JP2006018394A - Cell identification device, cell identification method, program for cell identification, and cell analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify each cell from the image of an imaged cell. <P>SOLUTION: Edge extraction processing is executed to the phase difference image of a cell (a step S20), and the outline of the cell is obtained (a step S30). Also, the fluorescence image of a nucleus is binarized (a step S40), and edge extraction processing is executed (a step S50), and the outline of the nucleus is obtained (a step S60). Then, the outline of the nucleus is overlapped with a phase difference image obtained by extracting the outline of the cell (a step S70). Cells in which two or more nucleuses are present, respectively are extracted (a step S80), and divided so that one nucleus can be included in the outline of one cell (a step S90), and the outline of the nucleus including the outline of the nucleus is recognized as one cell (a step S110). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell identification device, a cell identification method, a cell identification program, and a cell identification program for identifying a cell by extracting a cell outline from the captured cell image, and processing the captured image The present invention relates to a cell analysis apparatus that performs analysis.

  Patent Document 1 discloses a cell image processing method that extracts an outline of a cell by binarizing an image obtained by imaging a cell.

JP-A-5-274422

  However, in the cell image processing method described in Patent Document 1, there has been a problem that two or more cells connected to each other on the image may be erroneously extracted as one cell.

The cell identification device according to claim 1 extracts a cell contour candidate to be identified as one cell from the first image captured by the first imaging optical system, and also the second image captured by the second imaging optical system. A contour extracting unit for extracting a nucleus from the cell, and an identifying unit for identifying a cell contour candidate having one nucleus as one cell based on the cell contour candidate and the nucleus extracted by the contour extracting unit. And
The cell identification device according to claim 2 further includes an image superimposing unit that superimposes the cell contour candidate extracted by the contour extraction unit and the nucleus in the cell identification device according to claim 1, wherein the identification unit includes image superimposition. Based on the image superimposed by the means, a judging means for judging whether there is only one nucleus or a plurality of nuclei in one cell contour candidate, and a nucleus in one cell contour candidate by the judging means. And dividing means for dividing the corresponding cell contour candidate so that one nucleus is present in one cell contour candidate, and the identifying means is one divided by the dividing means. One cell contour candidate surrounding the nucleus is identified as one cell.
According to a third aspect of the present invention, in the cell identification device according to the second aspect, when the dividing unit determines that a plurality of nuclei exist in one cell contour candidate by the determining unit, One cell contour candidate is divided so as to include one nucleus by a straight vertical bisector connecting centroids of nuclei included in one cell contour candidate.
A cell identification device according to claim 4 is the cell identification device according to claim 2, wherein the cell contour candidate and the nucleus extracted by the contour extraction means are displayed in one cell contour candidate by the judgment means. When it is determined that a plurality of nuclei are present, the user further has an input means for inputting a dividing line for dividing the cell contour candidate, and the display means includes the determination means within one cell contour candidate. When it is determined that there are a plurality of nuclei, the cell contour candidate is displayed, and the dividing means is surrounded by the dividing line input by the user via the input means and the outline of the cell contour candidate. A region including a single nucleus is divided as cell contour candidates.
The cell identification device according to claim 5 is characterized in that, in the cell identification device according to claim 4, the display means enlarges and displays the cell contour candidate determined by the determination means as having a plurality of nuclei. To do.
The cell identification device according to claim 6 is the cell identification device according to any one of claims 1 to 5, wherein the first image captured by the first imaging optical system is a cell phase difference image or differential interference. The second image captured by the second imaging optical system is a nuclear fluorescence image.
A cell analysis device according to claim 7, the cell identification device according to any one of claims 1 to 6, and a plurality of images of cells at predetermined time intervals, and the plurality of images captured And analyzing means for executing time-series image processing and analyzing time-series changes of individual cells.
The cell identification method according to claim 8, wherein a cell contour candidate to be identified as one cell is extracted from a first image imaged by the first imaging optical system, and a second image imaged by the second imaging optical system. Nuclei are extracted from the cell contour candidates, and based on the extracted cell contour candidates and nuclei, a cell contour candidate in which one nucleus exists is identified as one cell.
The cell identification method according to claim 9 is the cell identification method according to claim 8, wherein the extracted cell contour candidate and the nucleus are superimposed, and based on the superimposed image, the nucleus is included in one cell contour candidate. If there is only one or more than one, and if it is determined that there are multiple nuclei in one cell contour candidate, then there is one nuclei in one cell contour candidate The cell contour candidate to be divided is divided, and one cell contour candidate surrounding one divided nucleus is identified as one cell.
The cell identification method according to claim 10 is the cell identification method according to claim 9, wherein when it is determined that there are a plurality of nuclei in one cell contour candidate, the nuclei included in one cell contour candidate The cell is divided so that one nucleus is included in one cell outline candidate by a straight vertical bisector connecting the centroids of each cell.
The cell identification method according to claim 11 is the cell identification method according to claim 9, wherein when it is determined that a plurality of nuclei exist in one cell contour candidate, the cell displayed on the display means A region including one nucleus surrounded by a dividing line input by a user via an input unit and an outline of a cell outline candidate is divided as a cell outline candidate.
A cell identification method according to a twelfth aspect is the cell identification method according to the eleventh aspect, wherein the cell contour candidates determined to have a plurality of nuclei are enlarged and displayed on the display means.
The cell identification method according to claim 13 is the cell identification method according to any one of claims 8 to 12, wherein the first image captured by the first imaging optical system is a phase difference image or differential interference of a cell. The second image captured by the second imaging optical system is a nuclear fluorescence image.
The cell identification program according to claim 14 extracts a cell contour candidate to be identified as one cell from the first image captured by the first imaging optical system, and the second image captured by the second imaging optical system. A contour extraction process for extracting a nucleus from an image, and an identification process for identifying a cell contour candidate having one nucleus as one cell based on the cell contour candidate and the nucleus extracted by the contour extraction process. Features.
The cell identification program according to claim 15 further includes an image superimposing process for superimposing the cell contour candidate and the nucleus extracted by the contour extraction process in the cell identification program according to claim 14, wherein the identification process includes: Based on the image superimposed in the image superimposition process, a determination process for determining whether there is only one nucleus or a plurality of nuclei in one cell contour candidate, and within one cell contour candidate in the determination process And dividing the corresponding cell contour candidate so that one nucleus exists in one cell contour candidate. One cell contour candidate surrounding one nucleus is identified as one cell.
The cell identification program according to claim 16, in the cell identification program according to claim 15, when the division processing determines that a plurality of nuclei exist in one cell contour candidate in the determination processing, A region including one nucleus surrounded by the dividing line input by the user via the input unit and the contour of the cell contour candidate for the cell contour candidate displayed on the display unit is divided as the cell contour candidate. It is characterized by doing.
The cell identification program according to claim 17 is the cell identification program according to any one of claims 14 to 16, wherein the first image captured by the first imaging optical system is a phase difference image of a cell or It is a differential interference image, and the second image captured by the second imaging optical system is a nuclear fluorescence image.

  According to the present invention, two adjacent cells can be identified as two individual cells.

  FIG. 1 is a block diagram showing a configuration of an embodiment of a cell identification device according to the present invention. The cell identification device 100 includes an input device 110, a control device 120, a monitor 130, and an image memory 140. The cell identification device 100 is connected to a microscope 200 with a camera that captures an enlarged image of the cell. The cell image observed by the user and captured by the microscope 200 is transferred to the control device 120, and the image is displayed. It is stored in the memory 140 and displayed on the monitor 130.

  The microscope 200 includes a CCD 210 as an imaging device, and includes a phase difference observation optical system using transmitted light and a fluorescence observation optical system using epifluorescence. In the present embodiment, the cells are identified based on the phase difference image captured by the phase difference observation optical system and the nucleus fluorescence image captured by the fluorescence observation optical system, and the identified cells are observed by the fluorescence observation optical system. .

  Cell identification device 100 in the present embodiment identifies individual cells included in the cell image captured by microscope 200. When imaging the cell with the microscope 200, the cell is previously stained with a fluorescent dye, such as Hoechst, for fluorescent staining of the nucleus of the cell and a fluorescent dye, such as DiBAC, for fluorescent staining of the cell. And the enlarged image of the cell dye | stained with these pigment | dyes is imaged with the microscope 200. FIG. At this time, by changing the wavelength of the excitation light that irradiates the illumination optical system or the cell, any one of a phase difference image, a fluorescent image in which the cell is fluorescently stained with a fluorescent dye added in advance, and a fluorescent image in which the nucleus is fluorescently stained Can be imaged.

  In the present embodiment, an inverted microscope is used as the microscope 200, light of a predetermined wavelength is irradiated from the upper illumination system, and a phase difference image of the cell is captured as shown in FIG. Thereafter, the illumination is changed to epi-illumination, and light having a wavelength that excites the dye that fluorescently stains the cell nucleus is irradiated, and a fluorescence image of the cell nucleus shown in FIG. 3 is captured. At this time, the phase difference image of the cell shown in FIG. 2 and the fluorescence image of the nucleus shown in FIG. 3 are imaged so that their fields of view are the same.

  The phase difference image of the cell imaged by the microscope 200 and the image data of the fluorescence image of the nucleus are transferred to the control device 120 and stored in the image memory 140. In the control device 120, the phase difference image of the cell and the fluorescence image of the nucleus are processed to identify individual cells present in the image as described below. Edge extraction processing is performed on the phase difference image of the cell, and the outline of the cell is extracted by connecting adjacent ones of the extracted edges with a straight line. As described later, each cell is identified by using the extracted cell contour as one cell contour candidate.

  FIG. 4 shows a specific example of an algorithm for extracting the outline of a cell based on the result of the edge extraction process. Each point in FIG. 4 represents an edge extracted by the edge extraction process. In FIG. 4 (a), arbitrary two edges A and B are connected by a straight line 4a, and an edge C farthest from the straight line 4a in the vertical direction and an end point of the straight line 4a, that is, the edges A and B are respectively connected to the straight line 4b and Tie with 4c.

  Next, as shown in FIG. 4B, the edges D and E that are farthest in the vertical direction from the straight lines 4b and 4c and the end points of the straight lines 4b and 4c are connected by straight lines. As a result, the edges A, D, C, E, and B are connected by straight lines, and the contours are extracted. Then, in FIG. 4 (c), the same process as shown in FIG. 4 (b) is performed, and then the process is repeated until all the points are connected by straight lines, thereby extracting the outline of the cell. Can do.

  Next, image processing performed on the nuclear fluorescence image will be described. First, the fluorescence image of the nucleus is binarized. Then, edge extraction is performed on the binarized fluorescence image of the nucleus in the same manner as performed on the phase difference image of the cell to obtain the contour of the nucleus.

  Thereafter, the extracted outline of the cell is overlapped (superposed) with the extracted outline of the cell to generate an image displaying the outline 5a of the cell and the outline 5b of the nucleus shown in FIG. Then, in the generated image, a cell outline 5a in which two or more nucleus outlines 5b exist in one cell outline 5a is extracted. When there are two or more nuclear outlines 5b in one cell outline 5a, a phase difference image of the cells is taken in a state where the two cells are adjacent to each other, and as a result of edge extraction, one cell It is thought that the contour has been extracted. Therefore, when there are two or more nucleus contours 5b in one cell contour 5a, the cell contour 5a is such that one nucleus contour 5b exists in one cell contour 5a. Split.

  In dividing the cell outline 5a, for example, as shown in FIG. 6, the centroids of the outline 5b of the nucleus included in one cell outline 5a are connected by a straight line 6a, and the perpendicular bisector 6b of the straight line is obtained. To divide the cell outline 5a. FIG. 7 shows the result of dividing the cell outline 5a on the image displaying the cell outline 5a and the nucleus outline 5b shown in FIG. Thereby, the cell outline 5a can be divided so that one nucleus outline 5b exists within one cell outline 5a. Then, in the image obtained as a result of the division processing, the cell outline 5a including one nucleus outline 5b is recognized as one cell, and individual cells are identified.

  That is, by identifying one cell contour candidate surrounding one nuclear contour candidate divided by a vertical bisector (partition line) 6b as one cell, one cell contour candidate having one nuclear contour candidate is defined as one cell contour candidate. Identify as one cell. Each identified cell is given an ID (identification code) for uniquely identifying the individual cell, and the given ID indicates the outline of the identified cell, that is, the shape of the cell in the image. The position, for example, the coordinate value of the center of gravity of the nucleus when the inside of the image is an XY coordinate system is stored in a memory (not shown) as identification data of each cell. Then, the observed images of the identified individual cells can be image-processed in units of cells based on the cell identification data.

  In the present embodiment, for example, as shown in FIGS. 8 and 9, image processing is performed on an observation image of each cell using a microscope 200, and the observation result is obtained. That is, in the microscope 200, the wavelength of the excitation light applied to the cell is changed to a wavelength that excites a dye (for example, DiBAC) that fluorescently stains the cell, and the fluorescence image of the cell shown in FIG. Take an image. Then, the fluorescence intensity of each cell in the fluorescence image is measured, and a graph showing the time series change is displayed on the monitor 130 as shown in FIG.

  FIG. 9 is a graph showing a time-series change in the average value of the fluorescence intensity in the contours of the two cells 8a and 8b identified in FIG. In FIG. 9, it can be seen that when the reagent is added at time t1, the average value of the fluorescence intensity within the contours of the cells 8a and 8b increases. For example, when the dye that fluorescently stains cells is DiBAC, it can be seen that the membrane potential of the cells changes.

  FIG. 10 is a flowchart showing the operation of the cell identification device 100 in the present embodiment. When starting observation of cells using the microscope 200, the user sets a petri dish containing cells that have been fluorescently stained in advance by adding the above-described fluorescent dye to the cells to be observed and sets the observation magnification, etc. Make preparations. The process shown in FIG. 10 is executed by the control device 120 as a program to be started when these preparations are completed and the user instructs the execution of the cell identification process.

  In step S <b> 10, an enlarged image captured by the microscope 200 is stored in the image memory 140 and displayed on the monitor 130. Then, it progresses to step S11 and it is judged whether the image imaged with the microscope 200 is a phase difference image of a cell, or a fluorescence image of a nucleus. If the image captured by the microscope 200 is a cell phase difference image, the process proceeds to step S20. In step S20, edge extraction processing is performed on the cell phase difference image. Then, it progresses to step S30 and the extracted edge is connected and the outline 5a of a cell is obtained. Thereafter, the process proceeds to step S61.

  On the other hand, if it is determined that the image captured by the microscope 200 is a nuclear fluorescence image, the process proceeds to step S40. In step S40, the fluorescence image of the nucleus is binarized, and then the process proceeds to step S50, where edge extraction processing is performed on the image obtained as a result of binarization to extract the edge of the nucleus. Thereafter, the process proceeds to step S60, where the extracted edges are connected to obtain the outline 5b of the nucleus. Thereafter, the process proceeds to step S61.

  In step S61, it is determined whether or not the image processing for the imaged phase difference image of the cell and the fluorescence image of the nucleus has been completed. If it is determined that the image processing has been completed, the process proceeds to step S70. In step S70, the outline 5a of the cell extracted from the phase contrast image of the cell and the outline 5b of the nucleus extracted from the fluorescence image of the nucleus are superimposed to display the outline of the cell and nucleus as shown in FIG. An image is generated, the process proceeds to step S71, and the generated image is displayed on the monitor 130.

  Thereafter, the process proceeds to step S80, where a cell outline 5a in which two or more nucleus outlines 5b exist within one cell outline 5a is extracted, and the process proceeds to step S90. In step S90, the centroid of the outline 5b of the nucleus included in the outline 5a of one outline is connected with a straight line as described above for the outline 5a of the cell having two or more nuclei in one cell, A straight vertical bisector 6b divides the cell so that one cell contour 5a includes one nucleus contour 5b.

  Thereafter, the process proceeds to step S100, and it is determined whether or not the division process has been completed for all the cell outlines 5a in which two or more nucleus outlines 5b exist within the extracted cell outline 5a. If it is determined that the dividing process has not been completed for the extracted outlines 5a of the cells, the dividing process is repeated until all the outlines 5a are completed. If it is determined that the division process has been completed for all extracted cell outlines 5a, the process proceeds to step S110. In step S110, the cell outline 5a including one outline of the nucleus is recognized as one cell, an ID number is assigned to each recognized cell, and the above-described cell identification data is stored in the memory. Thereafter, the process ends.

According to the present embodiment described above, the following effects can be obtained.
(1) An image displaying the outlines of cells and nuclei is generated by superimposing the extracted cell outlines and the extracted nucleus outlines, and 2 nuclei outlines are included in one cell outline from the generated image. It was decided to extract and divide the outline of two or more existing cells. This makes it possible to accurately determine that the extracted outline of one cell is actually composed of a plurality of cells, and to appropriately divide the outline of the cell.
(2) When two or more nuclei exist in one cell, the centroids of the nuclei contained in one outline are connected with a straight line, and one cell is formed by the perpendicular bisector of the straight line. The outline of the cell is divided so that one nucleus is included, and the outline of the cell including one outline of the nucleus is recognized as one cell. This makes it possible to accurately recognize individual cells present in the phase difference image of the cells imaged in the same visual field and the fluorescence image of the nucleus.

  In the present embodiment described above, there are two or more nucleus contours in one cell contour based on an image generated by superimposing the extracted cell contour and nucleus contour. The outline of the cell is extracted, and the outline of the cell extracted by the control device 120 is divided by a straight vertical bisector connecting the centroids of the outlines of the nuclei. However, a cell in which two or more nuclei contours exist within the extracted contour of one cell is displayed on the monitor 140 in an enlarged manner, and the user uses the input device 110 to display a cell dividing line by, for example, a drag operation. You may enable it to input. In this case, the control device 120 identifies an area including one nucleus surrounded by a dividing line and a cell outline input by the user as one cell.

  In the embodiment described above, the edges extracted by the edge extraction process are connected by the algorithm described in FIG. 4 to obtain the outline of the cell and the nucleus. However, the present invention is not limited to this, and other algorithms are used. Edges may be joined together.

  In the above-described embodiment, the enlarged image captured by the microscope 200 is taken into the cell identification device 100 and image processing is performed to identify individual cells present in the image. May be taken into a general-purpose PC equipped with a cell identification program, and individual cells existing in the image may be identified on the general-purpose PC.

  In the embodiment described above, the outline of the cell is extracted based on the phase difference image of the cell imaged by the microscope 200. However, a differential interference image of a cell may be captured by the microscope 200, and a cell outline may be extracted based on the captured differential interference image of the cell.

  In the above-described embodiment, individual cells are identified based on an image generated by superimposing the outline of the nucleus extracted from the fluorescence image of the nucleus on the outline of the cell extracted from the phase difference image of the cell. However, without superimposing the images, for example, based on the coordinate values of each point constituting the contour of the cell when the inside of the image is an XY coordinate system and the coordinate value of the center of gravity of the nucleus, The outline of a cell in which one nucleus is present may be determined, and the outline of a cell containing one nucleus may be identified as one cell. Individual cells may be identified by other algorithms.

  The correspondence between the constituent elements of the claims and the embodiment will be described. The control device 120 corresponds to a contour extracting unit, an image superimposing unit, an identifying unit, a determining unit, and a dividing unit. The input device 110 corresponds to input means, and the monitor 130 corresponds to display means. Note that the present invention is not limited to the configurations in the above-described embodiments as long as the characteristic functions of the present invention are not impaired.

It is a block diagram which shows the structure of one Embodiment of the cell identification device by this invention. It is a figure which shows the phase difference image of a cell. It is a figure which shows the fluorescence image of the nucleus of a cell. The example of the algorithm which extracts the outline of a cell based on the result of having performed the edge extraction process is illustrated in figure. It is a figure which shows the phase difference image which displayed the outline of the cell and the nucleus. It is a figure which shows the specific example which divides | segments the outline of a cell, when two or more nuclei exist in one cell. It is a figure which shows the result of having performed the division | segmentation process of the outline of a cell. It is a 1st figure which shows the specific example of the observation method of a cell. It is a 2nd figure which shows the specific example of the observation method of a cell. It is a flowchart figure which shows the operation | movement of the cell identification device 100 in this Embodiment.

Explanation of symbols

100 Cell Identification Device 110 Input Device 120 Control Device 130 Monitor 140 Image Memory 200 Microscope 210 CCD

Claims (17)

A contour extracting means for extracting a cell contour candidate to be identified as one cell from the first image captured by the first imaging optical system, and extracting a nucleus from the second image captured by the second imaging optical system;
A cell identification device comprising: identification means for identifying a cell contour candidate having one nucleus as one cell based on the cell contour candidate and the nucleus extracted by the contour extraction means. The cell identification device according to claim 1,
Further comprising image superimposing means for superimposing the cell contour candidate and the nucleus extracted by the contour extracting means,
The identification means includes
Based on the image superimposed by the image superimposing means, a determination means for determining whether there is only one nucleus or a plurality of nuclei in one cell contour candidate;
Dividing means for dividing the corresponding cell contour candidate so that one nucleus exists in one cell contour candidate when it is determined by the determining means that there are a plurality of nuclei in one cell contour candidate. ,
The cell identification device characterized in that the identification means identifies one cell contour candidate surrounding one nucleus divided by the division means as one cell. The cell identification device according to claim 2,
When the determining unit determines that there are a plurality of nuclei in one cell contour candidate, the dividing unit is a straight vertical bisector that connects the centroids of the nuclei included in one cell contour candidate. By this, the cell identification device is divided so as to include one nucleus in one cell contour candidate. The cell identification device according to claim 2,
Display means for displaying cell contour candidates and nuclei extracted by the contour extraction means;
When it is determined by the determining means that there are a plurality of nuclei in one cell contour candidate, the user further has an input means for inputting a dividing line for dividing the cell contour candidate,
When the determining means determines that a plurality of nuclei are present in one cell contour candidate, the display means displays the cell contour candidate,
The dividing unit divides a region including one nucleus surrounded by a dividing line input by a user through the input unit and a contour of a cell contour candidate as a cell contour candidate. Identification device. The cell identification device according to claim 4, wherein
The cell identification apparatus characterized in that the display means enlarges and displays cell contour candidates determined by the determination means as having a plurality of nuclei. In the cell identification device according to any one of claims 1 to 5,
The first image captured by the first imaging optical system is a phase difference image or differential interference image of a cell, and the second image captured by the second imaging optical system is a nuclear fluorescence image, Cell identification device. The cell identification device according to any one of claims 1 to 6,
A cell having a plurality of images of a cell at a predetermined time interval, and performing analysis on the captured images to analyze a time-series change of each cell. Analysis device. Extracting a cell contour candidate identified as one cell from the first image captured by the first imaging optical system, and extracting a nucleus from the second image captured by the second imaging optical system;
A cell identification method, wherein a cell contour candidate in which one nucleus exists is identified as one cell based on the extracted cell contour candidate and the nucleus. The cell identification method according to claim 8, wherein
The extracted cell contour candidate and the nucleus are superimposed,
Based on the superimposed image, determine whether there is only one nucleus or multiple nuclei in one cell contour candidate,
If it is determined that there are multiple nuclei in one cell contour candidate, the corresponding cell contour candidate is divided so that one nucleus exists in one cell contour candidate,
A cell identification method characterized in that one cell contour candidate surrounding one divided nucleus is identified as one cell. The cell identification method according to claim 9, wherein
If it is determined that there are multiple nuclei in a single cell contour candidate, a straight vertical bisector connecting the centroids of the nuclei contained in one cell contour candidate will be included in one cell contour candidate. A cell identification method characterized by dividing so as to include one nucleus. The cell identification method according to claim 9, wherein
When it is determined that there are a plurality of nuclei in one cell contour candidate, the dividing line and the cell contour candidate entered by the user via the input unit with respect to the cell contour candidate displayed on the display unit A cell identification method, wherein a region including one nucleus surrounded by a contour line is divided as a cell contour candidate. The cell identification method according to claim 11, wherein
A cell identification method characterized by enlarging and displaying cell contour candidates determined to have a plurality of nuclei on the display means. In the cell identification method according to any one of claims 8 to 12,
The first image captured by the first imaging optical system is a phase difference image or differential interference image of a cell, and the second image captured by the second imaging optical system is a nuclear fluorescence image, Cell identification method. A contour extraction process for extracting a cell contour candidate to be identified as one cell from the first image captured by the first imaging optical system, and extracting a nucleus from the second image captured by the second imaging optical system;
A computer-executed cell identification comprising: identifying processing for identifying a cell contour candidate having one nucleus as one cell based on the cell contour candidate and the nucleus extracted by the contour extraction processing Program. In the cell identification program according to claim 14,
Further comprising an image superimposing process for superimposing the cell contour candidate and the nucleus extracted in the contour extracting process;
The identification process includes
Based on the image superimposed in the image superimposition process, a determination process for determining whether there is only one nucleus or a plurality of nuclei in one cell contour candidate;
If it is determined in the determination process that there are a plurality of nuclei in one cell contour candidate, a division process for dividing the corresponding cell contour candidate so that one nucleus exists in one cell contour candidate,
The identification process identifies a single cell contour candidate surrounding a single nucleus divided by the division process as a single cell. In the cell identification program according to claim 15,
In the dividing process, when it is determined in the determination process that a plurality of nuclei exist in one cell contour candidate, the user can input the cell contour candidate displayed on the display unit via the input unit. A cell identification program characterized by dividing a region including one nucleus surrounded by an input dividing line and a contour of a cell contour candidate as a cell contour candidate. In the cell identification program according to any one of claims 14 to 16,
The first image captured by the first imaging optical system is a phase difference image or differential interference image of a cell, and the second image captured by the second imaging optical system is a nuclear fluorescence image, Cell identification program.

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