Patent Publication Number: US-11035845-B2

Title: Observation apparatus, observation method, observation system, program, and cell manufacturing method

Description:
RELATED APPLICATION 
     The present application is a continuation application of U.S. patent application Ser. No. 15/319,336 filed Feb. 27, 2017, which in turn is a U.S. national stage application of PCT/JP2014/065916 filed Jun. 16, 2014. Each of these prior applications is incorporated herein by reference in their entireties. 
    
    
     TECHNICAL FIELD 
     The present invention relates to an observation apparatus, an observation method, an observation system, a program, and a cell manufacturing method. 
     BACKGROUND 
     In general, a technology of evaluating a cell incubation state is a base technology in a broad range of fields including an advanced medical field such as regenerative medicine and screening of pharmaceutical products. For example, the regenerative medical field includes a process of cell proliferation and cell differentiation in vitro. In the process, in order to manage success and failure of cell differentiation and presence or absence of cell canceration and infection, it is indispensable to accurately evaluate the cell incubation state. As an example, an evaluation method of a cancer cell using a transcription factor as a marker is disclosed (refer to Patent Document 1). 
     A stem cell such as an ES (Embryonic Stem) cell or an iPS (induced Pluripotent Stem) cell can be substantially infinitely proliferated theoretically while maintaining differentiation pluripotency that the cell can differentiate into substantially all tissues and therefore has been drawing attention in pharmaceutical development and regenerative medical applications. 
     RELATED ART DOCUMENTS 
     Patent Documents 
     [Patent Document 1] U.S. Pat. No. 7,060,445 
     SUMMARY OF INVENTION 
     Problems to be Solved by the Invention 
     When such a stem cell is applied to drug discovery research or regenerative medicine, it is necessary to incubate a stem cell having a good state (the size of a colony is a moderate size, and the density of a cell that is present in the colony is a moderate density), and therefore, it is required to accurately determine the maturity degree of a cell line during incubation. However, in the related art, the maturity degree of a cell line is determined according to visual observation of a researcher, and therefore, there is a problem that it is impossible to improve the determination accuracy of the maturity degree of a cell line. 
     In view of the foregoing, a problem to be solved by the present invention is to provide an observation apparatus, an observation method, an observation system, a program, and a cell manufacturing method capable of improving the determination accuracy of the maturity degree of a cell line. 
     Means for Solving the Problem 
     [1] In order to solve the problem, an aspect of the present invention is an observation apparatus including: an area calculation unit that calculates a colony area based on an image in which a cell colony is captured; a cell number calculation unit that calculates, based on the image, the number of cells included in a target colony of which an area is calculated by the area calculation unit; and a density calculation unit that calculates, based on the area of the target colony calculated by the area calculation unit and the number of the cells included in the target colony calculated by the cell number calculation unit, a density of the cells included in the target colony. 
     [2] Further, in order to solve the problem, an aspect of the present invention is an observation system including: an imaging unit that captures an image of a cell during incubation; and an observation apparatus described above. 
     [3] Further, in order to solve the problem, an aspect of the present invention is an observation method including: an area calculation step of calculating a colony area based on an image in which a cell colony is captured; a cell number calculation step of calculating, based on the image, the number of cells included in a target colony of which an area is calculated according to the area calculation step; and a density calculation step of calculating, based on the area of the target colony calculated according to the area calculation step and the number of the cells included in the target colony calculated according to the cell number calculation step, a density of the cells included in the target colony. 
     [4] Further, in order to solve the problem, an aspect of the present invention is a program that causes a computer to execute: an area calculation step of calculating a colony area based on an image in which a cell colony is captured; a cell number calculation step of calculating, based on the image, the number of cells included in a target colony of which an area is calculated according to the area calculation step; and a density calculation step of calculating, based on the area of the target colony calculated according to the area calculation step and the number of the cells included in the target colony calculated according to the cell number calculation step, a density of the cells included in the target colony. 
     [5] Further, in order to solve the problem, an aspect of the present invention is a cell manufacturing method including: a cell incubation step of incubating a cell; an area calculation step of imaging a cell colony incubated in the incubation step and calculating a colony area based on a captured image of the colony; a cell number calculation step of calculating, based on the image, the number of cells included in a target colony of which an area is calculated according to the area calculation step; and a density calculation step of calculating, based on the area of the target colony calculated according to the area calculation step and the number of the cells included in the target colony calculated according to the cell number calculation step, a density of the cells included in the target colony. 
     Advantage of the Invention 
     According to the present invention, it is possible to improve the determination accuracy of the maturity degree of a cell line. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a block diagram showing an example of a configuration of an observation apparatus according to an embodiment of the present invention. 
         FIG. 2  is a block diagram showing an example of a configuration of a control device included in the observation apparatus of the present embodiment. 
         FIG. 3  is a front view of the observation apparatus of the present embodiment. 
         FIG. 4  is a plan view of the observation apparatus of the present embodiment. 
         FIG. 5  is a graph showing an example of a temporal change of a colony area for each cell line of the present embodiment. 
         FIG. 6  is a graph showing an example of a relationship between a colony area and a cell number for each cell line of the present embodiment. 
         FIG. 7  is a graph showing an example of a relationship between a cell colony area and the density of the cell included in the colony. 
         FIG. 8  is a schematic diagram showing an example of an entire observation image captured by an imaging device of the present embodiment. 
         FIG. 9  is a graph showing an example of a relationship between the temporal change of the colony area and a first calibration curve of the present embodiment. 
         FIG. 10  is a flowchart showing an example of a calibration curve registration operation according to the observation apparatus of the present embodiment. 
         FIG. 11  is a flowchart showing an example of a cell number estimation operation according to the observation apparatus of the present embodiment. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Embodiment 
     Hereinafter, an embodiment of the present invention will be described with reference to the drawings. First, with reference to  FIG. 1  to  FIG. 4 , an outline of a configuration of an incubator (observation apparatus) according to the embodiment of the present invention is described.  FIG. 1  is a block diagram showing an example of a configuration of an incubator  11  according to the embodiment of the present invention.  FIG. 2  is a block diagram showing an example of a configuration of a control device  41  included in the incubator  11  of the present embodiment.  FIG. 3  and  FIG. 4  are front and plan views of the incubator  11  of the present embodiment. 
     The incubator  11  is an apparatus used for incubating a cell and observing the state of the cell by imaging the incubated cell using a microscope camera. The incubator  11  has an upper casing  12  and a lower casing  13 . In an assembled state of the incubator  11 , the upper casing  12  is arranged above the lower casing  13 . The internal spaces of the upper casing  12  and the lower casing  13  are separated in the vertical direction by a base plate  14 . 
     First, an outline of a configuration of the upper casing  12  is described. A constant temperature room  15  in which a cell is incubated is formed inside the upper casing  12 . The constant temperature room  15  has a temperature adjustment device  15   a  and a humidity adjustment device  15   b , and the inside of the constant temperature room  15  is maintained to be an environment (for example, an atmosphere at a temperature of 37° C. and a humidity of 90%) suitable for cell incubation (the temperature adjustment device  15   a  and the humidity adjustment device  15   b  are not shown in  FIG. 3  and  FIG. 4 ). 
     A large door  16 , a medium door  17 , and a small door  18  are arranged on the front surface of the constant temperature room  15 . The large door  16  covers the front surfaces of the upper casing  12  and the lower casing  13 . The medium door  17  covers the front surface of the upper casing  12  and isolates the circumstance of the constant temperature room  15  from the external circumstance when the large door is opened. The small door  18  is attached to the medium door  17  and is a door used for carrying in and out an incubation container  19  in which a cell is incubated. As the incubation container  19  is carried in and out of the small door  18 , it is possible to prevent environmental changes in the constant temperature room  15 . The airtightness of the large door  16  is maintained by a packing P 1 , the airtightness of the medium door  17  is maintained by a packing P 2 , and the airtightness of the small door  18  is maintained by a packing P 3 . 
     A stocker  21 , an observation unit  22 , a container carry device  23 , and a carry table  24  are arranged in the constant temperature room  15 . Here, the carry table  24  is arranged in front of the small door  18  and is used to carry in and out the incubation container  19  from the small door  18 . 
     The stocker  21  is arranged on the left side of the constant temperature room  15  when seen from the front surface (lower side of  FIG. 4 ) of the upper casing  12 . The stocker  21  has a plurality of shelves, and each shelf of the stocker  21  can store a plurality of incubation containers  19 . A cell as a target of incubation together with a culture medium is stored in each of the incubation containers  19 . The stocker  21  is not essential. 
     The observation unit  22  is arranged on the right side of the constant temperature room  15  when seen from the front surface of the upper casing  12 . A time lapse observation of a cell in the incubation container  19  can be performed using the observation unit  22 . 
     The observation unit  22  is arranged to be fitted in an opening part of the base plate  14  of the upper casing  12 . The observation unit  22  has a sample table  31 , a stand arm  32  that projects above the sample table  31  and on which an illumination light source is arranged, and a main body part  33  that includes an observation system and an imaging device  34 . The sample table  31  and the stand arm  32  are arranged in the constant temperature room  15 . On the other hand, the main body part  33  is stored in the lower casing  13 . 
     The sample table  31  is formed of a translucent material, and the incubation container  19  can be arranged on the sample table  31 . The sample table  31  is configured to be movable in the horizontal direction and can adjust the position of the incubation container  19  arranged on the upper surface of the sample table  31 . The stand arm  32  includes a LED light source  39 . The imaging device  34  images, via a microscope optical system, a cell in the incubation container  19  that is illuminated according to transmission illumination from the upper side of the sample table  31  by the stand arm  32  and thereby can obtain a microscope image of the cell. 
     The container carry device  23  is arranged at the center of the constant temperature room  15  when seen from the front surface of the upper casing  12 . The container carry device  23  exchanges the incubation container  19  among the stocker  21 , the sample table  31  of the observation unit  22 , and the carry table  24 . When the stocker  21  is not provided as described above, the container carry device  23  is also unnecessary. 
     As shown in  FIG. 4 , the container carry device  23  has a vertical robot  38  having a multijoint arm, a rotation stage  35 , a mini stage  36 , and an arm unit  37 . The rotation stage  35  is attached rotatably by 180° in the horizontal direction to the front end part of the vertical robot  38  via a rotation shaft  35   a . Therefore, the rotation stage  35  can cause the arm unit  37  to face each of the stocker  21 , the sample table  31 , and the carry table  24 . 
     The mini stage  36  is attached slidably in the horizontal direction with respect to the rotation stage  35 . The arm unit  37  that grips the incubation container  19  is attached to the mini stage  36 . 
     Next, an outline of a configuration of the lower casing  13  is described. The main body part  33  of the observation unit  22  and the control device  41  of the incubator  11  are stored inside the lower casing  13 . 
     The control device  41  is connected to each of the temperature adjustment device  15   a , the humidity adjustment device  15   b , the observation unit  22 , and the container carry device  23 . The control device  41  controls overall the units of the incubator  11  in accordance with a predetermined program. 
     As an example, the control device  41  controls each of the temperature adjustment device  15   a  and the humidity adjustment device  15   b  and maintains the inside of the constant temperature room  15  to be a predetermined environmental condition. The control device  41  controls, based on a predetermined observation schedule, the observation unit  22  and the container carry device  23  and automatically performs an observation sequence of the incubation container  19 . The control device  41  performs, based on the image acquired in the observation sequence, an incubation state evaluation process in which the incubation state of the cell is evaluated. 
     [Calibration Curve (First Calibration Curve) Indicating Temporal Change of Cell Colony Area] 
     With reference to  FIG. 5 , a temporal change of a cell colony area is described. 
       FIG. 5  is a graph showing an example of a temporal change of a colony area for each cell line. The cells proliferate as time elapses, and therefore, the area of a colony including these cells (hereinafter, simply referred to also as a colony area) increases according to the elapse of time. The temporal change of the colony area differs for each cell line. As an example, the temporal change of the colony area of a cell line A is indicated by a calibration curve L 1 A in  FIG. 5 . The temporal change of the colony area of a cell line B is indicated by a calibration curve L 1 B in  FIG. 5 . The calibration curve L 1 A and the calibration curve L 1 B are collectively referred to as a first calibration curve L 1 . When the cell line is specified, it is possible to estimate the change of the colony area of a cell line in accordance with the elapse of time based on a predetermined relation shown in  FIG. 5 , that is, the first calibration curve L 1 . 
     By observing the temporal change of the colony area, it is possible to determine whether or not the colony is proliferating normally. That is, when the temporal change amount of the colony area of one colony is larger than a standard temporal change amount of the colony area (for example, when the colony area abnormally increases), there is a possibility that the cell included in this colony differentiates. That is, in a colony of which the colony area abnormally increases, it can be deemed that the cell differentiates, and it is possible to determine that the colony is an abnormal colony. On the other hand, when the temporal change amount of the colony area of one colony is smaller than the standard temporal change amount of the colony area (for example, when the colony area does not temporally change), there is a possibility that the cells included in this colony are dead. That is, in a colony of which the colony area does not temporally change, it can be deemed that the cells are dead, and it is possible to determine that the colony is an abnormal colony. The first calibration curve L 1  is registered (stored) in advance for each cell line in the incubator  11  of the present embodiment, and thereby, the incubator  11  determines whether or not the colony is proliferating normally. 
     [Calibration Curve (Second Calibration Curve) Indicating Relationship Between Cell Colony Area and Cell Number Included in this Colony] 
     With reference to  FIG. 6 , the relationship between a cell colony area and a cell number is described. The unit of the horizontal axis in  FIG. 6  is μm 2  (micro·square meter), and the unit of the vertical axis is number. 
       FIG. 6  is a graph showing an example of a relationship between a colony area of one colony for each cell line (hereinafter, colony area) and a cell number in one colony (hereinafter, colony cell number). The user selects a colony suitable for a calibration curve in appearance as a colony which is a target when a calibration curve is prepared. The point of selection is, for example, that a colony which is not adhered to another colony and which is present independently as an individual colony is preferable. The colony size is visually determined, and a wide variety of colonies from a colony having a small colony size to a colony having a large colony size are selected. Such selection may be performed visually according to user&#39;s determination, or automatic determination also can be made according to an image analysis by storing the selection condition in advance. 
     There is a predetermined relationship between a cell colony area and a cell number included in the colony. The predetermined relationship differs for each cell line. As an example, the relationship between the colony area of a cell line A and the cell number included in the colony is indicated by a calibration curve L 2 A in  FIG. 6 . The relationship between the colony area of a cell line B and the cell number included in the colony is indicated by a calibration curve L 2 B in  FIG. 6 . The calibration curve L 2 A and the calibration curve L 2 B are collectively referred to as a second calibration curve L 2 . When the cell line is specified, it is possible to estimate the cell number included in the colony from the colony area of the cell line based on the second calibration curve L 2  shown in  FIG. 6 . The second calibration curve L 2  is registered (stored) in advance for each cell line in the incubator  11  of the present embodiment, and thereby, the incubator  11  estimates (calculates) the cell number included in the colony from the colony image. Specifically, with respect to a certain cell line, the relationship between the cell number and the colony area is measured based on a phase difference image and a fluorescence image which is an image of a fluorescently-stained cell line. The measured relationship between the cell number and the colony area is stored in advance as the second calibration curve L 2 . 
     An image used for measuring the cell number is, for example, an image of a cell in a colony captured in a state where the cell is fluorescently stained. Then, with respect to the acquired image, the cell number of cells indicating a predetermined brightness value is measured. At this time, it is possible to apply a smoothing process with respect to the brightness value data obtained from the image, and based on the data, it is possible to specify the cell indicating the predetermined brightness value as a cell to be counted. By controlling the process level when the smoothing process is performed, it is possible to adjust the sensitivity for specifying the cell. 
     [Calibration Curve (Third Calibration Curve) Indicating Relationship between Cell Colony Area and Cell Density Included in this Colony] 
     With reference to  FIG. 7 , the relationship between a cell colony area and the density of the cell included in the colony is described. 
       FIG. 7  is a graph showing an example of a relationship between a cell colony area and the density of the cell included in the colony. The density of the cell included in the colony increases in response to the cell maturity in accordance with the elapse of time. When the cell maturity reaches a certain maturity, the change with the elapse of time of the density of the cell included in the colony is decreased. This means that in accordance with the increase of the colony area, the cell number in the colony is increased, but the change of the area per individual one cell in the colony is decreased. 
     Accordingly, by observing the density of the cell included in the colony, it is possible to determine the cell maturity. 
     Specifically, the density of the cell included in the colony is represented by a relationship between the colony area and the area per one cell included in the colony. The colony proliferates over time, and therefore, the colony area increases. When the maturity of the cell included in the colony is increased, the inside of the colony becomes a packed state. That is, when the cell included in the colony is matured, the density of the cell inside the colony is increased. When the cell further proliferates in a state where the inside of the colony is packed, the density of the cell reaches an upper limit to suppress the increase of the density, and the colony area is increased. Accordingly, by observing the temporal change of the density of the cell inside the colony, it is possible to determine the maturity of the cell included in the colony. 
     The density of the cell included in the colony differs for each cell line. As an example, the relationship between the colony area of a cell line A and the area per one cell included in the colony is indicated by a calibration curve L 3 A in  FIG. 7 . The relationship between the colony area of a cell line B and the area per one cell included in the colony is indicated by a calibration curve L 3 B in  FIG. 7 . The calibration curve L 3 A and the calibration curve L 3 B are collectively referred to as a third calibration curve L 3 . When the cell line is specified, it is possible to determine the maturity of the cell based on the third calibration curve L 3  shown in  FIG. 7 . Specifically, with respect to the cell line A, when the area per one cell included in the colony reaches a threshold value ThA of the calibration curve L 3 A shown in  FIG. 7 , it is determined that the cell line A is matured. With respect to the cell line B, when the area per one cell included in the colony reaches a threshold value ThB of the calibration curve L 3 B shown in  FIG. 7 , it is determined that the cell line B is matured. 
     That is, the third calibration curve L 3  is registered (stored) in advance for each cell line in the incubator  11  of the present embodiment, and thereby, the incubator  11  determines the cell maturity from the colony image. Specifically, when a calibration curve is prepared, with respect to a certain cell line, the relationship between the colony area and the density of the cell included in the colony is measured based on a phase difference image and a fluorescence image which is an image of a fluorescently-stained cell line. The measured relationship between the colony area and the density of the cell included in the colony is stored in advance as the third calibration curve L 3 . 
     With reference back to  FIG. 1 , the configuration of the control device  41  is described. The control device  41  has a control unit  42 , a storage unit  43 , and an input unit  44 . 
     The storage unit  43  is formed of a hard disk, a non-volatile storage medium such as a flash memory, a volatile storage medium such as a DRAM and a SRAM, or the like. Management data regarding each incubation container  19  stored in the stocker  21 , data of an entire observation image captured by an imaging device, and data of a microscope image are stored in the storage unit  43 . A program executed by the control unit  42  is stored in the storage unit  43 . A variety of calculation results by the control unit  42  are temporarily stored in the storage unit  43 . 
     The management data described above includes (a) index data indicating an individual incubation container  19 , (b) a storage position of the incubation container  19  in the stocker  21 , (c) the type and shape (well plate, dish, flask, or the like) of the incubation container  19 , (d) the type (information by which a cell line is identified) of the cell incubated in the incubation container  19 , (e) an observation schedule of the incubation container  19 , (f) an imaging condition (a magnification of an objective lens, an observation point in the container, or the like) during a time lapse observation, and the like. With respect to the incubation container  19  in which cells can be incubated simultaneously at a plurality of small containers such as a well plate, each of the management data is generated for each of the plurality of small containers. 
     In the present example, different types of cell lines are observed as the cell line to be observed. In this case, information by which the cell line is identified is required. However, when the cell line which is observed is one cell line, and it is unnecessary to identify the cell line, the cell line identification information is not essential. Even when the cell line which is observed is one cell line, information indicating the cell line may be input. 
     The colony area and calibration curve information indicating the relationship between the colony area and the number of cells included in the colony are associated with each other and stored in the storage unit  43 . 
     When different types of cell lines are observed, cell line information by which a cell line of a cell is identified is stored in the storage unit  43  and is preferably associated with each of information to be stored. Feature amount information indicating a feature amount of the colony area is stored and is preferably associated with each of information to be stored. 
     The input unit  44  includes an input device such as a keyboard and a mouse. A variety of information such as cell line information are input to the input unit  44  according to the operation of the user. 
     Next, with reference to  FIG. 2 , the configuration of the control unit  42  is described. The control unit  42  includes an image read unit  4203 , an area calculation unit  4211 , a write control unit  4221 , a cell number calculation unit  4222 , and a density calculation unit  4223 . 
     In  FIG. 2 , a case in which it is determined whether the colony is good or bad based on the first calibration curve L 1  is supposed, and a configuration in which a quality determination unit  4224  is included is shown. In  FIG. 2 , a case in which the cell maturity is determined based on the third calibration curve L 3  is supposed, and a configuration in which a maturity determination unit  4225  is included is shown. In the incubator  11  of the present embodiment, the quality determination unit  4224  and the maturity determination unit  4225  are not essential. 
     The control unit  42  is, for example, a processor that performs a variety of calculation processes of the control device  41 . The control unit  42  may function as each of the image read unit  4203 , the area calculation unit  4211 , the quality determination unit  4224 , the maturity determination unit  4225 , the write control unit  4221 , and the cell number calculation unit  4222  by executing a program. 
     The write control unit  4221  controls writing, in the storage unit  43 , of information output by each unit of the control device  41 . 
     The image read unit  4203  reads the image data of the microscope image or the entire observation image captured by the imaging device  34  and supplies the read image data to each unit of the control device  41 . The image read unit  4203  reads the image data of the microscope image or the entire observation image stored in the storage unit  43  and supplies the read image data to each unit of the control device  41 . 
     In two cases which are a case in which the first calibration curve L 1  is stored in the storage unit  43  and a case in which it is determined whether the colony is good or bad based on the first calibration curve L 1  and the colony image captured by the imaging device  34 , the area calculation unit  4211  calculates the colony area. Here, first, the case in which the first calibration curve L 1  is stored in the storage unit  43  is described, and then, the case in which it is determined whether the colony is good or bad based on the first calibration curve L 1  is described. 
     The area calculation unit  4211  calculates the colony area and causes the storage unit  43  via the write control unit  4221  to store, as calibration curve information, the first calibration curve L 1  in which the calculated colony area and the number of cells included in the colony are associated with each other. That is, the area calculation unit  4211  causes the storage unit  43  to store the first calibration curve L 1 . In case of a plurality of cell lines, the storage unit  43  is caused to store cell line information (cell line ID) via the input unit  44 , and the storage unit  43  is caused to store calibration curve information for each cell line. 
     In case of a plurality of types of cell lines, it is required to input information (cell line ID) indicating a cell line type. With respect to the input of information indicating a cell line type, the user understanding the types of observed cell lines may input the information. Further, by using a technology in which the cell line type is automatically determined using a matching technique or the like of identifying a cell according to the morphology, brightness, and the like of the observed cell, the information indicating a cell line type can be also automatically generated to be input. 
     In the present example, a case in which the user inputs the information (cell line ID) indicating the cell line via the input unit  44  is described. 
     The area calculation unit  4211  calculates the colony area based on the image in which the cell colony is captured. A specific example of the image of which the colony area is calculated by the area calculation unit  4211  is described with reference to  FIG. 8 . 
       FIG. 8  is a schematic diagram showing an example of an entire observation image captured by the imaging device  34  of the present embodiment. Among the entire observation image shown in  FIG. 8 , an entire observation image PIC 01  is an image (frame  1 ) of a colony detection result after one day elapses from the start of incubation. Entire observation image PIC 02  to entire observation image PIC 05  (frame  1  to frame  5 ) are images of colony detection results after two to five days elapse from the start of incubation. 
     The entire observation image PIC 01  includes an image of a first colony CO 1  and an image of a second colony CO 2  after one day elapses from the start of incubation. The entire observation image PIC 02  includes an image of the first colony CO 1  and an image of the second colony CO 2  after two days elapse from the start of incubation. As shown in  FIG. 8 , the first colony CO 1  after two days elapse from the start of incubation proliferates compared to the first colony CO 1  after one day elapses from the start of incubation, and the area is increased. As shown in  FIG. 8 , the second colony CO 2  after two days elapse from the start of incubation proliferates compared to the second colony CO 2  after one day elapses from the start of incubation, and the area is increased. 
     The entire observation image PIC 03  includes an image of the first colony CO 1 , an image of the second colony CO 2 , and an image of a third colony CO 3  after three days elapse from the start of incubation. As shown in  FIG. 8 , the first colony CO 1  after three days elapse from the start of incubation proliferates compared to the first colony CO 1  after two days elapse from the start of incubation, and the area is increased. As shown in  FIG. 8 , the second colony CO 2  after three days elapse from the start of incubation proliferates compared to the second colony CO 2  after two days elapse from the start of incubation, and the area is increased. 
     The temporal change of the colony area is described by sorting the entire observation image PIC 01  to the entire observation image PIC 03  in a time series and comparing the images. In this case, the area of the first colony CO 1  monotonously increases in the entire observation image PIC 01  to the entire observation image PIC 03 . On the other hand, with respect to the area of the second colony CO 2 , the increase amount in the entire observation image PIC 01  to the entire observation image PIC 02  greatly differs from the increase amount in the entire observation image PIC 02  to the entire observation image PIC 03 . That is, the increase amount of the area of the second colony CO 2  in the entire observation image PIC 02  to the entire observation image PIC 03  is larger than the increase amount of the area of the second colony CO 2  in the entire observation image PIC 01  to the entire observation image PIC 02 . That is, the area of the second colony CO 2  drastically increases after three days elapse from the start of incubation. This indicates that there is a possibility that the second colony CO 2  differentiates after three days elapse from the start of incubation and abnormally proliferates. 
     With reference to  FIG. 9 , the relationship between the temporal change of the colony area and the first calibration curve is described. 
       FIG. 9  is a graph showing an example of the relationship between the temporal change of the colony area and the first calibration curve of the present embodiment. In  FIG. 9 , the horizontal axis represents time, and the vertical axis represents colony area.  FIG. 9(A)  shows the area of the colony included in the entire observation image PIC 01  obtained by acquiring the image of the colony. That is,  FIG. 9(A)  shows the area of the first colony CO 1  and the area of the second colony CO 2  after one day elapses from the start of incubation. As shown in  FIG. 9(A) , both an area CO 11  of the first colony CO 1  and an area CO 21  of the second colony CO 2  are plotted on the first calibration curve L 1 . That is, after one day elapses from the start of incubation, both the area CO 11  of the first colony CO 1  and the area CO 21  of the second colony CO 2  show a normal value. 
     Each of  FIG. 9(B)  to (E) shows the area of the colony included in each of the entire observation image PIC 02  to the entire observation image PIC 05  obtained by acquiring the image of the colony.  FIG. 9(B)  shows an area CO 12  of the first colony CO 1  and an area CO 22  of the second colony CO 2  after two days elapse from the start of incubation. As shown in  FIG. 9(B) , both the area CO 12  of the first colony CO 1  and the area CO 22  of the second colony CO 2  are plotted on the first calibration curve L 1 . That is, after two days elapse from the start of incubation, both the area CO 12  of the first colony CO 1  and the area CO 22  of the second colony CO 2  show a normal value. 
       FIG. 9(C)  shows an area CO 13  of the first colony CO 1  and an area CO 23  of the second colony CO 2  after three days elapse from the start of incubation. As shown in  FIG. 9(C) , the area CO 13  of the first colony CO 1  is plotted on the first calibration curve L 1 . On the other hand, the area CO 23  of the second colony CO 2  is not plotted on the first calibration curve L 1 . That is, after three days elapse from the start of incubation, the area CO 12  of the first colony CO 1  shows a normal value, and the area CO 23  of the second colony CO 2  shows an abnormal value. This indicates that there is a possibility that the second colony CO 2  differentiates after three days elapse from the start of incubation and abnormally proliferates. That is, it is possible to determine whether the colony area is a normal value or is an abnormal value based on whether or not the colony area is present on the first calibration curve L 1 . In this example, since the second colony CO 2  is in a state where the colony area is greater than that of a normal colony, and there is a possibility that the second colony CO 2  differentiates after three days elapse from the start of incubation and abnormally proliferates, the subsequent incubation of the second colony CO 2  is stopped (for example, remove the second colony CO 2  from the culture medium). 
     In the present embodiment, the quality determination unit  4224  determines whether the colony is good or bad. That is, the quality determination unit  4224  determines whether the colony is good or bad based on the change of the colony area according to the elapse of time calculated by the area calculation unit  4211 . Specifically, the quality determination unit  4224  determines whether the colony is good or bad based on the colony area calculated by the area calculation unit  4211  and the first calibration curve L 1  stored in the storage unit  43 . 
     With reference back to  FIG. 8 , after three days elapse from the start of incubation, the third colony CO 3  that has not been present before arises. This is because an iPS cell is different from an ordinary cell and the timing of adhesion is non-uniform. Specifically, the first colony CO 1  and the second colony CO 2  adhere and proliferate after one day elapses from the start of incubation. On the other hand, the third colony CO 3  does not adhere until two days elapse from the start of incubation and adhere after three days elapse from the start of incubation to start proliferating. In this way, the imaging device  34  generates an entire observation image by imaging multiple times in accordance with the elapse of time from the start of incubation. 
     An entire observation image PIC 04  (frame  4 ) includes an image of the first colony CO 1 , an image of the second colony CO 2 , and an image of the third colony CO 3  after four days elapse from the start of incubation. As shown in  FIG. 8 , the first colony CO 1  after four days elapse from the start of incubation proliferates compared to the first colony CO 1  after three days elapse from the start of incubation, and the area is increased. As shown in  FIG. 8 , the second colony CO 2  after four days elapse from the start of incubation proliferates compared to the second colony CO 2  after two days elapse from the start of incubation, and the area is increased. 
     That is, the imaging device  34  captures a time lapse image from the start of incubation. Thereby, the incubator  11  of the present embodiment can observe the state of colony with good accuracy even with respect to a cell of which the timing of adhesion is non-uniform such as an iPS cell. 
     With reference back to  FIG. 2 , the area calculation unit  4211  acquires the entire observation image captured by the imaging device  34  from the image read unit  4203 . The area calculation unit  4211  generates a colony detection result image (the entire observation image PIC 01  to the entire observation image PIC 05 ) shown in  FIG. 8  based on the entire observation image which was acquired. 
     The area calculation unit  4211  calculates the colony area of a cell based on the generated colony detection result image. Specifically, the area calculation unit  4211  masks a colony part using an object detection algorithm according to a known learning function, determines the masked part (region surrounded by a curve in the colony detection result image of  FIG. 8 ) as a region where the colony is present, and calculates the colony area from the masked region. The calculation method of the colony area is not limited thereto. 
     When the user inputs information (cell line ID) indicating a cell line via the input unit  44 , the cell number calculation unit  4222  searches calibration curve information stored by the storage unit  43  using the cell line ID as a search key and acquires the second calibration curve L 2  (relationship between the colony area and the cell number) which the cell line ID matches based on the search result. The cell number calculation unit  4222  calculates the cell number based on the colony area calculated by the area calculation unit  4211  using the acquired second calibration curve L 2 . That is, the cell number calculation unit  4222  calculates the number of cells included in the target colony of which the area is calculated by the area calculation unit  4211  based on the image captured by the imaging device  34 . 
     The density calculation unit  4223  calculates the density of cells included in the colony based on the colony area calculated by the area calculation unit  4211  and the number of cells included in the colony calculated by the cell number calculation unit  4222 . Specifically, the density calculation unit  4223  calculates the area per one cell included in the colony based on the colony area calculated by the area calculation unit  4211  and the cell number calculated by the cell number calculation unit  4222 . 
     The maturity determination unit  4225  determines the maturity of the cell included in the colony based on the density of the cell calculated by the density calculation unit  4223 . Specifically, with respect to the cell line A, when the area per one cell included in the colony reaches the threshold value ThA of the calibration curve L 3 A shown in  FIG. 8 , the maturity determination unit  4225  determines that the cell line A is matured. With respect to the cell line B, when the area per one cell included in the colony reaches the threshold value ThB of the calibration curve L 3 B shown in  FIG. 8 , the maturity determination unit  4225  determines that the cell line B is matured. 
     [Operation of Incubator (Observation Apparatus)] 
     Next, an example of an operation of the incubator  11  is described. The incubator  11  estimates the number of cells included in the colony from the cell colony area based on the registered calibration curve to thereby calculate the number of cells. Here, the calibration curve is information indicating the relationship between the cell colony area and the number of cells included in the colony. First, an operation in which the calibration curve is registered is described, and then, an operation in which the number of cells is calculated based on the registered calibration curve is described. 
     [Operation of Calibration Curve Registration] 
     First, an operation of calibration curve registration is described with reference to  FIG. 10 . 
       FIG. 10  is a flowchart showing an example of an operation of calibration curve registration by the incubator  11  (observation apparatus) of the present embodiment. The incubator  11  stores a calibration curve (first calibration curve L 1 , second calibration curve L 2 , and third calibration curve L 3 ) for each cell line. 
     As an example, based on the image data described above, the incubator  11  detects a colony image in the image data and calculates the area of the colony image. The incubator  11  associates the calculated colony area with the elapsed time from the start of incubation for each cell line to thereby register a first calibration curve L 1  with respect to a certain cell line. 
     The incubator  11  associates the calculated colony area with the number of cells in the colony counted according to a fluorescently-stained observation image for each cell line to thereby register a second calibration curve L 2  with respect to a certain cell line as calibration curve information. 
     The incubator  11  associates the calculated colony area with an area per one cell in the colony for each cell line to thereby register a third calibration curve L 3  with respect to a certain cell line as calibration curve information. 
     Before the operation start of calibration curve registration, the control unit  42  preliminarily accepts a command of a calibration curve registration operation input via the input unit  44  by the user. The command of the calibration curve registration operation includes information (cell line ID) indicating a cell line of which the calibration curve is to be registered (when the observed cell line is one cell line, the input of information (cell line ID) indicating a cell line of which the calibration curve is to be registered is not essential). The storage unit  43  preliminarily stores observation start times as an observation schedule of management data such that an observation is started after each day elapses from the elapse of one day since the start of incubation until the elapse of five days. When the method by which the user counts the number of cells in the colony is a method in which a cell in the incubation container  19  is destructed and is observed such as a counting method in which cells are fluorescently-stained and are counted according to a fluorescent observation, incubation containers  19  are prepared corresponding to the number of observation times. For example, when the observation is performed after the elapse of each of one day, two days, and three days from the start of incubation, at least three incubation containers  19  are stored in the stocker  21  of the constant temperature room  15 . The same cell line is incubated in each of the incubation containers  19 . By observing the incubation containers  19  one by one per one day, the cell line after the elapse of one day from the start of incubation to the cell line after the elapse of five days from the start of incubation are observed. The specific operation of preparation of the calibration curve performed by the incubator  11 , that is, calculation of the colony area and registration of the calibration curve is described below. In the present embodiment, the following operation is performed by the control unit  42  included in the incubator  11 ; however, the following operation may be performed by a control unit externally provided on the incubator  11 . 
     Step S 101 : The control unit  42  determines whether or not the observation start time of the incubation container  19  has come by comparing the observation schedule of the management data of the storage unit  43  and the current date and time. When it is the observation start time (YES), the control unit  42  forwards the process to Step S 102 . On the other hand, when it is not the observation time (NO), the control unit  42  waits until the next observation schedule time. 
     Step S 102 : The control unit  42  commands the container carry device  23  to carry the incubation container  19  corresponding to the observation schedule. Then, the container carry device  23  carries out the commanded incubation container  19  from the stocker  21  and places the incubation container  19  on the sample table  31  of the observation unit  22 . At the timing when the incubation container  19  is placed on the sample table  31 , the entire observation image of the incubation container  19  is captured by a bird view camera (not shown) embedded in the stand arm  32 . Thereby, the image of the incubation container  19  including a colony image is captured. As described above, the stocker  21  is not essential. When there is no stocker  21 , the step regarding the container carry is unnecessary. 
     Step S 103 : The image read unit  4203  of the control unit  42  stores the entire observation image captured in Step S 102 . The area calculation unit  4211  of the control unit  42  detects a colony image from the stored entire observation image and calculates the sum of areas of colony cells in the incubation container  19  or the sum of areas of colonies as the colony area. 
     When the calibration curve is prepared, the image of the incubation container including the colony image may be acquired, the cell number of each colony may be counted (or the cell number of each colony may be summed) from the fluorescent observation image, and the colony area may be calculated from the phase difference image. Alternatively, the total number of cells in each colony may be counted by a hemocytometer, and the colony area may be calculated from the phase difference image. 
     Step S 104 : The control unit  42  commands the container carry device  23  to carry the incubation container  19  to the small door  18  after the observation schedule is finished. Then, the container carry device  23  carries the commanded incubation container  19  to the position of the small door  18  from the sample table  31  of the observation unit  22 . 
     Step S 105 : The user opens the small door  18  and takes out the incubation container  19 . The user counts the number of cells according to a known method with respect to the incubation container  19  which is taken out. For example, the user counts the number of cells according to fluorescence stain. The user inputs the counted number of cells to the input unit  44 . Here, the user calculates the cell density in the colony and inputs the calculated density to the input unit  44 . 
     Step S 106 : The write control unit  4221  of the control unit  42  associates the cell number in the colony and the cell density in the colony input to the input unit  44  with the colony area calculated in Step S 103  and the information (cell line ID) indicating the cell line to be stored in the storage unit  43 . Thereby, the colony area, the cell number, and the cell line ID are associated with one another and stored in the storage unit  43  as calibration curve information (in a case of observation of one cell line, the association with the cell line ID is not essential). Then, the control unit  42  finishes the observation sequence and causes the process to return to Step S 101 . 
     The calibration curve information is stored in the storage unit  43  by repeating Step S 101  to Step S 106  in this way. By repeating Step S 101  to Step S 106  with respect to a plurality of cell lines, the calibration curve information with respect to each of the plurality of cell lines is stored in the storage unit  43 . Specifically, by repeating Step S 101  to Step S 106  with respect to each of the cell line A and the cell line B, each of the calibration curve information of the cell line A and the calibration curve information of the cell line B is stored in the storage unit  43 . 
     [Operation of Cell Number Estimation and Cell Quality Determination] 
     Next, with reference to  FIG. 11 , an example of the cell number estimation operation of the incubator  11  is described. 
       FIG. 11  is a flowchart showing an example of a cell number estimation operation according to the incubator  11  (observation apparatus) of the present embodiment. In the present example, data (referred to as contrast data) regarding the number of cells against the change of the colony area of a specific cell line which is to be an observation target is acquired and is stored in advance. From the data, the determination of the quality of the observed cell is in an unknown state. 
     The incubator  11  performs a time lapse observation of the incubation container  19  carried in the constant temperature room  15  in accordance with the registered observation schedule. A plurality of specific types of cell lines are incubated in the incubation container  19 . For example, the cell line A is incubated in an incubation container  19 A of the incubation containers  19 . The cell line B is incubated in an incubation container  19 B of the incubation containers  19 . In accordance with the observation schedule, the incubator  11  sequentially carries the incubation container  19 A and the incubation container  19 B to the vertical robot  38  and the observation unit  22  and captures an entire image (entire observation image) of the incubation container  19  and a microscope image in which part of the incubation container  19  is magnified. According to the registration sequence of the calibration curve information described above, the calibration curve information of the cell line A and the calibration curve information of the cell line B are stored in the storage unit  43  in advance. The operation of cell number estimation in the time lapse observation of the incubator  11  is described. 
     Step S 201 : The control unit  42  determines whether or not the observation start time of the incubation container  19  has come by comparing the observation schedule of the management data of the storage unit  43  and the current date and time. When it is the observation start time (YES), the control unit  42  forwards the process to Step S 202 . On the other hand, when it is not the observation time of the incubation container  19  (NO), the control unit  42  waits until the next observation schedule time. 
     Step S 202 : The control unit  42  commands the container carry device  23  to carry the incubation container  19  corresponding to the observation schedule. Then, the container carry device  23  carries out the commanded incubation container  19  from the stocker  21  and places the incubation container  19  on the sample table  31  of the observation unit  22 . At the timing when the incubation container  19  is placed on the sample table  31 , the entire observation image of the incubation container  19  is captured by a bird view camera (not shown) embedded in the stand arm  32 . 
     In the present example, the observation apparatus shown in  FIGS. 1, 3, and 4  is an apparatus including a constant temperature room in which a cell to be observed is incubated. Accordingly, the cell imaged in Step S 202  is a cell incubated in a constant temperature room of the observation apparatus. Accordingly, the present step can be also started from the step of incubating a cell. The apparatus may not be an apparatus in which the constant temperature room is included in the observation apparatus like the present example. The apparatus may be an apparatus in which the constant temperature room for incubating a cell is separated from the observation apparatus. 
     Step S 203 : The cell number calculation unit  4222  that calculates the cell number in the colony acquires the information (cell line ID) indicating the cell line from the management data stored in the storage unit  43 . 
     Step S 204 : The cell number calculation unit  4222  searches calibration curve information stored by the storage unit  43  using the acquired cell line ID as a search key and acquires calibration curve information which the cell line ID matches. 
     Step S 205 : The image read unit  4203  acquires an image captured in Step S 202 . The image includes an image of a colony. 
     Step S 206 : The area calculation unit  4211  calculates, based on the image acquired in Step S 205 , the area of the colony included in the image. 
     Step S 207 : The cell number calculation unit  4222  estimates (calculates) the number of cells based on the area of the colony calculated in Step S 206  and a second calibration curve L 2  of the calibration curve information acquired in Step S 204 . 
     The area calculation unit  4211  calculates the area of the recognized colony in the acquired image. 
     This step may be performed when a state in which observation can be made is realized after the incubation container is carried to the observation unit (S 202 ). In this case, after the colony area is calculated, the information indicating a cell line is acquired. 
     Step S 208 : The density calculation unit  4223  calculates a cell density based on the area of the colony calculated in Step S 206  and the number of cells calculated in Step S 207 . 
     Step S 209 : When the quality determination unit  4224  is included, the quality determination unit  4224  determines whether the colony is good or bad based on the colony area calculated from the acquired image and a first calibration curve L 1  of the calibration curve information acquired in Step S 204 . When the quality determination unit  4224  is not included, the user determines whether the colony is good or bad based on the calculated colony area and the first calibration curve L 1  of the calibration curve information acquired in Step S 204 . 
     When the maturity determination unit  4225  is included, the maturity determination unit  4225  determines the cell maturity based on the colony area calculated in Step S 206 , the cell density calculated in Step S 208 , and a third calibration curve L 3  of the calibration curve information acquired in Step S 204  by using the acquired image. When the maturity determination unit  4225  is not included, the user determines the cell maturity based on the colony area calculated in Step S 206 , the cell density calculated in Step S 208 , and the third calibration curve L 3  of the calibration curve information acquired in Step S 204 . 
     Step S 210 : The control unit  42  commands the container carry device  23  to carry the incubation container  19  after the observation schedule is finished. Then, the container carry device  23  carries the commanded incubation container  19  from the sample table  31  of the observation unit  22  to a predetermined storage position of the stocker  21 . Then, the control unit  42  finishes the observation sequence and causes the process to return to S 201 . 
     In addition, after it can be understood whether or not a cell is in a good state as a result of determination in Step S 209 , it is possible to take out a cell which is determined that the cell is in a good state from the observation apparatus and, for example, supply the cell as a cell used for drug discovery research or regenerative medicine. 
     By repeating Step S 201  to Step S 210  for each cell line in this way, the estimation result of the number of cells in each observation schedule is stored in the storage unit  43 . Further, it is possible to determine whether the cell is good or bad based on the estimation result of the number of cells. Further, it is possible to supply only the selected good cell to a research institute or the like. 
     As described above, the incubator  11  (observation apparatus) of the present embodiment includes the cell number calculation unit  4222  that calculates, based on the calibration curve information and the colony area on the basis of the non-invasively obtained image, the number of cells included in the colony. Thereby, the incubator  11  can calculate the number of cells according to a non-invasive method and can improve the accuracy of the calculated number of cells. Further, an image obtained by a non-invasive observation (for example, phase difference observation) is used for the maturity determination or quality determination, and therefore, the determined cell can be subsequently used without obstacle for drug discovery research or regenerative medicine as a subsequent process. 
     The control device  41  may treat a plurality of microscope images captured during the same period of observation time of a plurality of points (for example, five points of observation or the entire incubation container  19 ) of the same incubation container  19  as an image of one time of the time lapse observation. 
     The embodiment is described using an example in which the area calculation unit  4211  detects the colony image based on the entire observation image; however, the embodiment is not limited thereto. The area calculation unit  4211  may detect the colony image by image processing of a phase difference microscope image. 
     A program for executing each process of the incubator  11  (observation apparatus) according to the embodiments of the invention may be recorded in a computer-readable recording medium, and the program recorded in the recording medium may be read into and executed on a computer system to thereby perform a variety of processes described above. 
     It is assumed that the “computer system” used herein includes an OS or hardware such as peripherals. It is also assumed that the term “computer system” includes a homepage provision environment (or a display environment) when a WWW system is used. The term “computer-readable recording medium” refers to a recordable non-volatile memory such as a flexible disk, a magneto-optical disk, a ROM, or a flash memory, a portable medium such as a CD-ROM, or a storage device such as a hard disk embedded in the computer system. 
     It is also assumed that the term “computer-readable recording medium” includes a medium which holds a program for a given time such as a volatile memory (for example, a DRAM (Dynamic Random Access Memory)) in the computer system which becomes a server or a client when a program is transmitted through a network such as the Internet or a communication line such as a telephone line. The program may be transmitted from the computer system which stores the program in the storage device or the like to other computer systems through a transmission medium or through transmission waves in the transmission medium. The term “transmission medium” which transmits the program refers to a medium which has a function of transmitting information, for example, a network (communication network) such as the Internet or a communication line such as a telephone line. The program may be a program which can realize part of the above-described functions. The program may be a so-called differential file (differential program) which can realize the above-described functions by a combination with a program already recorded in the computer system. 
     Although the embodiments of the invention has been described in detail with reference to the drawings, a specific configuration is not limited to the embodiments, and designs or the like without departing from the scope of the invention are also included. 
     DESCRIPTION OF THE REFERENCE SYMBOLS 
     
         
         
           
               11 : Observation apparatus 
               41 : Control device 
               4211 : Area calculation unit 
               4222 : Cell number calculation unit 
               4223 : Density calculation unit 
               4224 : Quality determination unit 
               4225 : Maturity determination unit 
               43 : Storage unit