problem to be solved by one embodiment of the present disclosure is to provide a method for determining cell life and death using the optical properties of cells.
  • a problem to be solved by another embodiment of the present disclosure is to provide a cell life/death determination device using the above cell life/death determination method.
  • a problem to be solved by another embodiment of the present disclosure is to provide a cell life-and-death determination system including the cell life-and-death determination device.
  • the present disclosure includes the following aspects. ⁇ 1> Acquiring an image of the cell captured with a plurality of focal planes including the focal plane of the cell from the direction opposite to the light-irradiated side of the cell irradiated with light; Obtaining an image piece containing the central part and the peripheral part of the cell from each image; connecting the image pieces in order of the imaging direction of the focal plane to create a connected image for analysis; a step of extracting a feature amount from the concatenated image for analysis; and a step of judging whether a cell is alive or dead based on the feature amount of the concatenated image for analysis and a predetermined range of the feature amount;
  • a method for determining cell life and death comprising: ⁇ 2> The method for determining cell life and death according to ⁇ 1>, wherein the determination of cell life and death includes determining whether or not the cell is a target cell.
  • ⁇ 3> The cell according to ⁇ 1> or ⁇ 2>, wherein a machine learning device determines whether the cell is alive or dead based on the feature amount of a known reference connected image of the cell and the determination result of whether or not the cell is a living cell. method of judging life and death.
  • the feature amount of the concatenated image for analysis is selected from the group consisting of a feature amount related to lens effect of cells, a feature amount related to average refractive index of cells, a feature amount related to cell diameter, and a feature amount related to specific gravity of cells.
  • the method for determining cell life and death according to any one of ⁇ 1> to ⁇ 3>, including one or more.
  • ⁇ 5> The method for judging cell life and death according to any one of ⁇ 1> to ⁇ 4>, wherein a plurality of cells in a cell suspension are judged for life and death.
  • ⁇ 6> The cell viability determination method according to ⁇ 5>, comprising the step of determining the viable cell concentration of the cells in the cell suspension based on the results of the viability determination.
  • ⁇ 7> The method for judging cell life and death according to ⁇ 5> or ⁇ 6>, comprising the step of determining the cell viability of the cells in the cell suspension based on the result of the life and death judgment.
  • An image acquisition unit that acquires an image of a cell captured with a plurality of focal planes including the focal plane of the cell from the direction opposite to the side of the cell irradiated with light, an image piece acquisition unit that acquires an image piece including the central part and the peripheral part of a cell from each image; an analytical connected image creating unit that creates an analytical connected image by connecting image pieces in order of the imaging direction of the focal plane; A feature quantity extraction unit for extracting a feature quantity from the concatenated image for analysis; A cell life-and-death determination device.
  • the life-and-death determination unit is configured by a machine learning device, and performs life-and-death determination based on the feature amount of the known reference connected image of the cell and the determination result as to whether or not the cell is a live cell.
  • cell viability determination device according to. ⁇ 10> ⁇ 8> or ⁇ 9, comprising a viable cell concentration determination unit that determines the viable cell concentration of cells in the cell suspension based on the results of viability determination of a plurality of cells in the cell suspension.
  • cell viability determination device a viable cell concentration determination unit that determines the viable cell concentration of cells in the cell suspension based on the results of viability determination of a plurality of cells in the cell suspension.
  • the device for judging cell life and death according to ⁇ 10> comprising a cell viability determination unit that determines the cell viability of cells in the cell suspension based on the result of the life and death determination.
  • a cell viability determination unit that determines the cell viability of cells in the cell suspension based on the result of the life and death determination.
  • ⁇ 12> Equipped with a light source for emitting light, an imaging device for imaging cells, means for changing a focal plane, and the device for determining cell life and death according to any one of ⁇ 8> to ⁇ 11>.
  • cell viability determination system ⁇ 13>
  • the method according to ⁇ 12>, wherein the means for changing the focal plane is a stage movement mechanism that changes the distance between the cells and the imaging device by moving the stage on which the holding container holding the cells is placed. Cell viability determination system.
  • the cell viability determination system according to ⁇ 12>, wherein the means for changing the focal plane is an imaging device moving mechanism that moves the imaging device to change the distance between the cell and the imaging device.
  • the imaging device includes a liquid lens as means for changing the focal plane.
  • a cell viability determination method using optical properties of cells is provided.
  • a cell viability determination device using the above cell viability determination method is provided.
  • a cell life/death determination system comprising the cell life/death determination device described above.
  • FIG. 1 is a schematic diagram showing an example of a cell viability determination system.
  • FIG. 2 is a schematic diagram showing an example of a focal plane.
  • FIG. 3 is a schematic diagram showing an example of an image obtained by imaging a living cell while changing the focal plane.
  • FIG. 4 is a schematic diagram showing an example of an image obtained by imaging dead cells while changing the focal plane.
  • FIG. 5 is a schematic diagram showing an example of a connected image obtained from living cells.
  • FIG. 6 is a schematic diagram showing an example of a connected image obtained from dead cells.
  • FIG. 7 is a schematic diagram showing an example of processing in the learning phase and operation phase of the machine learner.
  • FIG. 1 is a schematic diagram showing an example of a cell viability determination system.
  • FIG. 2 is a schematic diagram showing an example of a focal plane.
  • FIG. 3 is a schematic diagram showing an example of an image obtained by imaging a living cell while changing the focal plane.
  • FIG. 4 is a schematic diagram showing an example of an
  • FIG. 8 is a flowchart showing an example of a life-or-death judgment flow of the life-or-death judgment process.
  • FIG. 9 is a block diagram showing an example of a control unit that constitutes a cell life/death determination device.
  • FIG. 10 is a block diagram showing an example of processing of the device for determining cell life and death.
  • a numerical range indicated using “to” means a range including the numerical values before and after “to” as the minimum and maximum values, respectively.
  • upper or lower limits described in a certain numerical range may be replaced with upper or lower limits of other numerical ranges described step by step.
  • upper or lower limits described in a certain numerical range may be replaced with values shown in Examples.
  • a combination of two or more preferred aspects is a more preferred aspect.
  • the amount of each component means the total amount of the multiple types of substances unless otherwise specified when there are multiple types of substances corresponding to each component.
  • the term "process” includes not only an independent process, but also if the intended purpose of the process is achieved, even if it cannot be clearly distinguished from other processes. .
  • the cell viability determination method includes: A step of acquiring an image of a cell captured with a plurality of focal planes including the focal plane of the cell from the direction opposite to the side of the cell irradiated with light (hereinafter referred to as "image acquisition step ”), a step of acquiring an image piece including the central part and the peripheral part of the cell from each image (hereinafter sometimes referred to as “image piece acquisition step”); a step of connecting the image pieces in order of the imaging direction of the focal plane to create a concatenated image for analysis (hereinafter sometimes referred to as a “concatenated image for analysis step”); A step of extracting a feature amount from the concatenated image for analysis (hereinafter sometimes referred to as a “feature amount extraction step”), and based on the feature amount of the concatenated image for analysis and a predetermined range of the feature amount, a step of judging cell life and death (hereinafter sometimes referred to as
  • Japanese National Publication of International Patent Application No. 2013-517460 describes that cells are imaged in different focal planes, and then the life and death of cells is judged based on the brightness of the image.
  • the technique disclosed in Japanese Patent Application Publication No. 2013-517460 may not be sufficient to determine whether cells are alive or dead.
  • the optical properties of cells are used to create and analyze a connected image from images of cells captured in different focal planes, thereby determining cell life and death. Is going.
  • living cells Since living cells are covered with cell membranes, they tend to be spherical in a cell suspension (this does not mean that they are completely spherical), and live cells are translucent. Therefore, living cells have the property of a spherical lens, which is called "lens effect". Therefore, when a living cell is irradiated with light, a focal point (hereinafter sometimes referred to as a “lens effect focal point") is formed in the direction opposite to the side of the living cell irradiated with light due to the lens effect. be done. When the living cell is imaged from the direction opposite to the side of the living cell on which the light is irradiated, an image including the focal point of the lens effect can be obtained at the focal plane of the living cell. Therefore, for living cells, a connected image created from images acquired in a plurality of focal planes including an in-focus plane contains information peculiar to living cells.
  • Dead cells do not have a spherical shape because the cell membrane has been ruptured, and the liquid medium of the cell suspension has flowed into the dead cells. Therefore, dead cells do not exhibit a lens effect. Therefore, even if a dead cell is irradiated with light, the focal point of the lens effect is not formed. It is not possible.
  • the connected image created from living cells has features unique to living cells. Therefore, it is possible to determine whether a cell is alive or dead based on the feature amount of the analytical connected image of the cell whose life or death is unknown and the predetermined range of the feature amount.
  • the cell life/death determination method by creating and analyzing a connected image, it is possible to appropriately determine cell life/death with further consideration of information on cell life/death.
  • the method for determining cell life and death according to the present disclosure uses the optical properties of cells as described above, it is possible to more easily determine the life and death of cells without using staining reagents.
  • Predetermined feature amount range means a threshold for distinguishing whether a cell is a living cell or not, and if the feature amount is within the above range, the cell is determined to be a living cell. can do.
  • the method for determining the “predetermined range of feature values” is not particularly limited, and may be determined appropriately so as to enable determination of cell life and death. In another embodiment, determination of whether a cell is alive or dead may be performed based on the feature amount of a known reference connected image of the cell and the determination result of whether or not the cell is a living cell. Such an aspect is preferable when judging whether a cell is alive or dead using a machine learning device.
  • Target cell means a specific type of cell that is subject to life-or-death determination, and may be, for example, a cell that is the target of cell culture or the like.
  • the concatenated image for analysis and the concatenated image for reference are sometimes simply called “concatenated image”.
  • the "target cell” may be simply referred to as "cell”.
  • the cell type is not particularly limited, and it is possible to determine the life and death of various living cells.
  • mesenchymal stem cells collected and isolated from living tissue may be used, and examples thereof include human synovium-derived mesenchymal stem cells.
  • Synovium-derived mesenchymal stem cells are also referred to as synovium-derived stem cells.
  • the conditions for creating the concatenated image for analysis shall be the same as the conditions for creating the concatenated image for reference, at least to the extent that determination of cell life and death and identification of cells is possible. Also, when comparing different analytical connected images, the conditions for creating these analytical connected images are the same as long as the comparison is at least possible with respect to determination of cell life and death and identification of cells.
  • Image acquisition process In the image acquisition step, an image of the cell captured in a plurality of focal planes including the focal plane of the cell is acquired from a direction opposite to the light-irradiated side of the cell.
  • Images of cells can be obtained by imaging the cell suspension with an imaging device.
  • the means for changing the focal plane is not particularly limited, and examples include a method for changing the distance between the cell and the imaging device. Further, as a method of capturing an image while keeping the distance between the cell and the imaging device constant, there is a method of changing the focal point of the liquid lens using an imaging device equipped with a liquid lens. The focal plane moves as the focal length changes.
  • a holding container 40 containing cells (cell suspension) C is arranged between the light source 10 and the imaging device 20, and the stage 30 on which the holding container 40 is placed is moved.
  • Cells may be imaged while changing the focal plane by changing the focal plane.
  • a holding container containing cells may be arranged between the light source and the imaging device, and the cells may be imaged while changing the focal plane by moving the imaging device.
  • a holding container containing cells is placed between the light source and the imaging device to change the focus of the liquid lens provided in the imaging device without moving the imaging device and the stage. Cells may be imaged while thus changing the focal plane.
  • the cells may be imaged while moving the focal plane from the light source 10 toward the imaging device 20 , or the cells may be imaged while changing the focal plane from the imaging device 20 toward the light source 10 .
  • the imaging device consists of a combination of an imaging lens and an area sensor.
  • the imaging lens may be a telecentric lens, a microscope objective, or the like.
  • the type of lens, aperture angle, magnification, and the like of the imaging device are not particularly limited, but they can affect the convergence and divergence of light in the combined image, so they may be appropriately selected so as to properly capture the image.
  • a CCD Charge-Coupled Device
  • a CMOS Complementary Metal Oxide Semiconductor
  • the resolution of the area sensor is preferably set such that, for example, one pixel is about 1 ⁇ m to 3 ⁇ m in consideration of the lens magnification.
  • the light source is not particularly limited, for example, an LED (Light Emitting Diode) can be used.
  • the light source irradiate parallel light.
  • the positions and number of multiple focal planes for imaging cells are not particularly limited as long as they include the focal planes of the cells, but it is preferable to set them so that condensed light and divergence of light can be seen in the combined image.
  • cells may be imaged while moving the focal plane over a wide range from below to above the holding container, including from the bottom to the top of the holding container.
  • the distance between adjacent focal planes is preferably constant.
  • the position of the stage 30 may be specified by an encoder, and cells may be imaged while moving at equal intervals.
  • the movement interval of the focal plane is, for example, about 0.01 mm.
  • the moving distance of the focal plane in the cell suspension is shorter than the moving distance of the stage due to the influence of the refractive index of the cell suspension. is preferred.
  • an image may be acquired by focusing on the bottom surface (or the vicinity thereof) of the holding container holding the cells and used as the reference plane.
  • the concatenated image can be created under the same conditions, which is useful for analysis of the concatenated image.
  • the liquid thickness can be about 0.1 mm and the cell concentration can be about 1 ⁇ 10 6 cells/ml. Any holding container may be used.
  • the cell suspension contains other substances (e.g., other cells, red blood cells, oil droplets, etc.)
  • image processing using image processing software will confirm that the diameter is within a predetermined range and the shape
  • a material having a circularity within a predetermined range may be selected in advance.
  • the cell C A plurality of images may be acquired by imaging the cell C from the direction opposite to the light irradiation side.
  • the focal plane Pn is the n -th focal plane in the direction of the imaging device 20 from the focal plane P0 of the cell C.
  • the focal plane P 1 ⁇ m is the m-th focal plane in the direction of the light source 10 from the focal plane P 0 of the cell C.
  • FIG. At least one of m and n is an integer of 1 or more, and m+n is an integer of 1 or more.
  • FIG. 2 shows an example where m is 2 or more and n is 4 or more.
  • the cell C is a living cell
  • the figure 3 live cell images I 7 to I ⁇ 2 are obtained.
  • the image I0 was taken at the in-focus plane P0 of the living cell, and the outline of the living cell is clear. In addition, in the center of the living cell, there is a focal point with an indistinct lens effect.
  • the image I1 is taken at the first focal plane P1 in the direction of the imaging device 20 from the focal plane P0 of the living cell, and the outline of the living cell is unclear.
  • focal plane P1 is an example of the focal plane of the focal point of the lens effect.
  • Image I2 and image I3 were taken from the live cell focal plane P0 toward the imaging device 20 at the second focal plane P2 and the third focal plane P3, respectively. Contours are vague. In addition, in the center of the living cell, there is a focal point with an indistinct lens effect.
  • Images I 4 to I 7 are respectively taken from the focal plane P 0 of the living cell in the direction of the imaging device 20 at the fourth focal plane P 4 to the seventh focal plane P 7 . Contours are vague. Also, no focal point of the lens effect is seen.
  • Image I -1 was taken from the living cell focal plane P 0 toward the light source 10 at the first focal plane P -1 , and the outline of the living cell is unclear. In addition, in the center of the living cell, there is a focal point with an indistinct lens effect.
  • Image I -2 was taken at the second focal plane P -2 in the direction of the light source 10 from the focal plane P 0 of the live cell, and the outline of the live cell is unclear. Also, no focal point of the lens effect is seen.
  • the outline of the living cell tends to become larger as the focal plane is further away from the focal plane P 0 of the living cell, as shown in, for example, images I7 to I - 2 .
  • the size of the focal point of the lens effect is, on the side where the focal point of the lens effect is formed (that is, the imaging device 20 side ), for example, as shown in the image I1 and the image I2 . There is a tendency that the farther the focal plane is from the focal plane P1 of the condensing point, the larger the focal plane becomes.
  • the size of the lens effect focal point becomes smaller, as shown in image I3 , and further away from the focal plane P3 At P4 , no focal point can be seen.
  • the size of the focal point of the lens effect is such that on the side opposite to the side where the focal point of the lens effect is formed (that is, on the side of the light source 10), for example, as shown in the image I -1 , the lens It tends to be smaller away from the focal plane P1 of the focus of the effect. Then, at the further distant focal plane P 1 ⁇ 2 , the focal point of the lens effect is no longer seen.
  • the cell is a dead cell, for example, by imaging the dead cell at a plurality of focal planes P7 to P-2 , including the focal plane P0 of the dead cell, while changing the focal plane, FIG. As shown in , image I'7 to image I' -2 of dead cells are obtained.
  • the image I'0 was taken at the focal plane P0 of the dead cells, and the outlines of the dead cells are clear.
  • Image I′ 1 was taken at the first focal plane P 1 in the direction of the imaging device 20 from the focal plane P 0 of the dead cells, and the outline of the dead cells is unclear.
  • Image I'2 is taken at the second focal plane P2 in the direction of imaging device 20 from the focal plane P0 of the dead cells, and the outline of the dead cells is unclear.
  • dead cells are opaque compared to live cells because the cell membrane is broken and light scatters on the outer peripheral surface of dead cells.
  • a phenomenon similar to the diffraction caused by a shielding object placed on the plane wave of light occurs, and near the center of the dead cell in the direction opposite to the side of the dead cell irradiated with light. A brightened area is formed.
  • a focal point of the diffracted light is present in the center of the dead cell in image I'2 , and the outline of the focal point is clear. That is, the focal plane P2 is an example of the focal plane of the condensing point of the diffracted light.
  • Images I′ 3 to I′ 7 are respectively taken from the focal plane P 0 of the dead cell in the direction of the imaging device 20 at the third focal plane P 3 to the seventh focal plane P 7 .
  • Cell outlines are unclear.
  • Image I' -1 and image I' -2 were taken at the first and second focal planes P -1 and P- 2 , respectively, in the direction of the light source 10 from the focal plane P 0 of the dead cells. dead cells, and the outline of dead cells is unclear.
  • the outline of dead cells tends to increase with increasing distance from the focal plane P 0 of the dead cells, as shown, for example, in images I 7 to I -2 .
  • the size of the focal point of the diffracted light for example, as shown in images I'7 to I'2 , increases with the distance from the focal plane P2 of the focal point of the diffracted light.
  • the condensing point of the lens effect and the condensing point of the diffracted light are sometimes simply called "condensing points”.
  • image piece acquisition process In the image piece acquisition step, an image piece including the central portion and the peripheral portion of the cell is acquired from each image obtained in the image acquisition step.
  • the image piece acquisition process may be performed, for example, by image processing using image processing software.
  • the position where the image piece is acquired from the cell image is not particularly limited as long as it includes the central part and the peripheral part of the cell.
  • the image piece preferably includes a portion where the length of the cell is the maximum (that is, a portion where the length of a straight line connecting any two points on the outline of the cell is the maximum).
  • image pieces S 7 to S ⁇ 2 including the portion where the length of the living cell is the maximum may be obtained, and the portion where the length of the dead cell is the maximum may be acquired.
  • Image strips S′ 7 through S′ ⁇ 2 may be obtained.
  • each image may be preprocessed prior to acquiring image slices from each image of cells.
  • preprocessing for example, concentric circles with different radii centering on the center of the cell are set in pixel units, and the average value of brightness on the circumference is obtained for each concentric circle, and the brightness on the circumference is the above average value.
  • There is a process of reconstructing an image of a cell by drawing a certain concentric circle that is, the brightness on the circumference is averaged and has the same radius as the original concentric circle. This makes it possible to obtain a symmetrical image with respect to the center of the cell. Therefore, when acquiring image fragments, it is possible to eliminate the influence of the direction in which the image fragments are cut out.
  • a symmetrical concatenated image (for example, as shown in FIGS. 5 and 6, a symmetrical concatenated image) can be obtained. It becomes easier to extract the feature amount from the connected image in the feature amount extraction step.
  • Step of creating linked images for analysis In the analysis connected image creation step, the image pieces obtained in the image piece acquisition step are connected in order of the imaging direction of the focal plane to create an analysis connection image.
  • the process of creating a connected image for analysis may be performed, for example, by image processing using image processing software.
  • the method of connecting the image pieces is not particularly limited as long as they are connected in order of the imaging direction of the focal plane. It is preferable to connect the image pieces so that the long sides of the image pieces are in contact with each other along a straight line connecting the centers of the cells of each image piece.
  • the image pieces S 7 to S ⁇ 2 of the living cells acquired from FIG. By concatenating along the lines, a concatenated image L for analysis of living cells is obtained. Also, for example, image pieces S′ 7 to S′ ⁇ 2 of dead cells obtained from FIG. are connected along a line connecting , a connected image L′ for analysis of dead cells is obtained.
  • Feature quantity extraction process In the feature quantity extraction step, a feature quantity is extracted from the analysis connected image obtained in the analysis connected image creation step.
  • the feature quantity extraction process may be performed, for example, by image processing using image processing software.
  • the connected image contains various information about the cells, and for example, feature quantities 1 to 11 below can be obtained.
  • the concatenated image contains information about cell viability based on the presence or absence of the focal point of the lens effect.
  • the merged image contains image slices of the cell's in-focus plane, where live cells have a lensing focal point, while dead cells have a lensing focal point. do not do.
  • the connected image L of the living cell shown in FIG. 5 has the focal point of the lens effect at the image piece S0 of the focal plane P0 of the living cell.
  • the connected image L' of the dead cells shown in FIG. 6 does not have the focal point of the lens effect on the image piece S'0 of the focal plane P0 of the dead cells. Therefore, it is possible to determine whether a cell is alive or dead based on the presence or absence of the focal point of the lens effect.
  • the brightness of the central portion of the image piece of the in-focus plane of the cell may be defined as the feature quantity 1, and based on the predetermined range of the feature quantity 1, the life or death of the cell may be determined. Accordingly, when the feature amount 1 is within the range described above, it can be determined that the cell is a living cell for the analysis connected image. Further, in another embodiment, life-and-death determination is performed based on the feature amount 1 of the concatenated image for analysis, the feature amount 1 of the known concatenated image for reference of the cell, and the determination result of whether or not the cell is a living cell. you can
  • the stitched image contains information about cell viability based on the starting position of the focal spot.
  • "Starting position of the focal point” means the focal plane of the focal point of the lens effect or the diffracted light.
  • the "starting position of the focal point” means the image piece in the focal plane of the lens effect or the focal point of the diffracted light.
  • the starting position of the focal point of the lens effect reflects the average refractive index of the cell.
  • the focal plane P1 of the lensing focal point is the starting position of the lensing focal point.
  • the focal plane P2 of the condensing point of the diffracted light is the starting position of the condensing point of the diffracted light.
  • the starting position of the focal point of the lens effect is closer to the cell than the starting position of the focal point of the diffracted light.
  • the focal point of the lens effect is formed by the light passing through the living cell and concentrating near the living cell, whereas the focal point of the diffracted light passes through the vicinity of the outer peripheral surface of the dead cell. This is because the emitted light is focused relatively far from the dead cells and formed. That is, the focal point of the lens effect formed in the living cell is closer to the cell than the focal point of the diffracted light formed in the dead cell.
  • the starting position of the focal point of the lens effect is close to the focal plane P 0 of the living cell.
  • the starting position of the focal point of the diffracted light is far from the focal plane P0 of the dead cell. Therefore, it is possible to determine whether a cell is alive or dead based on the starting position of the focal point.
  • the feature amount 2 may be obtained by specifying the start position of the focal point based on the distance based on the focal plane of the cell. For example, the length along the direction in which the image strips are joined between the image strip at the focal plane of the cell and the image strip at the start position of the focal point (i.e., the focal plane of the focal point) is characterized.
  • a predetermined feature amount 2 range may be used to determine the life or death of a cell. Thereby, when the feature quantity 2 is within the above range, it can be determined that the cell is a living cell for the analysis connected image.
  • life and death determination is performed based on the feature amount 2 of the concatenated image for analysis, the feature amount 2 of the known concatenated image for reference of the cell, and the determination result of whether or not the cell is a living cell. you can
  • the concatenated image contains information about cell viability based on the duration of the focal spot.
  • focal point duration is meant the distance from the starting position of the focal point of the lens effect or diffracted light to the focal plane where the focal point vanishes.
  • continuous length of the focal point is the shortest length from the image piece on the focal plane of the focal point of the lens effect or diffracted light to the image piece on the focal plane where the focal point disappears.
  • the focal point continues over focal planes further away from in-focus plane P0 than focal plane P4.
  • the condensed points of the lens effect have a continuous length of the condensed points. Also, for the focal point of the lens effect, the intensity of the light decreases at short distances due to divergence.
  • the blue component short wavelength component
  • the red component long wavelength component
  • the focal point of the diffracted light is formed by condensing the light passing through the vicinity of the outer peripheral surface of the dead cell, and the interference pattern changes over a relatively long distance depending on the distance from the dead cell. . Therefore, the focal point of the diffracted light has a continuous length of the focal point that is longer than the lens effect. That is, the live cell focal spot has a shorter focal spot duration than the dead cell focal spot.
  • the focal point of the lens effect is continuous from the image piece S 1 ⁇ 1 to the image piece S 3 .
  • the focal points of the diffracted light are continuous from the image piece S'2 to the image piece S7.
  • the duration of the focal spot of the live cell concatenated image L is shorter than the focal spot duration of the dead cell concatenated image L′. Therefore, it is possible to determine whether a cell is alive or dead based on the continuous length of the focal point.
  • the length along the direction in which the image pieces are connected is defined as the feature quantity 3, and based on the predetermined range of the feature quantity 3, cell life/death determination may be performed.
  • life-and-death determination is performed based on the feature amount 3 of the concatenated image for analysis, the feature amount 3 of the known concatenated reference image of the cell, and the determination result of whether or not the cell is a living cell. you can
  • the concatenated image contains information regarding cell identification based on the starting position of the focal spot.
  • the starting position of the focal point of the lens effect reflects the average refractive index of the cell, and other substances with different average refractive indices from the target cell (e.g., other cells, red blood cells, oil droplets, etc.) etc.), the starting position of the focal point is different from that of the target cell. Therefore, based on the starting position of the focal point, it is possible to determine whether or not the cell to be determined for life or death is the target cell.
  • the starting position of the focal point may be specified as the feature quantity 4 based on the distance based on the focal plane of the cell.
  • the length along the direction in which the image strips are joined between the image strip at the focal plane of the cell and the image strip at the start position of the focal point i.e., the focal plane of the focal point
  • Quantity 4 and based on the predetermined range of the feature quantity 4 determination of life or death of the cell may be performed.
  • the characteristic value 4 of the analysis connected image is within the above range, it can be determined that the cell to be determined for life or death is the target cell.
  • life-and-death determination is performed based on the feature quantity 4 of the concatenated image for analysis, the feature quantity 4 of the known reference concatenated image of the cell, and the determination result of whether or not the cell is a living cell. you can
  • Feature quantity 4 may be the same as feature quantity 2.
  • the feature quantity 4 may be different from the feature quantity 2.
  • the feature quantity 4 is set so as to be more suitable for identifying cells, and the feature quantity 4 is set so as to be more suitable for judging whether cells are alive or dead. You may set the feature quantity 2 to .
  • the concatenated image contains information regarding cell identification based on the duration of the focal spot.
  • the duration of the focal point of the lens effect reflects the average refractive index of the cell, so other substances with different average refractive indices from the target cell (e.g., other cells, red blood cells, oil droplets, etc.) differ in the duration of the focal spot from the target cell. Therefore, it is possible to determine whether or not the cell to be determined for life or death is the target cell based on the duration of the focal point.
  • the length along the direction in which the image pieces are connected is defined as the feature value 5, and based on the predetermined range of the feature value 5, cell survival determination may be performed.
  • cell survival determination may be performed.
  • life-and-death determination is performed based on the feature amount 5 of the concatenated image for analysis, the feature amount 5 of the known concatenated image for reference of the cell, and the determination result of whether or not the cell is a living cell. you can
  • Feature quantity 5 may be the same as feature quantity 3.
  • the feature amount 5 may be different from the feature amount 3.
  • the feature amount 5 is set so as to be more suitable for identifying cells, and the feature amount 5 is set so as to be more suitable for determining whether cells are alive or dead.
  • a feature amount 3 may be set to .
  • red blood cells have a shape like a concave lens.
  • a light-condensing point is formed on the side, and a light-condensing point is also formed on the side irradiated with light.
  • erythrocytes may also differ from living cells in the starting position of focal spots and the duration of focal spots.
  • Concatenated images may contain information regarding cell identification based on cell size in the plane of focus.
  • the stitched image contains image pieces of the in-focus plane of the cell. Therefore, if the image piece of the in-focus plane of the cell contains the part where the length of the cell is the maximum (that is, the part where the length of the straight line connecting any two points on the outline of the cell is the maximum) , the length of the maximum portion can be used as the diameter of the cell. Then, for example, it is examined whether or not the diameter of the cell to be determined for life and death is about the same as the known diameter of the target cell, and if not, it is determined that the cell to be determined for life and death is not the target cell. can do. Therefore, based on the size of the cell in the in-focus plane, it is possible to determine whether or not the cell to be determined for life or death is the target cell.
  • the portion where the length of the cell is the maximum (that is, the portion where the length of the straight line connecting any two points on the outline of the cell is the maximum) ) is defined as the feature quantity 6, and based on a predetermined range of the feature quantity 6, determination of life or death of the cell may be performed.
  • the characteristic value 6 is within the above range, it can be determined that the cell to be determined for life or death is the target cell in the connected image for analysis.
  • life and death determination is performed based on the feature amount 6 of the concatenated image for analysis, the feature amount 6 of the known concatenated image for reference of the cell, and the determination result as to whether the cell is a living cell or not. you can
  • the merged image contains information regarding the identification of cells based on the position of the plane of focus.
  • a cell suspension containing target cells and other substances e.g., other cells, red blood cells, oil droplets, etc.
  • target cells and other substances e.g., other cells, red blood cells, oil droplets, etc.
  • the specific gravity of the cells is different from the specific gravity of the other substances, the cells and the other substances , there is a difference in how they sink (the degree of sinking) in the cell suspension, and the positions of the cells differ from the positions of other substances in the depth direction of the cell suspension. Therefore, when cells and other substances are imaged on the same focal plane and a connected image is created, the position of the image piece on the focal plane of the cell and the position of the image piece on the focal plane of the other substance (for example, , height in the connecting direction of the image pieces) are different.
  • the position of the image piece of the in-focus plane of the cell that is the target of life/death determination is comparable to the position of the image piece of the known in-focus plane of the target cell.
  • the feature value 7 is the length along the direction in which the image pieces are connected between the image piece of the focal plane and the image piece of the lowest focal plane of the cell that is the target of life-or-death determination. Based on the range of the feature value 7 obtained, determination of cell life and death may be performed. As a result, when the characteristic value 7 of the analysis connected image is within the above range, it can be determined that the cell to be determined for life or death is the target cell. Further, in another embodiment, life-and-death determination is performed based on the feature quantity 7 of the concatenated image for analysis, the feature quantity 7 of the known concatenated reference image of the cell, and the determination result of whether or not the cell is a living cell. you can
  • the connected image contains information regarding cell identification based on the connected shape of the focal points.
  • the “connected shape of condensing points” means the shape of a portion formed by connecting condensing points in a connected image.
  • the image of the portion where the focal points are connected is set as the feature amount 8
  • the feature amount 8 of the connected image for analysis and the feature amount 8 of the known connected image for reference of the cell are calculated by a machine learning device. It may be determined whether or not the cell to be determined for life or death is the target cell based on the result of determining whether or not the cell is a viable cell.
  • the connecting shape of the focal points is related to the size of the focal points in addition to the starting position of the focal points and the continuous length of the focal points. It reflects Therefore, other substances (eg, other cells, red blood cells, oil droplets, etc.) having a different average refractive index from the target cell have a different connection shape of the focal points from the target cell. Therefore, it is possible to determine whether or not the cell to be determined for life or death is the target cell based on the connected shape of the condensing points.
  • substances eg, other cells, red blood cells, oil droplets, etc.
  • the concatenated image may contain information regarding cell identification based on the color distribution of the foci.
  • the blue component short wavelength component
  • the red component long wavelength component
  • a distribution of colors can be seen.
  • the color distribution of the lensing focus reflects the average refractive index of the cell. Therefore, other substances (eg, other cells, red blood cells, oil droplets, etc.) having a different average refractive index from the target cell have a different color distribution of the focal point than the target cell. Therefore, based on the color distribution of the condensed points, it is possible to determine whether or not the cell to be determined for life or death is the target cell.
  • the color separation of the focal point of the lens effect as described above can be seen, showing a color distribution in which the vicinity of the cell is colored blue and the area relatively far away is colored red.
  • red blood cells for example, tend to have weak color separation at the focal points and appear relatively white due to their concave lens-like shape.
  • the image of the portion where the focal points are connected is set as the feature amount 9, and the feature amount 9 of the connected image for analysis and the feature amount 9 of the known connected image for reference of the cell are obtained by a machine learning device. It may be determined whether or not the cell to be determined for life or death is the target cell based on the result of determining whether or not the cell is a viable cell.
  • the merged image contains information regarding cell viability and cell identification, as described above. Therefore, based on the degree of matching between the analysis connected image and the reference connected image, it is possible to determine whether the cell is alive or dead, and whether or not the cell to be determined for life or death is the target cell. .
  • the concatenated image for analysis itself is used as a feature quantity 10
  • a machine learning device uses the feature quantity 10 of the concatenated image for analysis, the feature quantity 10 of a known reference concatenated image of a cell, and determination of whether or not it is a living cell. Based on the results, it may be determined whether or not the cells to be determined for life and death are viable cells and whether or not they are target cells.
  • Concatenated images contain life-or-death information based on the amount of lensing penetration of cells.
  • the “lens effect transmission amount” of a cell means the amount of light transmitted through the cell when the cell has a lens effect.
  • the "transmission amount of the lens effect” of the cell is the average brightness of the "multiple image pieces in the imaging direction" starting from the image piece of the focal plane of the cell when the cell has the lens effect, It means the weighted sum of the average brightness of "multiple image pieces in the direction of the light source” starting from the image piece on the focal plane of the cell.
  • the weighting factor is not particularly limited and may be set as appropriate.
  • the dead cells immediately after the cell membrane is broken, as an intermediate state they may have a focal point due to the lens effect, similar to the living cells. Since the dead cells scatter light on the outer peripheral surface due to the breakage of the cell membrane, the amount of transmission due to the lens effect is lower than that of the live cells. Therefore, it is possible to determine whether a cell is alive or dead based on the amount of light transmitted by the lens effect. It is assumed that the output of the light source is kept constant.
  • the average brightness of the image piece in the imaging direction with respect to the focal plane of the cell is, for example, the average brightness of the image piece S 0 on the focal plane P 0 to the image piece S n on the focal plane P n shown in FIG. .
  • the sum may be used as a feature quantity 11, and cell survival may be determined based on a predetermined range of the feature quantity 11.
  • a connected image is created for each color, and the average brightness of "multiple image pieces in the imaging direction" and " By combining the average luminosity of a plurality of image pieces in the direction of the light source from different colors, determination of life and death of cells may be performed.
  • a multi-wavelength light source e.g., white LED
  • an imaging device capable of spectroscopy e.g., RGB color camera
  • the feature amounts 1 to 11 described above are examples of feature amounts obtained from the connected images, and it is possible to determine the life and death of cells and identify cells based on other feature amounts.
  • Feature quantity 1 and feature quantity 11 relate to the lens effect of cells. Further, feature quantities 2 to 5 relate to the average refractive index of cells. Moreover, the feature value 6 relates to the diameter of the cell. Moreover, the feature value 7 relates to the specific gravity of cells. Also, the feature amount 8 and the feature amount 9 relate to the form of the connected image itself. Furthermore, the feature quantity 10 relates to all of the average refractive index of cells, the diameter of cells, and the specific gravity of cells. In this way, since many feature values can be obtained from the concatenated image, the concatenated image is effective not only for judging the life and death of cells but also for identifying cells.
  • the feature quantity used to determine the life and death of the cell is one or more selected from the group consisting of feature quantity 1 to feature quantity 11, that is, the feature quantity related to the lens effect of the cell, the feature quantity related to the average refractive index of the cell, the cell's It is preferable to include one or more selected from the group consisting of a feature amount related to diameter and a feature amount related to cell specific gravity.
  • life-and-death determination is performed based on the feature amount of the analysis connected image and a predetermined range of the feature amount. In another embodiment, determination of whether a cell is alive or dead may be performed based on the feature amount of a known reference connected image of the cell and the determination result of whether or not the cell is a living cell.
  • determination of whether or not a cell to be determined for life and death is a target cell can also be performed based on a predetermined range of feature values. , When determining whether or not the cell that is the target of life-and-death determination is the target cell, based on the feature amount of the known reference connected image of the target cell and the determination result of whether or not it is a live cell cells may be determined for viability.
  • Determining whether a cell is alive or dead can include determining whether the cell to be determined is a target cell, that is, identifying the cell.
  • Such an aspect includes, for example, a case where cell life-or-death determination is performed by the feature quantity 2 (or feature quantity 4) based on the starting position of the focal point, and the feature quantity 2 is also based on the starting position of the focal point.
  • the feature quantity 4 or the feature quantity 2
  • 4 or feature quantity 3
  • the feature value 10 based on the matching degree of the connected images is used, and in addition to the determination of life and death of the cell, determination of whether or not the cell that is the target of the determination of life and death is the target cell is also performed at the same time.
  • any one of feature amounts 1 to 3 and feature amount 11 may be used to determine whether a cell is alive or dead.
  • cell life-or-death determination may be performed by combining two or more of feature amounts 1 to 3 and feature amount 11.
  • Any one of the feature values 4 to 9 may be used to identify cells (that is, to determine whether or not a cell that is the target of life-and-death determination is a target cell). From the viewpoint of further improving the accuracy of cell identification, cell identification may be performed by combining two or more of the feature quantities 4 to 9. FIG.
  • determination and identification of cell life and death may be performed at the same time.
  • Cell viability determination (which in some embodiments may include cell identification) may be performed using a machine learning machine.
  • the machine learning device may be constructed by any one method of neural network, support vector machine and boosting. It is preferable that the machine learning device is constructed by a neural network and performs deep learning.
  • the teacher data consists of the known feature values of connected reference images of cells (hereinafter sometimes referred to as "learning feature values”) and the determination result of whether or not the cell is a live cell corresponding to the learning feature values ( hereinafter sometimes referred to as "correct life-or-death judgment result").
  • learning feature values are input to the machine learning device.
  • the machine learning device outputs learning life-or-death judgment results for the learning feature values. Based on this life-and-death determination result for learning and the correct life-and-death determination result, the loss calculation of the machine learner using a loss function is performed. Various coefficients of the machine learning device are updated according to the result of the loss calculation, and the machine learning device is updated according to the update settings.
  • the process is repeated while teaching data is exchanged.
  • the repetition of the above series of processes is terminated when the prediction accuracy of the learning life-death judgment result with respect to the correct life-death judgment result reaches a predetermined set level.
  • the machine learning device whose prediction accuracy has reached the set level in this way is used in the operation phase, and outputs a life-and-death determination result in response to the input of the feature amount of the connected image for analysis.
  • life-and-death determination may be performed for multiple cells in the cell suspension.
  • a connected image for analysis of target cells and other substances is created. Then, based on the feature amount of these concatenated images for analysis, the feature amount of the known concatenated reference image of the target cell, and the determination result of whether or not the target cell is a living cell, the target cell is identified and at the same time, the target cell life-or-death judgment may be performed.
  • target cells and other substances eg, other cells, red blood cells, oil droplets, etc.
  • the life-and-death determination flow shown in FIG. 8 is an example of identifying a target cell using feature quantities 4 to 7 and performing life-and-death determination using feature quantities 4 and 5 .
  • Step S10- One concatenated image for analysis is selected from a plurality of concatenated images for analysis (S10).
  • Step S12- It is determined whether or not the feature amount 6 (diameter size) of the analysis connected image is within a predetermined range of the feature amount 6 (S12). If the feature amount 6 of the analysis connected image is within the above range, it is determined that the cell may be the target cell, and the process proceeds to the next step (S14). If the feature quantity 6 of the analysis connected image is not within the above range, it is determined that the cell is not the target cell (S22).
  • Step S14- It is determined whether or not the feature amount 7 (the position of the in-focus plane) of the analysis connected image is within the range of the predetermined feature amount 7 (S14). If the feature value 7 of the analysis connected image is within the above range, it is determined that the cell may be the target cell, and the process proceeds to the next step (S16). If the feature amount 7 of the analysis connected image is not within the above range, it is determined that the cell is not the target cell (S22).
  • Step S16- It is determined whether or not the characteristic amount 4 (starting position of the condensing point) of the analysis connected image is within the range of the predetermined characteristic amount 4 (S16). If the feature value 4 of the analysis connected image is within the above range, it is determined that the target cell may be a living cell (hereinafter sometimes referred to as a "target living cell"), and the process proceeds to the next step (S18). If the characteristic value 4 of the analysis connected image is not within the above range, it is determined that the target cell is not a viable cell (S22).
  • Step S18- It is determined whether or not the characteristic amount 5 (continuation length of the condensing point) of the analysis connected image is within the range of the predetermined characteristic amount 5 (S18). If the feature value 5 of the combined image for analysis is within the above range, it is determined that the target cell is a viable cell (S20). If the feature value 5 of the analysis connected image is not within the above range, it is determined that the target cell is not a living cell (S22).
  • Step S20 If it is determined to be a viable target cell (S20), proceed to the next step (S24), and if determined not to be the target cell (S22), proceed to the next step (S26). .
  • Step S24- Count the number of connected images determined to be live cells of the target cell, and proceed to the next step (S28).
  • Step S26- Count the number of connected images determined to be non-live cells of the target cell, and proceed to the next step (S28).
  • Step S28- If there is an analysis connected image for which life or death has not been determined, the process returns to step S10, and if life or death determination has been performed for all the analysis connected images, the life or death determination flow ends.
  • the cell viability determination method includes a step of determining the viable cell concentration of cells in the cell suspension based on the results of the viability determination of a plurality of cells in the cell suspension (hereinafter referred to as "viable cell concentration determination step”). may be called).
  • the viable cell concentration of cells means the number of viable cells per unit volume [cells/ml].
  • the viable cell concentration of cells can be determined, for example, as follows.
  • a cell suspension is housed in a holding container, and all cells present between the bottom of the holding container and the liquid surface of the cell suspension (substances other than the target cells (e.g., , other cells, erythrocytes, oil droplets, etc.) are created to determine life and death, and the number of analysis connected images determined to be live cells is the number of live cells.
  • the number of viable cells is the product of the volume of the cell suspension used for measurement (that is, the area of the field of view and the height of the cell suspension (height from the bottom of the holding container to the liquid surface) ) to determine the viable cell concentration of the cells in the cell suspension.
  • the method for determining cell life and death according to the present disclosure can determine cell life and death, and can determine the viable cell concentration of cells.
  • the cell viability determination method includes a step of determining the cell viability of the cells in the cell suspension based on the results of viability determination of a plurality of cells in the cell suspension (hereinafter referred to as "cell viability determination step”). may be called).
  • the cell viability is the value obtained by dividing the number of viable cells by the total number of cells (the sum of the number of viable cells and the number of dead cells).
  • the number of analysis connected images determined to be living cells is the number of living cells, and the total number of analysis connection images (for example, steps S24 and S26) can be calculated as the total cell number.
  • the cell type contained in the cell suspension is only one type of target cell, so the cell viability obtained in this case is the ratio of the target viable cells to the total target cells. Become.
  • the cell life-and-death determination device includes: An image acquisition unit that acquires an image of a cell captured with a plurality of focal planes including the focal plane of the cell from the direction opposite to the side of the cell irradiated with light, an image piece acquisition unit that acquires an image piece including the central part and the peripheral part of a cell from each image; an analytical connected image creating unit that creates an analytical connected image by connecting image pieces in order of the imaging direction of the focal plane; A feature quantity extraction unit for extracting a feature quantity from the concatenated image for analysis; a life-and-death determination unit that determines the life and death of cells; Prepare.
  • the life-and-death determination unit may be composed of a machine learning device.
  • the cell viability determination device may include a viable cell concentration determination unit that determines the viable cell concentration of cells in the cell suspension based on the results of viability determination of a plurality of cells in the cell suspension.
  • the cell viability determination device may include a cell viability determination unit that determines the cell viability of cells in the cell suspension based on the results of viability determination of a plurality of cells in the cell suspension.
  • the image acquisition unit, the image piece acquisition unit, the analytical connected image creation unit, the feature extraction unit, the life/death determination unit, the viable cell concentration determination unit, and the cell viability determination unit in the cell life/death determination device are each an image of the cell life/death determination method. It corresponds to an acquisition process, an image piece acquisition process, an analytical connection image creation process, a feature quantity extraction process, a life/death determination process, a viable cell concentration determination process, and a cell viability determination process.
  • the cell viability determination device may operate as a cell viability determination system with other devices for acquiring images of cells at multiple focal planes.
  • a cell life/death determination system includes a light source that emits light, an imaging device that images cells, and means for changing a focal plane.
  • the means for changing the focal plane is not particularly limited, and may be, for example, a stage movement mechanism that changes the distance between the cells and the imaging device by moving the stage on which the holding container holding the cells is placed.
  • the cell life/death determination system 200 includes a light source that irradiates cells C with light, an imaging device 20 that images cells C, and a holding container 40 that holds cells (cell suspension) C. , a stage 30 (stage moving mechanism) that moves the holding container 40 to change the distance between the cell C and the imaging device 20, and the cell survival determination device 100 according to the present disclosure.
  • the means for changing the focal plane may be an imaging device moving mechanism that moves the imaging device to change the distance between the cell and the imaging device.
  • the imaging device may include a liquid lens as means for changing the focal plane.
  • the cell life/death determination system does not include such an imaging device or the like, and may include, for example, an external storage device in which cell images are stored. Images of cells may be obtained from
  • the cell life/death determination system may include an input device for inputting data and a display device for displaying cell life/death determination results.
  • a keyboard for example, can be used as the input device, and a monitor, for example, can be used as the display device.
  • the cell life/death determination device according to the present disclosure will be described more specifically, taking the cell life/death determination system 200 shown in FIG. 1 as an example.
  • FIG. 9 is a block diagram showing an example of the control unit 106 that constitutes the cell life/death determination device 100 shown in FIG.
  • a CPU (Central Processing Unit) 101 provided in the control unit 106 is a processor that controls the cell life/death determination system 200 as a whole.
  • the CPU 101 reads a system program stored in a ROM (Read Only Memory) 102 via a bus 105, and controls the entire cell life/death determination apparatus 100 according to the system program.
  • a RAM (Random Access Memory) 103 temporarily stores calculation data, display data for the display device 60, various data input via the input device 50, and the like.
  • non-volatile memory 104 for example, an SRAM (Static Random Access Memory), an SSD (Solid State Drive), or the like backed up by a battery (not shown) is used. Data obtained from the stage 30, data input from the input device 50, and the like are stored. Data, programs, and the like stored in the nonvolatile memory 104 may be developed in the RAM 103 when used. In addition, the ROM 102 is pre-written with various algorithms required for image analysis of image data acquired from the imaging device 20, system programs for executing other required processes, and the like.
  • SRAM Static Random Access Memory
  • SSD Solid State Drive
  • the result of determining whether the cell is alive or dead is output to the display device 60.
  • FIG. 10 is a block diagram showing an example of processing of the cell life/death determination device shown in FIG. Each functional block shown in FIG. 10 is implemented by CPU 101 provided in cell life/death determination apparatus 100 shown in FIG.
  • the control unit 106 controls the light source 10 , the imaging device 20 and the stage 30 based on the imaging program stored in the nonvolatile memory 104 to image the cells C.
  • the control unit 106 turns on the light source 10 to irradiate the cells C with light, moves the holding container 40 holding the cells (cell suspension) C, and drives the stage 30 so as to change the focal plane.
  • the control unit 106 commands the imaging device 20 to perform an imaging operation.
  • the control unit 106 images the cell on a plurality of focal planes including the focal plane of the cell from the direction opposite to the light-irradiated side of the cell irradiated with light, according to the imaging program.
  • control unit 106 and the imaging program is unnecessary.
  • the image acquisition unit 107 acquires images of cells captured by the imaging device 20 .
  • a plurality of images obtained by imaging one cell may be collectively managed as one set of image data group.
  • the image piece acquiring unit 108 performs image processing on the cell image acquired by the image acquiring unit 107, and acquires an image piece including the central part and the peripheral part of the cell from the cell image.
  • a plurality of image pieces obtained by imaging one cell may be collectively managed as one set of image piece data group.
  • the analysis connected image creation unit 109 performs image processing on the image pieces obtained by the image piece acquisition unit 108, connects the image pieces in order of the imaging direction of the focal plane, and creates an analysis connection image.
  • the feature quantity extraction unit 110 performs image processing on the analysis connected image obtained by the analysis connected image creation unit 109, and extracts a feature quantity from the analysis connected image.
  • the life-and-death determination unit 111 determines whether a cell is alive or dead based on the feature amount of the analysis connected image extracted by the feature amount extraction unit 110 and a predetermined range of the feature amount. In another embodiment, the life-and-death determination unit 111 compares the feature amount of the analysis connected image extracted by the feature amount extraction unit 110, the feature amount of the known reference connected image of the cell, and whether or not the cell is a living cell. Based on the determination result, the life and death determination of the cell is performed. The life-and-death determination unit 111 outputs the cell life-and-death determination result generated by the determination to the display device 60 .
  • the viable cell concentration determining unit 112 determines the viable cell concentration of cells based on the result of the life and death determination, that is, the number of analysis connected images determined to be viable cells.
  • the viable cell concentration determination unit 112 outputs the viable cell concentration of cells to the display device 60 .
  • the cell viability determination unit 113 determines the cell viability based on the results of life and death determination, that is, the number of analysis connected images determined to be living cells and the total number of analysis connected images.
  • the cell viability determination unit 113 outputs the cell viability to the display device 60 .
  • the life-death determination device As described above, by using the cell life-death determination device according to the present disclosure, the life-death determination of cells can be performed, and the viable cell concentration of cells can be obtained.

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PCT/JP2022/017730 2021-04-28 2022-04-13 細胞生死判定方法、細胞生死判定装置及び細胞生死判定システム Ceased WO2022230672A1 (ja)

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AU2022263866A AU2022263866B2 (en) 2021-04-28 2022-04-13 Cell life-and-death determination method, cell life-and-death determination device, and cell life-and-death determination system
EP22795586.1A EP4317970A4 (en) 2021-04-28 2022-04-13 METHOD FOR DETERMINING CELL LIFETIME/DESATH, DEVICE FOR DETERMINING CELL LIFETIME/DESATH AND SYSTEM FOR DETERMINING CELL LIFETIME/DESATH
CN202280030722.4A CN117203525A (zh) 2021-04-28 2022-04-13 细胞生死判定方法、细胞生死判定装置及细胞生死判定系统
JP2023517433A JPWO2022230672A1 (https=) 2021-04-28 2022-04-13
KR1020237036082A KR20230159871A (ko) 2021-04-28 2022-04-13 세포 생사 판정 방법, 세포 생사 판정 장치 및 세포 생사 판정 시스템
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