JP2014217353A - Observation apparatus, observation method, observation system, program thereof, and method for producing cells - Google Patents

Abstract

An object of the present invention is to improve the calculation accuracy of the number of cells. An observation apparatus includes an area calculation unit that calculates an area of a cell colony based on an image of a captured cell, and a calibration that indicates a relationship between the area of the cell colony and the number of cells included in the colony. A cell number calculating unit that calculates the cell number based on the line information and the area of the colony calculated by the area calculating unit. [Selection] Figure 2

Description

  The present invention relates to an observation apparatus, an observation method, an observation system, a program thereof, and a method for manufacturing a cell.

  In general, techniques for evaluating the culture state of cells have become fundamental technologies in a wide range of fields including advanced medical fields such as regenerative medicine and drug screening. For example, in the field of regenerative medicine, there are processes for growing and differentiating cells in vitro. And in said process, in order to manage the success or failure of cell differentiation, the canceration of a cell, and the presence or absence of infection, it is indispensable to evaluate the culture state of a cell exactly. As an example, a cancer cell evaluation method using a transcription factor as a marker has been disclosed (see Patent Document 1).

  Stem cells such as ES (Embryonic Stem) cells or iPS (induced Pluripotent Stem) cells theoretically proliferate almost infinitely while maintaining the pluripotency of differentiation into all tissues As a result, attention has been focused on pharmaceutical development and application to regenerative medicine.

U.S. Pat. No. 7,060,445

  When such stem cells are applied to regenerative medicine, it is necessary to culture stem cells in good condition. For this purpose, it is required to accurately calculate the number of stem cells in culture. However, conventionally, since the occupancy ratio of stem cells (the ratio occupied by cells in the culture vessel) has been calculated by a researcher's visual observation, there has been a problem that the exact number of cells cannot be calculated.

  Therefore, the present invention has been made in view of the above problems, and provides an observation apparatus, an observation method, an observation system, a program therefor, and a cell production method that can improve the calculation accuracy of the number of cells. Is an issue.

  In order to solve the above problem, an aspect of the present invention includes an area calculation unit that calculates the area of the colony of the cell based on the captured image of the cell, the area of the colony of the cell, and the colony. An observation apparatus comprising calibration curve information indicating a relationship with the number of cells and a cell number calculation unit that calculates the number of cells based on the area of the colony calculated by the area calculation unit.

  In order to solve the above problem, one embodiment of the present invention is an observation system including an imaging unit that captures an image of a cell in culture and the above-described observation device.

  In order to solve the above problem, one embodiment of the present invention includes an area calculation procedure for calculating an area of a colony of the cell based on an image in which the cell is imaged, an area of the colony, and an area included in the colony. Read out the calibration curve information indicating the relationship with the number of cells to be read, the calibration curve information read out in the readout procedure, and the area of the colony calculated in the area calculation procedure And a cell number calculating procedure for calculating the number.

  In order to solve the above-described problem, according to one embodiment of the present invention, the computer calculates an area calculation procedure for calculating the area of the colony of the cell based on an image obtained by imaging the cell, the area of the colony, Based on a reading procedure for reading calibration curve information indicating the relationship with the number of cells contained in the colony, the calibration curve information read in the reading procedure, and the area of the colony calculated in the area calculation procedure And a cell number calculation procedure for calculating the cell number.

  In order to solve the above-described problem, one embodiment of the present invention provides a cell culture procedure for culturing cells, images of cells cultured in the culture procedure, and the cells based on an image obtained by imaging the cells. An area calculation procedure for calculating the area of the colony, a readout procedure for reading out calibration curve information indicating the relationship between the area of the colony and the number of cells contained in the colony, and the calibration curve read out in the readout procedure A procedure for calculating the number of cells based on information and the area of the colony calculated in the area calculation procedure, and a determination procedure for determining the state of the imaged cell with reference to the calibration curve information And a method for producing a cell.

  According to the present invention, the calculation accuracy of the number of cells can be improved.

It is a block diagram which shows an example of a structure of the observation apparatus by the 1st Embodiment of this invention. It is a block diagram which shows an example of a structure of the control apparatus with which the observation apparatus of this embodiment is provided. It is a front view of the observation apparatus of this embodiment. It is a top view of the observation apparatus of this embodiment. It is a graph which shows an example of the relationship between the colony area for every cell strain of this embodiment, and the number of cells. It is a schematic diagram which shows an example of the whole observation image imaged by the imaging device of this embodiment. It is a schematic diagram which shows an example of the image in which the predetermined relationship is not materialized between the colony area of this embodiment, and the number of cells. It is a graph which shows an example of the relationship between the colony area and cell number for every cell strain in the case where the feature-value of this embodiment is normal, and the case of abnormality. It is a schematic diagram which shows an example of the whole observation image imaged by the imaging device of this embodiment. It is a schematic diagram which shows an example of the analysis process by the co-occurrence matrix which the feature-value calculation part of this embodiment calculates. It is a schematic diagram which shows an example of the co-occurrence matrix which the feature-value calculation part of this embodiment calculates. It is a flowchart which shows an example of the operation | movement of the calibration curve registration by the observation apparatus of this embodiment. It is a flowchart which shows an example of the operation | movement of cell number estimation by the observation apparatus of this embodiment. It is a block diagram which shows an example of a structure of the observation apparatus which concerns on the 2nd Embodiment of this invention. It is a schematic diagram which shows an example of the plot screen of the feature-value which concerns on the modification of this invention.

[First Embodiment]
Embodiments of the present invention will be described below with reference to the drawings. First, the outline of the configuration of the incubator (observation apparatus) according to the first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a block diagram showing an example of the configuration of the incubator 11 according to the first embodiment of the present invention. FIG. 2 is a block diagram illustrating an example of the configuration of the control device 41 included in the incubator 11 according to the present embodiment. Moreover, FIG. 3, FIG. 4 is the front view and top view of the incubator 11 of this embodiment.

  The incubator 11 is an apparatus for culturing cells and imaging the cultured cells with a microscope camera to observe the state of the cells. The incubator 11 has an upper casing 12 and a lower casing 13. In the assembled state of the incubator 11, the upper casing 12 is placed on the lower casing 13. Note that the internal space between the upper casing 12 and the lower casing 13 is vertically divided by a base plate 14.

  First, an outline of the configuration of the upper casing 12 will be described. A constant temperature chamber 15 for culturing cells is formed inside the upper casing 12. The temperature-controlled room 15 includes a temperature adjusting device 15a and a humidity adjusting device 15b, and the temperature-controlled room 15 is maintained in an environment suitable for cell culture (for example, an atmosphere having a temperature of 37 ° C. and a humidity of 90%) ( In addition, illustration of the temperature adjusting device 15a and the humidity adjusting device 15b in FIGS. 3 and 4 is omitted).

  A large door 16, a middle door 17 and a small door 18 are arranged on the front surface of the temperature-controlled room 15. The large door 16 covers the front surfaces of the upper casing 12 and the lower casing 13. The middle door 17 covers the front surface of the upper casing 12 and isolates the environment between the temperature-controlled room 15 and the outside when the large door 16 is opened. The small door 18 is a door for carrying in and out a culture vessel 19 for culturing cells, and is attached to the middle door 17. It is possible to suppress environmental changes in the temperature-controlled room 15 by carrying the culture container 19 in and out of the small door 18. The large door 16, the middle door 17, and the small door 18 are maintained airtight by the packings P1, P2, and P3, respectively.

  In the temperature-controlled room 15, a stocker 21, an observation unit 22, a container transfer device 23, and a transfer table 24 are arranged. Here, the conveyance stand 24 is disposed in front of the small door 18, and carries the culture container 19 in and out of the small door 18.

  The stocker 21 is disposed on the left side of the temperature-controlled room 15 when viewed from the front surface of the upper casing 12 (the lower side in FIG. 4). The stocker 21 has a plurality of shelves, and each shelf of the stocker 21 can store a plurality of culture vessels 19. Each culture container 19 contains cells to be cultured together with a medium.

  The observation unit 22 is disposed on the right side of the temperature-controlled room 15 when viewed from the front surface of the upper casing 12. The observation unit 22 can execute time-lapse observation of cells in the culture vessel 19.

  Here, the observation unit 22 is disposed by being fitted into the opening of the base plate 14 of the upper casing 12. The observation unit 22 includes a sample stage 31, a stand arm 32 projecting above the sample stage 31, and a main body portion 33 containing a microscopic optical system for phase difference observation and an imaging device 34. The sample stage 31 and the stand arm 32 are disposed in the temperature-controlled room 15, while the main body portion 33 is accommodated in the lower casing 13.

  The sample stage 31 is made of a translucent material, and the culture vessel 19 can be placed thereon. The sample stage 31 is configured to be movable in the horizontal direction, and the position of the culture vessel 19 placed on the upper surface can be adjusted. The stand arm 32 includes an LED light source 39. And the imaging device 34 can acquire the microscope image of a cell by imaging the cell of the culture container 19 permeate | transmitted and illuminated by the stand arm 32 from the upper side of the sample stand 31 via a microscopic optical system.

  The container transport device 23 is disposed in the center of the temperature-controlled room 15 when viewed from the front surface of the upper casing 12. The container transport device 23 delivers the culture container 19 between the stocker 21, the sample table 31 of the observation unit 22, and the transport table 24.

  As shown in FIG. 4, the container transport device 23 includes a vertical robot 38 having an articulated arm, a rotary stage 35, a mini stage 36, and an arm unit 37. The rotary stage 35 is attached to the tip of the vertical robot 38 so as to be capable of rotating 180 ° in the horizontal direction via a rotary shaft 35a. Therefore, the rotary stage 35 can make the arm portions 37 face the stocker 21, the sample table 31, and the transport table 24.

  The mini stage 36 is attached to the rotation stage 35 so as to be slidable in the horizontal direction. An arm part 37 that holds the culture vessel 19 is attached to the mini stage 36.

  Next, the outline of the configuration of the lower casing 13 will be described. Inside the lower casing 13, the main body portion 33 of the observation unit 22 and the control device 41 of the incubator 11 are accommodated.

  The control device 41 is connected to the temperature adjustment device 15a, the humidity adjustment device 15b, the observation unit 22, and the container transport device 23, respectively. The control device 41 comprehensively controls each part of the incubator 11 according to a predetermined program.

  As an example, the control device 41 controls the temperature adjustment device 15a and the humidity adjustment device 15b, respectively, to maintain the inside of the temperature-controlled room 15 at a predetermined environmental condition. The control device 41 controls the observation unit 22 and the container transport device 23 based on a predetermined observation schedule, and automatically executes the observation sequence of the culture vessel 19. Furthermore, the control device 41 executes a culture state evaluation process for evaluating the culture state of the cells based on the image acquired in the observation sequence.

[Relationship between cell colony area and cell count]
Here, with reference to FIG. 5, the relationship between the cell colony area and the number of cells will be described.
FIG. 5 is a graph showing an example of the relationship between the colony area and the number of cells for each cell line. There is a predetermined relationship between the cell colony area and the number of cells contained in the colony. Further, this predetermined relationship differs for each cell line. As an example, the relationship between the colony area of the cell line A and the number of cells contained in the colony is indicated by a calibration curve L1 in FIG. Further, the relationship between the colony area of the cell line B and the number of cells contained in the colony is indicated by a calibration curve L2 in FIG. Further, the relationship between the colony area of the cell line C and the number of cells contained in this colony is indicated by a calibration curve L3 in FIG. Therefore, if a cell line is specified, the number of cells contained in this colony can be estimated from the colony area of this cell line based on the predetermined relationship shown in FIG. The incubator 11 of the present embodiment estimates (calculates) the number of cells included in the colony from the colony image by registering (storing) the predetermined relationship, that is, the calibration curve L, in advance for each cell line. To do.

  Returning to FIG. 1, the configuration of the control device 41 will be described. The control device 41 includes a control unit 42, a storage unit 43, and an input unit 44.

  The storage unit 43 includes a non-volatile storage medium such as a hard disk or a flash memory, and a volatile storage medium such as a DRAM or SRAM. The storage unit 43 stores management data related to each culture vessel 19 stored in the stocker 21, data of the entire observation image captured by the imaging device, and data of the microscope image. Further, the storage unit 43 stores a program executed by the control unit 42. The storage unit 43 temporarily stores various calculation results by the control unit 42.

  The management data includes (a) index data indicating individual culture containers 19, (b) the storage position of the culture container 19 in the stocker 21, and (c) the type and shape of the culture container 19 (well plate, (Dish, flask, etc.), (d) the type of cells cultured in the culture vessel 19 (information identifying the cell line), (e) the observation schedule of the culture vessel 19, and (f) imaging conditions (objective) during time-lapse observation Lens magnification, observation point in the container, etc.). In addition, for the culture container 19 that can simultaneously culture cells in a plurality of small containers such as a well plate, management data is generated for each small container.

  In this example, different types of cell lines are observed as cell lines to be observed. In this case, information for identifying the cell line is required. However, when there is only one cell line to be observed and it is not necessary to identify the cell line, the cell line identification information is not essential. Of course, even if one cell line is observed, information indicating the cell line may be input.

Further, the storage unit 43 stores therein calibration curve information indicating the relationship between the area of the colony and the number of cells included in the colony.
Moreover, when observing different types of cell lines, it is preferable to store cell line information for identifying cell lines of cells in the storage unit 43 and store them in association with each information. Further, it is preferable to store feature amount information indicating the feature amount of the area of the colony, and store the feature amount information in association with each piece of information.

  The input unit 44 includes input devices such as a keyboard and a mouse. Various information such as cell line information is input to the input unit 44 by user operation.

  Next, the configuration of the control unit 42 will be described with reference to FIG. The control unit 42 includes an image reading unit 4203, an area calculation unit 4211, a writing control unit, and a cell number calculation unit 4222. FIG. 2 illustrates a configuration including a feature amount calculation unit 4212 assuming that a feature amount of a colony area is calculated. The control unit 42 is, for example, a processor that executes various arithmetic processes of the control device 41. Note that the control unit 42 may function as an image reading unit 4203, an area calculating unit 4211, a feature amount calculating unit 4212, a writing control unit, and a cell number calculating unit 4222 by executing the program.

  The writing control unit controls writing of information output from each unit of the control device 41 to the storage unit 43.

  The image reading unit 4203 reads image data of a whole observation image or a microscope image captured by the imaging device 34 and supplies the read image data to each unit of the control device 41. The image reading unit 4203 reads the image data of the entire observation image or the microscope image stored in the storage unit 43 and supplies the read image data to each unit of the control device 41.

  The area calculation unit 4211 calculates the area of the cell colony based on the captured image of the cell. A specific example of an image for which the area calculation unit 4211 calculates the area of the colony will be described with reference to FIG.

  FIG. 6 is a schematic diagram illustrating an example of the entire observation image captured by the imaging device 34 of the present embodiment. Among the whole observation images shown in FIG. 6, the whole observation image PIC01 is an original image before colony detection after one day has elapsed since the start of culture. The overall observation image PIC11 is an image of a colony detection result after one day has elapsed from the start of culture. The overall observation image PIC02 is an original image before colony detection after the lapse of 2 days from the start of culture. The overall observation image PIC12 is an image of a colony detection result after two days have elapsed from the start of culture. The overall observation image PIC03 is an original image before colony detection after 3 days from the start of culture. The overall observation image PIC13 is an image of a colony detection result after 3 days from the start of culture.

The area calculation unit 4211 acquires the original image (overall observation image PIC01 to overall observation image PIC03) shown in the upper part of FIG. 6 from the image reading unit 4203. Moreover, the area calculation part 4211 produces | generates the colony detection result image (whole observation image PIC11-whole observation image PIC13) shown in the lower stage of FIG. 6 based on the acquired original image.
The area calculation unit 4211 calculates the area of the cell colony based on the generated colony detection result image. Specifically, the area calculation unit 4211 masks the colony portion by using an object detection algorithm based on a known learning function, and the masked portion (rectangular region dotted in the colony detection result image of FIG. 6) is colonized. The area of the colony is calculated from the masked area. In addition, the calculation method of a colony area is not restricted to this.

  The area calculation unit 4211 associates the calculated area of the colony with the number of cells counted by the user and causes the storage unit 43 to store it as calibration curve information via the write control unit 4221. That is, the area calculation unit 4211 stores calibration curve information. When there are a plurality of cell lines, cell line information (cell line ID) is stored in the storage unit 43 via the input unit 44, and calibration curve information is stored for each cell line.

When calculating the feature amount of the colony image, the feature amount calculation unit 4212 is required.
The feature amount calculation unit 4212 calculates the feature amount of the colony image included in the image based on the image data of the entire observation image read by the image reading unit 4203 or the image data of the microscope image. That is, the feature amount calculation unit 4212 calculates the feature amount of the colony image based on the image. The feature amount is an amount indicating a light and shade pattern of pixels constituting the image. This feature amount is also referred to as a texture in the following description.

Here, the feature amount of the colony image will be described with reference to FIG.
FIG. 7 is a schematic diagram illustrating an example of an image in which a predetermined relationship is not established between the colony area and the number of cells. The colony image included in the entire observation image or the microscope image captured by the imaging device 34 may include an image PIC1 in which a predetermined relationship is not established between the colony area and the number of cells. In the following description, a case where a predetermined relationship is not established between the colony area and the number of cells is also referred to as feature amount abnormality (or texture abnormality).

  FIG. 8 is a graph showing an example of the relationship between the colony area and the number of cells for each cell line when the feature amount is normal and abnormal. Of the calibration curve L shown in FIG. 8, the calibration curve L4 is a calibration curve when a predetermined relationship is established between the colony area and the number of cells, that is, when the feature value is normal. Of the calibration curve L shown in FIG. 8, the calibration curve L5 is a calibration curve when a predetermined relationship is not established between the colony area and the number of cells, that is, when the feature value is abnormal. As shown in FIG. 8, when there is an image PIC1 in which a predetermined relationship is not established between the colony area and the number of cells, the estimation accuracy in estimating the number of cells from the colony area based on the calibration curve L4 is high. May decrease. Therefore, when estimating the number of cells, if it is determined whether or not the image PIC1 is included in advance and the image PIC1 is excluded from the calculation for estimating the number of cells from the colony area, the estimation accuracy of the number of cells decreases. The degree can be reduced. There are various causes for the occurrence of the feature amount abnormality. Due to the difference in cause, various patterns as shown in FIG. 9 also appear in the shading pattern of the image PIC1.

  FIG. 9 is a schematic diagram illustrating an example of the entire observation image captured by the imaging device 34. Among these, FIG. 9A is an example of the entire observation image (image PIC0) when no feature amount abnormality has occurred. For example, when the cells are differentiated like EB in the process of culturing cells, an image (image PIC1A) in which a feature amount abnormality has occurred is obtained. In this image PIC1A, for example, as shown in FIG. 9B, a local black block-like image may appear in the image captured by the imaging device 34. For example, when the density of the cells in the colony is locally reduced, an image (image PIC1B) in which the feature amount abnormality has occurred is obtained. Specifically, as illustrated in FIG. 9C, the luminance pattern in the image captured by the imaging device 34 may be different from the luminance pattern in the normal case (FIG. 9A). is there.

  The feature amount calculation unit 4212 calculates the feature amount by analyzing the luminance pattern of the image captured by the imaging device 34. Specifically, the feature amount calculation unit 4212 obtains a co-occurrence matrix (GLCM: Gray Level Co-occurrence Matrix) for the image to be analyzed, thereby obtaining “uniformity”, “entropy”, “energy”, “inertia” of the image. ”And“ correlation ”are calculated. Here, the analysis using the co-occurrence matrix means, for example, the frequency at which the pixel value j is a pixel value j at a position separated by a certain displacement d = (r, θ) from the position of a certain target pixel (pixel value i). Is a method for calculating a feature value from the matrix. Here, r is a parameter indicating the distance between pixels. Θ is a parameter indicating the direction between pixels. A specific example will be described with reference to FIG.

  FIG. 10 is a schematic diagram illustrating an example of an analysis process using a co-occurrence matrix calculated by the feature amount calculation unit 4212 of this embodiment. As shown in FIG. 10A, the image PIC captured by the imaging device 34 is composed of grid-like pixels. The pixel value (for example, luminance) of each pixel is 0 to 3 in this example. Here, the case where r = 1 and θ = 180 ° in the displacement d = (r, θ) described above will be described below. Here, θ = 180 ° is the right direction of the drawing from a certain target pixel in FIG.

  In the pixel value pattern of FIG. 10A, when the pixel value i = 0, the pixel value j = 1, r = 1, and θ = 180 °, the position of a certain pixel of interest (pixel value i) is constant. The frequency at which the pixel value at the position separated by the displacement d = (r, θ) is the pixel value j is 8 (see FIG. 10B). Here, when the pixel value i = 0, the pixel value j = 1, r = 1, and θ = 180 °, the pixel value of the pixel adjacent to the right is the pixel value of the pixel of interest whose pixel value is 0 (zero). 1 indicates the frequency. That is, the frequency at which the pixel value of the pixel on the right is 1 is 8 for the pixel of interest whose pixel value is 0 (zero).

  In the pixel value pattern of FIG. 10A, a pixel value i (here, 0 to 3) and a pixel value j (here, 0 to 3) are calculated for each value, and the number of elements is calculated. When normalized, a co-occurrence matrix Pij shown in Expression (1) is obtained (in the example of FIG. 11, the number of elements of i and j (30)).

  FIG. 11 is a schematic diagram illustrating an example of a co-occurrence matrix calculated by the feature amount calculation unit 4212 of the present embodiment. The feature quantity calculation unit 4212 calculates a sum for each row and each column of the co-occurrence matrix Pij shown in the equation (1). Specifically, as shown in FIG. 11A, the feature amount calculation unit 4212 calculates the sum Px (m) (for example, Px (0) to Px (3)) for each row of the co-occurrence matrix Pij. Ask. Further, as shown in FIG. 11B, the feature amount calculation unit 4212 calculates a sum Py (n) (for example, sums Py (0) to Py (3)) for each column of the co-occurrence matrix Pij. .

  The feature amount calculation unit 4212 performs “uniformity”, “entropy”, “energy”, “inertia”, and “correlation” of this image based on the obtained co-occurrence matrix Pij, and the sum Px (m) and sum Py (n). Is calculated by the following equations (2) to (6).

  Here, in Expression (6), μx is the average of the sum of the co-occurrence matrices in the x direction, μ y is the average of the sum of the co-occurrence matrices in the y direction, σ x is the standard deviation of the co-occurrence matrix in the x direction, and σ y is y The standard deviation of the co-occurrence matrix of directions.

  Note that the image of the local luminance value change pattern such as type 2 shown in FIG. 9B described above includes the uniformity of equation (2), the entropy of equation (3), and the equation (4). Energy is suitable. In addition, the inertia of the equation (5) and the correlation of the equation (6) are suitable for an image of a change in the overall luminance value pattern such as type 3 shown in FIG. The feature amount calculation unit 4212 calculates the feature amount of the image by the various operations described above. The feature amount calculation unit 4212 stores the calculated feature amount in the storage unit 43 via the write control unit 4221.

  The feature amount is calculated by, for example, acquiring an image to be observed, automatically recognizing whether or not there is a portion indicating the feature whose feature amount is to be calculated in the acquired image, and Only when it is recognized that there is a feature amount may be calculated. As a method for recognizing a portion for which a feature amount is to be calculated from the observed image, luminance information and form information indicating an abnormal state are stored in advance, and the observation image is analyzed based on this information, and the feature amount is determined. It is also possible to determine whether there is a part to be calculated.

When there are a plurality of cell line types, it is necessary to input information indicating the cell line type (cell line ID). In addition, information indicating the type of cell line may be input by a user who knows the type of cell line to be observed, or by identifying cells such as the form and brightness of the cell to be observed, and matching technology. It is also possible to automatically create and input information indicating the type of cell line by using a technique for automatically determining the type of cell line by means of, for example.
In this embodiment, a case where information (cell line ID) indicating a cell line is input from the user via the input unit 44 will be described.

  When the cell number calculation unit 4222 receives information (cell line ID) indicating a cell line from the user via the input unit 44, the cell number calculation unit 4222 uses the cell line ID as a search key to obtain calibration curve information stored in the storage unit 43. A search is performed, and calibration curve information with a matching cell line ID is obtained based on the search result. The cell number calculation unit 4222 calculates the number of cells based on the acquired calibration curve information and the area of the colony calculated by the area calculation unit 4211. At this time, the cell number calculation unit 4222 determines whether there is a part to be excluded from the cell number calculation target in the colony image based on the feature amount calculated by the feature amount calculation unit 4212. As a result of this determination, if there is a part to be excluded from the cell number calculation target, the cell number calculation unit 4222 is based on the area obtained by removing the area of the part to be excluded from the area of the colony calculated by the area calculation unit 4211. Calculate the cell number. That is, the cell number calculation unit 4222 includes the calibration curve information stored in the storage unit 43 in association with the feature amount calculated by the feature amount calculation unit 4212 and the input cell line information, and the colony calculated by the area calculation unit 4211. The number of cells is calculated based on the area.

  In addition, the cell number calculation unit 4222 determines whether there is a part to be excluded from the cell number calculation target based on whether the feature amount calculated by the feature amount calculation unit 4212 exceeds a predetermined threshold. judge. Specifically, when the feature amount calculated for each pixel group of the colony image exceeds a predetermined threshold value (or less than the predetermined threshold value), the cell number calculation unit 4222 The image displayed by the pixel group is excluded from the cell number calculation target. That is, the cell number calculation unit 4222 calculates the number of cells based on whether or not the feature amount calculated by the feature amount calculation unit 4212 is within a predetermined range.

[Operation of incubator]
Next, an example of the operation of the incubator 11 will be described. The incubator 11 calculates the number of cells by estimating the number of cells contained in this colony from the area of the colony of cells based on the registered calibration curve. Here, the calibration curve is information indicating the relationship between the area of a cell colony and the number of cells contained in the colony. Here, an operation for registering a calibration curve will be described first, and then an operation for calculating the number of cells based on the registered calibration curve will be described.

[Operation of calibration curve registration]
First, the calibration curve registration operation will be described with reference to FIG.
FIG. 12 is a flowchart showing an example of the calibration curve registration operation by the incubator 11 (observation apparatus) of the present embodiment. The incubator 11 stores a calibration curve for each cell line. As an example, the incubator 11 calculates a colony area by detecting a colony image in the image data based on the image data described above and calculating the area of the colony image. The incubator 11 registers a calibration curve for a certain cell line by associating the calculated colony area with the number of cells counted by the user.

  Here, before the calibration curve registration operation starts, the control unit 42 receives in advance a calibration curve registration operation instruction that the user inputs via the input unit 44. The instruction for the calibration curve registration operation includes information (cell line ID) indicating the cell line to which the calibration curve is registered (if there is only one cell line to be observed, the calibration curve is registered). Input of information (cell line ID) indicating the target cell line is not essential, and the storage unit 43 starts observation after 1 day, 2 days, and 3 days from the start of culture, respectively. Each observation start time is stored in advance as an observation schedule for management data, and a method in which the user counts the number of cells is a method of counting cells using a hemocytometer, etc. In the case of this method, the number of culture vessels 19 corresponding to the number of observations is prepared, for example, when observing after 1 day, 2 days, and 3 days from the start of culture, respectively, at least 3 cultures Container 19 It is housed in the stocker 21 of the temperature-controlled room 15. The same cell line is cultured in each of these culture vessels 19. The culture starts by observing these culture vessels 19 one by one per day. The cell lines after the lapse of 1 day, 2 days, and 3 days are observed, and the specific operations of calculating the colony area and registering the calibration curve performed by the incubator 11 will be described below.

  Step S101: The control unit 42 compares the observation schedule of the management data in the storage unit 43 with the current date and time to determine whether or not the observation start time of the culture vessel 19 has arrived. When the observation start time is reached (YES side), the control unit 42 shifts the process to step S102. On the other hand, when it is not the observation time of the culture vessel 19 (NO side), the control unit 42 waits until the time of the next observation schedule.

  Step S102: The control unit 42 instructs the container transport device 23 to transport the culture container 19 corresponding to the observation schedule. Then, the container transport device 23 carries the instructed culture container 19 out of the stocker 21 and places it on the sample stage 31 of the observation unit 22. Note that, when the culture vessel 19 is placed on the sample stage 31, an entire observation image of the culture vessel 19 is captured by a bird view camera (not shown) built in the stand arm 32. Thereby, the image of the culture container 19 containing the image of a colony is imaged.

Step S103: The image reading unit 4203 of the control unit 42 reads the entire observation image captured in step S102. The area calculation unit 4211 of the control unit 42 detects a colony image from the read entire observation image, and calculates the total area of each colony in the culture vessel 19 as a colony area.
When creating a calibration curve, an image of a culture vessel containing a colony image may be obtained, cells may be counted from the fluorescence observation image, and the colony area may be calculated from the phase difference image. Alternatively, the cells may be counted using a hemocytometer and the colony area may be calculated from the phase difference image.

  Step S104: The control unit 42 instructs the container transport device 23 to transport the culture container 19 to the small door 18 after the observation schedule is completed. The container transport device 23 transports the instructed culture container 19 from the sample stage 31 of the observation unit 22 to the position of the small door 18.

  Step S105: The user opens the small door 18 and takes out the culture vessel 19. In addition, the user counts the number of cells of the extracted culture container 19 by a known method. For example, the user counts the number of cells using a hemocytometer. In addition, the user inputs the counted number of cells to the input unit 44.

  Step S106: The writing control unit 4221 of the control unit 42 associates the number of cells input to the input unit 44 with the colony area calculated in step S103 and the information (cell line ID) indicating the cell line, The data is stored in the storage unit 43. Thereby, the colony area, the number of cells, and the cell line ID are associated and stored as calibration curve information in the storage unit 43 (in the case of observation of one cell line, the association with the cell line ID is not essential). ). Then, the control part 42 complete | finishes an observation sequence and returns a process to step S101.

  Thus, the calibration curve information is stored in the storage unit 43 by repeating Steps S101 to S106. In addition, by repeating Steps S101 to S106 for a plurality of cell lines, calibration curve information for each of the plurality of cell lines is stored in the storage unit 43. Specifically, by repeating steps S101 to S106 for each of cell line A, cell line B, and cell line C, calibration curve information for cell line A, calibration curve information for cell line B, cell line C Calibration curve information is stored in the storage unit 43, respectively.

[Operation of cell number estimation and judgment of cell quality]
Next, an example of the cell number estimation operation of the incubator 11 will be described with reference to FIG.
FIG. 13 is a flowchart showing an example of the cell number estimation operation by the incubator 11 (observation apparatus) of the present embodiment. In this example, it is assumed that data (referred to as comparison data) regarding the number of cells with respect to the change in colony area of a specific cell line to be observed is acquired and stored in advance. From this data, it is unclear whether the observed cells are good or bad.

  The incubator 11 observes the culture container 19 carried into the temperature-controlled room 15 in a time-lapse manner according to a registered observation schedule. A plurality of specific cell lines are cultured in the culture container 19. For example, the cell line A is cultured in the culture container 19A among the culture containers 19. Among the culture vessels 19, the cell line B is cultured in the culture vessel 19B, and the cell line C is cultured in the culture vessel 19C. The incubator 11 sequentially conveys the culture vessels 19A to 19C to the vertical robot 38 observation unit 22 according to the observation schedule, and enlarges the entire image of the culture vessel 19 (entire observation image) and a part of the culture vessel 19. The microscope image is taken. In addition, the calibration curve information of the cell line A, the calibration curve information of the cell line B, and the calibration curve information of the cell line C are stored in the storage unit 43 in advance by the calibration curve information registration procedure described above. The operation of estimating the number of cells in the time lapse observation of the incubator 11 will be described below.

  Step S201: The control unit 42 compares the observation schedule of the management data in the storage unit 43 with the current date and time to determine whether or not the observation start time of the culture vessel 19 has come. When it is the observation start time (YES side), the control unit 42 shifts the process to step S202. On the other hand, when it is not the observation time of the culture vessel 19 (NO side), the control unit 42 waits until the time of the next observation schedule.

Step S202: The control unit 42 instructs the container transport device 23 to transport the culture container 19 corresponding to the observation schedule. Then, the container transport device 23 carries the instructed culture container 19 out of the stocker 21 and places it on the sample stage 31 of the observation unit 22. Note that, when the culture vessel 19 is placed on the sample stage 31, an entire observation image of the culture vessel 19 is captured by a bird view camera (not shown) built in the stand arm 32.
In this embodiment, the observation apparatus shown in FIGS. 1, 3 and 4 is an apparatus provided with a temperature-controlled room for culturing cells to be observed. Therefore, the cells imaged in step S202 are cultured in the temperature-controlled room of the observation apparatus. Therefore, this step can also be started from the step of culturing cells. Further, as in the present embodiment, the observation device may not be provided with a temperature-controlled room, and the temperature-controlled room for culturing cells may be a device separate from the observation device.

Step S203: The cell number calculation unit 4222 acquires information (cell line ID) indicating a cell line from the management data stored in the storage unit 43.
Step S204: The cell number calculation unit 4222 searches the calibration curve information stored in the storage unit 43 using the acquired cell line ID as a search key, and acquires the calibration curve information that matches the cell line ID.

  Step S205: The image reading unit 4203 acquires the image captured in step 202. This image includes a colony image.

  Step S206: The feature amount calculation unit 4212 calculates the feature amount of the image acquired in step S205. It should be noted that the feature amount calculation step is executed in response to the result of recognition and determination by a known image recognition technique as to whether or not the feature amount calculation step performed in step S206 is necessary for the image acquired in step S205. It is also possible to determine whether or not. If it is determined that it is not necessary to calculate the feature value (for example, there is no region or shape with an abnormal luminance value, there is no part with an abnormal size, and the state is specific to the particular cell line being observed) It is not necessary to go through step S206.

  Step S207: When the feature amount calculated for each pixel group of the colony image exceeds a predetermined threshold value (or less than the predetermined threshold value), the cell number calculation unit 4222 displays the pixel group. Are excluded from the cell count calculation target. Further, the cell number calculation unit 4222 estimates (calculates) the cell number based on the area excluding the area of the portion to be excluded from the area of the colony calculated by the area calculation unit 4211. The area calculation unit 4211 calculates the area of the recognized colony in the acquired image. Note that this step may be executed when observation is possible after the culture vessel is transported to the observation unit (S202). In this case, information indicating the cell line is obtained after calculating the colony area.

Step S208: The state of the cell is determined by comparing the number of cells calculated (estimated) in step S207 with, for example, the number of cells counted visually (contrast data). That is, when the contrast data matches or substantially matches the calibration curve, it is determined that the cell state is good. For example, the cell to be counted visually may be a cell imaged for calculating the area by the area calculation unit, or a new cell may be the observation target. However, it is essential to compare the cell lines of the data corresponding to the same calibration curve with the same type. The comparison result can be displayed on a monitor (not shown), for example. The determination of the cell state is processed by providing a cell state determination unit in the cell number calculation unit, for example.
It is also possible to grasp the degree of quality of the cell state by displaying the calibration curve on the monitor and displaying the contrast data on the monitor on which the calibration curve is displayed.
As in step S208, in a cell line in which the state of the cell line is unknown, a calibration curve can be obtained by acquiring in advance data relating to the number of cells with respect to changes in the colony area to be observed (data relating to newly measured cells). As well as calculating the number of cells, the cell status can be determined.

Step S209: The control unit 42 instructs the container transport device 23 to transport the culture container 19 after the observation schedule is completed. Then, the container transport device 23 transports the designated culture container 19 from the sample stage 31 of the observation unit 22 to a predetermined storage position of the stocker 21. Thereafter, the control unit 42 ends the observation sequence and returns the process to S201.
In addition, after determining whether or not the cells are in a good state as a result of the determination in step S208, the cells determined to be in a good state are taken out from the observation device, for example, cells used for drug discovery research Can be offered as.

  Thus, by repeating Step S201 to Step S209 for each cell line, the estimation result of the number of cells in each observation schedule is stored in the storage unit 43. Furthermore, it is possible to determine the quality of the cells based on the estimation result of the number of cells. Furthermore, only selected good cells can be provided to research institutions.

  As described above, the incubator 11 (observation apparatus) of the present embodiment includes the cell number calculation unit 4222 that calculates the number of cells included in the colony based on the calibration curve information and the area of the colony. . Thereby, the incubator 11 can calculate the number of cells by a non-invasive method and can improve the accuracy of the calculated number of cells.

[Second Embodiment]
Next, an incubator 11a (observation apparatus) according to a second embodiment of the present invention will be described with reference to FIG. The incubator 11a of this embodiment is different from the above-described embodiment in that the control unit 42a includes a colony identification unit 4204. In addition, about the structure same as embodiment mentioned above, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  FIG. 14 is a block diagram showing an example of the configuration of an incubator 11a (observation apparatus) according to the second embodiment of the present invention.

  The colony identifying unit 4204 identifies a plurality of colonies included in the image based on the image captured by the imaging device 34. Specifically, the colony identification unit 4204 extracts a colony image from the entire observation image captured by the imaging device 34, assigns a colony identification ID for each colony, and associates the colony image with the colony identification ID. Are output to the area calculation unit 4211 and the feature amount calculation unit 4212.

  The area calculation unit 4211 calculates the area of the colony for each colony identified by the colony identification unit 4204. The feature amount calculation unit 4212 calculates a feature amount for each colony identified by the colony identification unit 4204. In addition, the cell number calculation unit 4222 calculates the number of cells for each colony identified by the colony identification unit 4204.

  As described above, the incubator 11a (observation apparatus) of the present embodiment calculates the number of cells for each colony. Thereby, the incubator 11a (observation apparatus) can further improve the calculation (estimation) accuracy of the number of cells compared to the incubator 11 (observation apparatus) in the first embodiment.

Although the cell number calculation unit 4222 has been described as calculating the cell number by excluding this image from the cell number calculation target image when the feature amount of the image is abnormal, the present invention is not limited to this. I can't. For example, when the feature amount of the image is abnormal, the cell number calculation unit 4222 may calculate the cell number based on the calibration curve L ′ for abnormal values. More specifically, the cell number calculation unit 4222 calculates the number of cells based on the normal value calibration curve L for a portion where the image feature amount is normal, and the image feature amount is abnormal. For the portion, the number of cells is calculated based on the calibration curve L ′ for abnormal values. Thereby, the incubator 11 (observation apparatus) or the incubator 11a (observation apparatus) can further improve the calculation (estimation) accuracy of the number of cells.
In addition, as described above, whether the feature amount of the image is abnormal or normal, and further, the determination of the normal region and the abnormal region is automatically determined from the image, the calculation of the feature amount and the number of cells based on the determination result May be automatically executed.

  Further, as described above, the feature amount calculation unit 4212 of the control unit 42 (or the control unit 42a, which is the same in the following description) analyzes the luminance pattern of the read entire observation image, thereby calculating the feature amount. calculate. At this time, the control unit 42 may present the calculated feature amount to the user by displaying a plot screen of the calculated feature amount on a display unit (not shown). In this case, the user determines the normal and abnormal threshold values of the feature amount based on the displayed plot screen, and inputs the determined normal and abnormal threshold values of the feature amount to the input unit 44. The feature amount calculation unit 4212 generates a normal value calibration curve L and an abnormal value calibration curve L ′ based on the threshold value input to the input unit 44. By configuring in this way, the incubator 11 (observation apparatus) or the incubator 11a (observation apparatus) can further improve the calculation (estimation) accuracy of the number of cells.

Further, when the control unit 42 displays the feature amount plot screen on a display unit (not shown), the control unit 42 may be configured as shown in FIG.
FIG. 15 is a schematic diagram illustrating an example of a feature amount plot screen according to a modification of the present invention. As shown in FIGS. 15A to 15C, the control unit 42 causes the display unit to display a feature amount plot screen with the horizontal axis as the colony area and the vertical axis as the feature amount. Here, as shown in FIG. 15A, the control unit 42 plots the colony area calculated by the area calculation unit 4211 and the feature amount calculated by the feature amount calculation unit 4212 for each colony. Display on the screen. In this case, the control unit 42 detects a plot of a colony that is highly likely to have an abnormal feature amount as an abnormal point candidate plot CA (FIG. 15A). Next, when the user performs an operation for selecting the abnormal point candidate plot CA on the input unit 44, the control unit 42 displays the abnormal point candidate plot CA as the selected plot SL based on the selection operation. (FIG. 15B). In this case, the control unit 42 displays a colony image corresponding to the selected plot SL on a display unit (not shown). The user confirms the displayed image of the colony, determines whether or not this colony is abnormal in the feature amount, and inputs the determination result to the input unit 44. The control unit 42 generates a calibration curve L based on the determination result input to the input unit 44. More specifically, the control unit 42 generates a calibration curve L so that colonies with normal feature values and colonies with abnormal feature values are distinguished on the plot screen (FIG. 15C). ). With this configuration, the incubator 11 (observation apparatus) or the incubator 11a (observation apparatus) can automatically generate the calibration curve L and improve the accuracy of the calibration curve L.

  Note that the control device 41 treats a plurality of microscope images obtained by imaging a plurality of points (for example, five-point observation or the entire culture container 19) of the same culture container 19 in the same observation time period as an image for one time-lapse observation. It may be.

  In addition, although the area calculation part 4211 demonstrated the example which detects the image of a colony based on a whole observation image, it is not restricted to this. The area calculation unit 4211 may detect a colony image by performing image processing on the phase-contrast microscope image.

  Further, a program for executing each process of the incubator 11 (observation apparatus) in the first embodiment of the present invention or the incubator 11a (observation apparatus) in the second embodiment of the present invention is recorded on a computer-readable recording medium. The various processes described above may be performed by recording, reading the program recorded on the recording medium into a computer system, and executing the program.

  Here, the “computer system” may include an OS and hardware such as peripheral devices. Further, the “computer system” includes a homepage providing environment (or display environment) if a WWW system is used. The “computer-readable recording medium” means a flexible disk, a magneto-optical disk, a ROM, a writable nonvolatile memory such as a flash memory, a portable medium such as a CD-ROM, a hard disk built in a computer system, etc. This is a storage device.

  Further, the “computer-readable recording medium” means a volatile memory (for example, DRAM (Dynamic DRAM) in a computer system that 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. Random Access Memory)), etc., which hold programs for a certain period of time. The program may be transmitted from a computer system storing the program in a storage device or the like to another computer system via a transmission medium or by a transmission wave in the transmission medium. Here, the “transmission medium” for transmitting the program refers to a medium having a function of transmitting information, such as a network (communication network) such as the Internet or a communication line (communication line) such as a telephone line. The program may be for realizing a part of the functions described above. Furthermore, what can implement | achieve the function mentioned above in combination with the program already recorded on the computer system, what is called a difference file (difference program) may be sufficient.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design etc. of the range which does not deviate from the summary of this invention are included.

  DESCRIPTION OF SYMBOLS 11 ... Observation apparatus, 41 ... Control apparatus, 4211 ... Area calculation part, 4212 ... Feature-value calculation part, 4222 ... Cell number calculation part, 43 ... Memory | storage part

Claims (14)

Based on the captured image of the cell, an area calculation unit that calculates the area of the colony of the cell,
Calibration curve information indicating the relationship between the area of a colony of cells and the number of cells contained in the colony, and a cell number calculation unit that calculates the cell number based on the area of the colony calculated by the area calculation unit. Observation device. The observation apparatus according to claim 1, further comprising: a feature amount calculation unit that calculates a feature amount of the colony image based on the captured cell image. The observation apparatus according to claim 2, wherein the feature amount is information indicating at least one of uniformity, entropy, energy, inertia, or correlation of the image. The cell number calculation unit
The observation apparatus according to claim 2 or 3, wherein the number of cells is calculated based on whether or not the feature amount calculated by the feature amount calculation unit is within a predetermined range. Based on the image, a colony identifying unit for identifying a plurality of the colonies included in the image,
The area calculation unit
Calculate the area of the colony for each colony identified by the colony identification unit,
The feature amount calculation unit includes:
The feature amount is calculated for each colony identified by the colony identifying unit,
The cell number calculation unit
The observation apparatus according to any one of claims 2 to 4, wherein the number of cells is calculated for each colony identified by the colony identification unit. The cell number calculation unit
For each of the colonies identified by the colony identifying unit, calculating the number of cells included in the colony based on whether or not the feature amount calculated by the feature amount calculating unit is within a predetermined range. The observation apparatus according to claim 5, characterized in that: The observation apparatus according to claim 1, further comprising: an information writing unit that writes the calibration curve information and information input in association with the calibration curve information in a storage unit. The cell state determination part which determines the state of the imaged cell from data obtained by measuring the number of cells included in the colony of the imaged cell and the calibration curve information is provided. The observation apparatus according to claim 7. The calibration curve information is registered for each cell line of the cell, and the registered information includes cell line information for identifying the cell line of the cell. The observation apparatus according to one item. An imaging unit for capturing images of cells in culture;
An observation system comprising: the observation device according to any one of claims 1 to 9. An area calculation procedure for calculating the area of the colony of the cells based on the image of the cells,
A readout procedure for reading out calibration curve information indicating the relationship between the area of the colony and the number of cells contained in the colony,
An observation method comprising: a cell number calculating procedure for calculating the cell number based on the calibration curve information read in the reading procedure and the area of the colony calculated in the area calculating procedure. On the computer,
An area calculation procedure for calculating the area of the colony of the cells based on the image of the cells,
A readout procedure for reading out calibration curve information indicating the relationship between the area of the colony and the number of cells contained in the colony,
A program for executing the cell number calculation procedure for calculating the cell number based on the calibration curve information read in the read procedure and the area of the colony calculated in the area calculation procedure. The program according to claim 12, further comprising: a determination procedure that refers to the calibration curve information and determines the state of the imaged cell. A cell culture procedure for culturing cells;
Imaging the cells cultured in the culture procedure, an area calculation procedure for calculating the area of the colonies of the cells based on the image of the cells,
A readout procedure for reading out calibration curve information indicating the relationship between the area of the colony and the number of cells contained in the colony,
Refer to the calibration curve information read out in the readout procedure, the cell number calculation procedure for calculating the number of cells based on the area of the colony calculated in the area calculation procedure, and the calibration curve information, And a determination procedure for determining a state of the imaged cell.