JP2012163538A - Cell image analysis system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell image analysis system in which, while a microscopic image of a viable cell is captured, the cell is identified and a kind of the identified cell can be recognized.SOLUTION: A cell image analysis system includes a device 1 which time-sequentially captures an image of a viable cell into which a fluorescent protein that emits fluorescent light particularly in a specific term of a cell cycle is introduced, and an image analyzer 2 comprising a computer which uses the images to analyze the viable cell. The image analyzer 2 includes image analysis software which functions the computer as means 2a which specifies, as a cell region, a pixel group in which fluorescent luminance in the image exceeds a predetermined threshold or a luminance difference from neighboring luminance exceeds a predetermined threshold, means 2b which detects a term of a cell cycle of the viable cell in collation with a table in which terms of the cell cycle corresponding to the fluorescent light are stored, means 2c which measures the time until the term of the cell cycle of the viable cell changes into another term, and means 2h which uses the time of the term of the cell cycle to identify at least a kind of the cell.

Description

  The present invention relates to a cell image analysis system that acquires an image of a living cell and analyzes the cell using the acquired image.

In recent years, research on ES cells, iPS cells, cancer hepatocytes and the like has been active. These are cells called universal cells and can differentiate into various cells.
However, it is very difficult to visually confirm that these universal cells have differentiated and changed into other cells with a microscope. Differentiating cells are diverse, and it is difficult to distinguish the type of cells in the cell image only by observing the cell image with the naked eye.
Therefore, conventionally, in order to confirm that a universal cell has differentiated and changed to another cell, the differentiated cell is once taken out and proliferated, and molecular statistical analysis is performed on the proliferated cell. Therefore, it is necessary to use a technique of identifying the differentiated cell type, and it is difficult to identify the differentiated cell type while observing under a microscope.

  As a method for solving this problem, the present applicant specifies the cell cycle in the cultured cells, and the types of cells that differentiate depending on the time required for each phase in the specified cell cycle and the detailed phase within the M phase. Inspired to detect.

  Conventionally, as a method for specifying a cell cycle, for example, there are methods described in Non-Patent Documents 1 to 3 and Patent Document 1.

The method described in Non-Patent Document 1 is a method for analyzing a cell cycle using DAPI or PI which is a DNA intercalator, and is performed by the following procedure.
A DNA intercalator is a fluorescent reagent inserted between double strands of DNA. The cultured cells are stained with DAPI or PI which are DNA intercalators. For example, the stained cells are fixed on a slide or housed in a container such as a multiwell or petri dish to create a specimen, and an image is acquired via an imaging device equipped with a microscope.
A nucleus is recognized from the acquired image, each cell is specified, and the fluorescence amount in the nucleus is measured for each specified cell. For the measurement of the amount of fluorescence, a scatter diagram of the luminance is drawn from two parameters, the total luminance value for each image pixel in the nucleus and the maximum luminance value of the image pixel in the nucleus. Thereby, each cell is represented as a point on the scatter diagram. The cells can be stratified after the G1, S, G2, M, and M phases from the relative luminance values of individual cells in the cell population represented on the scatter diagram. Also, for each prepared specimen, calculate the ratio of cells corresponding to each of the G1, S, G2, M, and M periods, that is, calculate the ratio of cells corresponding to each period in the cell cycle. Is also possible.

This point will be described in detail.
When a DNA intercalator is used, the relationship of DNA amount = cell image brightness is established. For this reason, the cell cycle can be specified by using the following analysis method.
That is, the amount of DNA and the degree of nuclear aggregation differ at each stage of the cell cycle. The amount of DNA and the degree of aggregation of the nucleus are measured by the total luminance value for each image pixel in the nucleus and the maximum luminance value of the image pixel in the nucleus.
In the G1 phase, there are two chromosomes. The amount of DNA at this time is 2N. The S phase is in the DNA synthesis phase and is in the process of increasing from 2N to 4N. The G2 phase is in a preparation period before division, and the amount of DNA is doubled (4N) in the G1 phase. Further, in the M phase, the nuclei aggregate and become brighter.
Therefore, the G1, S, and G2 periods are specified by the amount of DNA (= sum of DAPI luminances). When a fluorescent reagent of a type that directly binds to DNA is used, the amount of fluorescence (the sum of the luminance of the nucleus region) = the amount of DNA.
The M period is judged by the brightness of the nucleus. Cells in division are bright and have nuclei aggregated. For this reason, the determination is made based on the maximum luminance in the nucleus region.

  However, in the method described in Non-Patent Document 1, DAPI used as a fluorescent reagent has cytotoxicity, and is killed so as not to cause an effect such as cell death or change in cell characteristics when staining. Although it is unavoidable to target fixed specimens, it is difficult to analyze while keeping cells alive, and it is possible to calculate the statistical proportion of cells corresponding to each phase of the cell cycle. It is impossible to grasp the rate of time of cell division and the situation of cell division in the cell division phase. In addition, since the method described in Non-Patent Document 1 needs to plot luminance values for several hundred or more cell populations in order to draw a scatter plot, the analysis is performed with the cells alive. However, since it takes too much time, it is not practical for analyzing the period of the cell cycle using living cells.

  On the other hand, with fluorescence microscopes (conventional microscopes, laser microscopes, etc.), time-lapse observation of live cells is possible, cell images can be acquired in sub-microsecond units, and the state of cell division in the M phase can be imaged. It is. By imaging such a state of cell division, cell shape information can be recognized. Further, if an image from when a cell is divided until the next division is acquired, the time required for one cycle of cell division (generation time) can be recognized from the cell shape information.

However, it is difficult to recognize each period in the cell cycle from a cell image only from cell shape information.
For example, Patent Document 1 shows that it is possible to specify the detailed period of cell division in the M phase from the shape of the cell or the like. However, the method described in Patent Document 1 is a method of measuring only the detailed period of cell division in the M phase, and cannot measure the time of each period in the cell cycle during cell culture. In addition, the method of Patent Document 1 has no idea about measuring the time of the detailed period of cell division in the M phase and the time of each detailed period.

In recent years, methods for visualizing the cell cycle, such as the methods described in Non-Patent Documents 2 and 3, have been proposed.
The method described in Non-Patent Document 2 introduces a fluorescent probe called FUCCI, which consists of two types of fluorescent proteins that can be fused to two types of proteins that exist only at specific times in the cell cycle, into the cell nucleus. This is a method for visualizing the cell cycle. A cell into which FUCCI has been introduced glows red in the G1 phase, and glows green in the S, G2, and M phases. If the method described in Non-Patent Document 2 is used, it is possible to measure the time of the G1 period and other periods. In addition, FUCCI, unlike fluorescent reagents such as DAPI, has no cytotoxicity, so not only the time of one cycle of the cell cycle (generation time) but also long-term measurement with cells remaining alive. Can do.

  However, the method described in Non-Patent Document 2 cannot distinguish between the S period, the G2 period, and the M period. Further, according to the method described in Non-Patent Document 2, it is impossible to measure the time of the detailed period of cell division in M phase and the time of each detailed period. In addition, the fluorescent protein has a weak fluorescent signal emitted from a living cell as compared with the fluorescent reagent described in Non-Patent Document 1 described above, and the signal is likely to vary from cell to cell. For this reason, in the method described in Non-Patent Document 2, it is difficult to analyze the cell cycle with high accuracy.

In the method of Non-Patent Document 3, the cell cycle is analyzed using a cell cycle marker (CCPM) in which a site emitting fluorescence in the cell moves from the cell nucleus to the cytoplasm according to the cell cycle.
However, in the method described in Non-Patent Document 3, since the cell cycle marker is toxic, the cell cannot be analyzed while it is alive, and each period in the cell cycle cannot be specified. In addition, the method described in Non-Patent Document 3 cannot measure the time of the detailed period of cell division in M phase and the time of each detailed period.

  Furthermore, each of these Patent Documents 1 and Non-Patent Documents 1 to 3 recognize each phase of the cell cycle, or a detailed phase in the M phase, and for each cell of a different type, each phase of the cell cycle, Since the time of the detailed period in the M period is not measured, the types of cells in the cell image cannot be determined by these methods.

JP 2008-164551 A

Product Catalog “Laser Scanning Cytometer LSC2”, Olympus Optical Co., Ltd. "Visualizing Spatiotemporal Dynamics of Multi cellular Cell-Cycle Progression", Cell 132, p487-498, February 8,2008, Elsevier inc. “G1 / S Cell Cycle Phase Maker Assay”, GE Healthcare, [online], http://www.gelifesciences.co.jp/technologies/cell-cycle/g1s.asp

As described above, in recent years, research on iPS cells and cancer stem cells for medical applications has been actively conducted in the observation of living cells. Also, iPS cell research has pointed out the problem of canceration after iPS cells are established.
For this reason, for example, identification of cells established with iPS cells and other cells, identification of cells established with iPS cells and cells differentiated after establishment, identification of cells differentiated after establishment of iPS cells, etc. In order to accurately grasp changes in the state of cells in the cell cycle, there is a growing need for quantification of time information related to cell division and cell tracking.

In addition, there are many needs to observe living cells in general, including differentiation analysis of iPS cells.
From the cells in the fixed state (dead state), it is impossible to accurately grasp the state of the cells at the time points before and after the fixed state, and the state of the cells at the time points before and after can generally be estimated statistically. On the other hand, if the cells are observed while they are alive, the state of the cells at the time points before and after the present time can be accurately grasped.
Stem cells including iPS cells repeat division and differentiation ("stem = original state" changes to a specific type of cell), but in particular, observe how differentiation proceeds. For this reason, living cells cannot be killed (fixed) during observation.
In addition, by observing living cells, it is possible to extract unique cells from the cell population. For example, in cancer cells, only a small number of cells may cause abnormalities during division. It is also known that iPS cells are likely to become cancerous depending on the preparation method. In order to extract and analyze specific cells within such a cell population, it is also necessary to analyze living cells in order to trace changes in the state of individual cells over time. is necessary.

  However, in the techniques described in Patent Document 1 and Non-Patent Documents 1 to 3, as described above, each period of the cell cycle or the detailed period in the M phase is recognized, It is difficult to specify different types of cells by these methods because the time of the detailed period in each period of the cycle and M period is not measured. For this reason, it is difficult to recognize in advance, for each identified type of cell, cells that are likely to continue cell division due to canceration or the like, and to accurately grasp changes in the state of cells in the cell cycle.

  In the first place, fluorescent reagents such as DAPI and CCPM as in the methods described in Non-Patent Documents 1 and 3 are toxic and are not suitable for observing living cells. Viable cells can be observed by using the fluorescent protein described in Non-Patent Document 2, but the fluorescence emitted from the fluorescent protein is weaker than the fluorescence emitted from a normal fluorescent reagent. Depending on the conditions such as the cell environment (temperature and Ph) and the number of surrounding cells, the fluorescence amount (luminance) of the fluorescent protein varies greatly. The G1, S, and G2 periods cannot be distinguished, and the detailed period in the M period cannot be classified. For this reason, in the conventional method, it is difficult to specify the period of the cell cycle and the detailed period in the M period while observing the living cells.

  The present invention has been made in view of such conventional problems. During the acquisition of a microscopic image of a living cell, each phase of the cell cycle and the detailed phase of the M phase are specified, and according to the specified phase. The cell type can be recognized by, for example, discriminating between cells established with iPS cells and other cells, distinguishing between cells established with iPS cells and cells differentiated after establishment, and every cell differentiated after iPS cells are established. It is an object of the present invention to provide a cell image analysis system that can accurately identify changes in the cell state in the cell cycle.

  In order to achieve the above object, a cell image analysis system according to the present invention includes a microscope for acquiring, in time series, images of living cells into which fluorescent proteins that specifically emit fluorescence in a specific phase of the cell cycle are introduced. A cell image analysis system comprising: an image acquisition device; and a cell image analysis device including a computer that performs a predetermined analysis on a living cell using images acquired in time series by the image acquisition device, wherein the cell The image analysis apparatus includes: a pixel group in which the fluorescence luminance exceeds a predetermined luminance threshold value in the image acquired by the image acquisition device; or a pixel group in which a luminance difference from adjacent luminance exceeds a predetermined luminance difference threshold value Cell region specifying means for specifying the cell region as a cell region, and collating it with a data table storing the period of the cell cycle corresponding to the fluorescence emitted from the fluorescent protein. Cell cycle period detection means for detecting the cell cycle period of a living cell, and the time of the cell cycle period until the cell cycle period of the living cell detected by the cell cycle period detection means changes to another period Image analysis software that functions as time measuring means for measuring the period of the cell cycle and identification means for identifying at least the cell type using the time of the cell cycle period.

  In the cell image analysis system of the present invention, the data table is the same type of cells as the living cells, in which the fluorescent protein is introduced in advance and stained with a predetermined fluorescent reagent different from the fluorescent protein, What is obtained by classifying the cell cycle period using fluorescence emitted from a predetermined fluorescent reagent and associating fluorescence emitted from the predetermined fluorescent reagent with fluorescence emitted from the fluorescent protein Is preferred.

  Further, in the cell image analysis system of the present invention, the fluorescent protein is two fluorescent proteins having different colors, and the cell cycle period detecting means includes the pixel group in the pixel group specified as the cell area by the cell area specifying means. The ratio of the total amount of fluorescent luminance between different colors emitted from the fluorescent protein and the amount of increase in the total amount of fluorescent luminance for each different color are preliminarily introduced with the fluorescent protein and stained with a predetermined fluorescent reagent different from the fluorescent protein. An image of a cell of the same type as the living cell is acquired through the image acquisition device and emitted from the predetermined fluorescent reagent in the data table creation pixel group specified as a cell region through the cell region specifying means. The period of the cell cycle is classified using the total amount of fluorescence brightness and the maximum value of fluorescence brightness, and the data table creation pixel group is classified. The ratio of the total amount of fluorescence brightness emitted from the predetermined fluorescent reagent and the maximum value of the fluorescence brightness and the total amount of fluorescence brightness emitted from the fluorescent protein in the data table creation pixel group and the fluorescence for each different color A cell corresponding to the ratio of the total amount of fluorescence brightness between different colors emitted from the fluorescent protein and the increase amount of the total amount of fluorescence brightness for each different color obtained by associating with the increase amount of the total amount of brightness It is preferable to detect the period of the cell cycle of the living cell in comparison with a data table in which the period of the cycle is stored.

  In the cell image analysis system of the present invention, the time of the cell cycle period is preferably a generation time until the cell cycle period of the living cell changes to the cell cycle period in the next generation. .

  In the cell image analysis system of the present invention, it is preferable that the time of the cell cycle period is a period of time until the cell cycle period of the living cell changes to the next period.

  In the cell image analysis system of the present invention, when the cell cycle period of the living cell detected by the cell cycle period detection means is M phase, the morphological characteristic element of the living cell is expressed as M phase. The detailed period detecting means in the M phase for detecting the detailed period in the M phase of the living cell by collating with the data table storing the detailed period in the M phase corresponding to the morphological characteristic element of the living cell in Is preferably further provided.

  In the cell image analysis system of the present invention, the detailed period within the M phase detected by the detailed period detecting means within the M period changes to the next detailed period or the next cell cycle period. It is preferable to further include time measuring means for the detailed period within the M period for measuring the time of the detailed period.

  In the cell image analysis system of the present invention, the time of the first period until the change from the first period of the living cell to the next period measured by the time measurement means of the period of the cell cycle, A time comparison means for comparing the period of the cell cycle with the time of the second period from the second period of the living cell measured by the time measurement means of the cell cycle period to the next period. Is preferred.

  In the cell image analysis system of the present invention, the first detailed period in the M phase of the live cell measured by the detailed time measuring means in the M period is the next detailed period or the next cell cycle. The time of the first detailed period until it changes to the period and the second detailed period in the M period of the living cell measured by the time measuring means of the detailed period in the M period are the next detailed period or the next detailed period It is preferable to further comprise a time comparison means for the detailed period within the M period for comparing the time of the second detailed period until the cell cycle is changed.

  In the cell image analysis system of the present invention, the identification unit measures the time of the cell cycle period of the living cell measured by the time measurement unit of the cell cycle period, and the time measurement of the detailed period within the M phase. The time of the detailed period within the M phase of the living cell measured by the means, the comparison value of the time of the cell cycle period of the living cell compared with the time comparison means of the cell cycle, the detailed period within the M phase It is preferable to identify at least the cell type using at least one of the comparison values of the detailed period within the M phase of the living cell compared by the time comparison means.

  Moreover, in the cell image analysis system of the present invention, the identification means further includes the time of the cell cycle period of the living cell measured by the time measurement means of the cell cycle period, and the detailed period within the M phase. The time of the detailed period within the M phase of the living cell measured by the time measuring means, the comparison value of the time of the cell cycle period of the living cell compared with the time comparison means of the cell cycle, By using at least one of the comparison values of the detailed period within the M phase of the living cell compared by the detailed period comparison means, cell division, cell growth, cell population stratification, cell properties It is preferable to identify at least one of them.

  Further, in the cell image analysis system of the present invention, the time measurement means in the cell cycle period measures generation times of a plurality of generations in one living cell, the image analysis software further includes the computer, It functions as a generation time comparison means for comparing generation times of different generations of the one living cell measured by the time measurement means in the period of the cell cycle, and the identification means is further compared with the generation time comparison means By comparing the comparison value of generation times of different generations of one living cell with a data table storing the change information of living cells corresponding to the comparison value of generation times of different generations of living cells, Preferably, one living cell change is detected.

  In the cell image analysis system of the present invention, the time measurement means in the cell cycle period measures generation times of a plurality of generations in each of two or more living cells, and the image analysis software includes the computer. Furthermore, the identification means further functions as a generation time comparison means for comparing generation times of different generations in each of the two or more living cells measured by the time measurement means in the period of the cell cycle, The comparison value of the generation time between different generations in each of the two or more living cells compared by the generation time comparison means, and the stratification information of the living cells corresponding to the comparison value of the generation times between the different generations of the living cells It is preferable to stratify the two or more living cells by collating with a data table in which is stored.

  In the cell image analysis system of the present invention, the time measurement means in the cell cycle period measures generation times of a plurality of generations in each of two or more living cells, and the image analysis software includes the computer. Furthermore, the identification means further functions as a generation time comparison means for comparing generation times of different generations in each of the two or more living cells measured by the time measurement means in the period of the cell cycle, The comparison value of the generation time between the different generations in each of the two or more living cells compared by the generation time comparison means, and the change information and layer of the living cell corresponding to the comparison value of the generation time between the different generations of the living cells By comparing with the data table in which the separate information is stored, changes in each of the two or more living cells are detected and the two Preferably stratify live cells above.

  In the cell image analysis system of the present invention, the image acquisition device is different in at least one of chemicals to be administered, light stimulation, genetic manipulation, environment such as temperature / humidity / pressure / PH, nutrient of medium, and the like. Images of living cells under the two measured conditions are acquired in time series, and the generation time comparison means is configured to generate the live time measured by the time measuring means in the cell cycle period under each of the two measurement conditions. It is preferred to compare cell generation times.

  In the cell image analysis system of the present invention, the living cells are preferably at least one of ES cells, iPS cells, and cancer stem cells.

  In the cell image analysis system of the present invention, the living cells are preferably at least one of ES cells, iPS cells, and cells differentiated from cancer stem cells.

  In the cell image analysis system of the present invention, the fluorescent protein is preferably FUCCI.

  According to the present invention, while acquiring a microscopic image of a living cell, it is possible to identify each phase of the cell cycle, the detailed phase of the M phase, identify the identified cell, and recognize the cell type according to the identified phase. For example, identification of cells established with iPS cells and other cells, identification of cells established with iPS cells and cells differentiated after establishment, identification of cells differentiated after establishment of iPS cells, and cell cycle A cell image analysis system capable of accurately capturing changes in the state of cells in a cell is obtained.

1 is a block diagram showing an overall configuration of a cell image analysis system according to an embodiment of the present invention. The figure shows the correlation between the fluorescence intensity emitted from the cells and the cell cycle. (A) shows the fluorescence intensity emitted from the fluorescent reagent staining the cells in the pixel group specified as the cell region in the captured cell image. An explanatory diagram showing the correlation between the total amount and the maximum value of fluorescence brightness and the cell cycle period of the cell, (b) is emitted from the fluorescent protein introduced into the cell in the pixel group specified as the cell region in the captured cell image Explanatory drawing which shows the time passage characteristic of the total amount of fluorescence luminance for each different color, (c) is a group of pixels identified as a cell region in the captured cell image classified by the association of (a) and (b) It is a figure which shows the correlation with the total amount of the fluorescence brightness | luminance for every different color emitted from the fluorescent protein introduce | transduced into the cell, and the period of the cell cycle of a cell. It is explanatory drawing which shows typically the state of the cell for every detailed period in M phase, (a) is a figure which shows the state of the cell in the first period, (b) is a figure which shows the state of the cell in the previous middle stage, (c) (D) is a figure which shows the state of the cell in a late stage, (e) is a figure which shows the state of the cell in a terminal stage, (f) is a figure which shows the state of cytokinesis. It is explanatory drawing which shows the outline of the whole process sequence from introduction | transduction of the fluorescent protein to the living cell as a process of the previous step using the cell image analysis system of this embodiment, and analysis of a living cell image. It is a flowchart which shows the process sequence of the cell image analyzer in the cell image analysis system of this embodiment. 1 is a block diagram showing the overall configuration of a cell image analysis system of Example 1. FIG. It is a graph which shows typically the generation time for every cell from which a kind differs. It is a block diagram which shows the whole structure of the cell image analysis system of Example 2. FIG. It is a graph which shows typically the correlation with the total value of the generation time according to the generation number for every cell from which a kind differs, and the generation number. It is a block diagram which shows the whole structure of the cell image analysis system of Example 3. It is a graph which shows typically the ideal value and measured value in the correlation with the total value of the generation time according to the generation number of a specific cell, and the generation number. It is a block diagram which shows the whole structure of the cell image analysis system of Example 4. It is a graph which shows typically the time of the detailed period in the M phase for every different type of cells. It is a block diagram which shows the whole structure of the cell image analysis system of Example 5. It is a graph which shows typically the time of each period of the cell cycle in one generation for every kind of different cells. It is a block diagram which shows the whole structure of the cell image analysis system of Example 6. FIG. 10 is a block diagram showing the overall configuration of a cell image analysis system of Example 7. FIG. 20 is a block diagram showing the overall configuration of a cell image analysis system of Example 18.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a block diagram showing the overall configuration of a cell image analysis system according to an embodiment of the present invention.
The cell image analysis system of this embodiment includes an image acquisition device 1, a cell image analysis device 2, a culture device (not shown), and an electric stage (not shown).
The image acquisition apparatus 1 is configured using a microscope that observes a fluorescence image of a cell, such as a fluorescence microscope or a confocal laser fluorescence microscope, and includes a microscope observation optical system 1a and an imaging unit 1b.
The microscope observation optical system 1a includes a general fluorescence microscope including a light source, an illumination lens, excitation light switching means including a plurality of types of excitation filters in a turret, an objective lens, an absorption filter, an imaging lens, and the like. It comprises an illumination optical system and an observation optical system (not shown) in a laser fluorescence microscope, and forms an image of the sample on the imaging surface of the imaging device 1b2.
The imaging unit 1b includes an imaging time control unit 1b1 and an imaging element 1b2. The imaging element 1b2 captures a cell image formed through the microscope observation optical system 1a. The imaging time control unit 1b1 controls the imaging element 1b2 to perform an imaging operation intermittently at a predetermined time interval.
With such a configuration, the image acquisition device 1 has two fluorescent proteins having different colors that emit fluorescence specifically in a specific period of the cell cycle, or one fluorescent protein that emits different colors depending on the period of the cell cycle. An image of a living cell into which is introduced is acquired in time series at predetermined time intervals.

  Note that the time interval by the imaging time control means 1b1 for acquiring time series images of live cells is a sampling time which is a unit for calculating the time of each period in the cell cycle and the detailed period within the M period. Become. For this reason, a shorter time interval is desirable. In the image acquisition device 1 in the cell image analysis system of the present embodiment, the imaging time control means is configured to acquire images at a predetermined time interval from a short time of ms to several hundred ms to a long time of about 1 hour. The time interval 1b1 captures images is controlled. On the other hand, since one cycle of the cell cycle is about one day, if the time interval at which the image acquisition device 1 in the cell image analysis system of the present embodiment acquires an image, It is possible to sufficiently maintain the accuracy of calculating the time of the detailed period within the M period.

A culture apparatus (not shown) cultivates a cell sample accommodated in a container such as a dish or a microplate while keeping temperature and humidity constant.
The electric stage (not shown) includes an XY stage and the like, and conveys a dish or microplate containing live cells to an imaging position in the image acquisition device 1.

The image acquisition device 1 in the cell image analysis system of the present embodiment is capable of acquiring images of a plurality of living cells at the same time as long as it is within one field of view in microscopic observation.
For living cells that do not fit in a single field of view under microscope observation, it is possible to acquire images of living cells over a wide range by using an XY stage or the like so that the living cells are positioned at the imaging position. It has become. Alternatively, instead of using the XY stage, the image pickup device 1b2 is configured by a line sensor type TDI (Time Delay Integration) camera so that an image of a living cell can be acquired over a wide range. Good.

The cell image analysis apparatus 2 includes a computer (for example, a personal computer) and image analysis software.
The image analysis software includes a computer, a cell region specifying unit 2a, a cell cycle period detection unit 2b, a cell cycle period time measurement unit 2c, a detailed period detection unit 2d within the M period, and a detailed period time within the M period. The measuring means 2e, the time comparison means 2f in the cell cycle period, the detailed time comparison means 2g in the M period, and the identification means 2h are configured to function.

  In the image acquired by the image acquisition device 1, the cell region specifying unit 2 a selects a pixel group in which the fluorescence luminance exceeds a predetermined luminance threshold value or a pixel group in which a luminance difference from adjacent luminance exceeds a predetermined luminance value threshold value. Specify as an area.

The cell cycle period detection means 2b is configured to determine the total amount of fluorescence luminances of different colors emitted from the fluorescent protein in the pixel group identified as the cell region by the cell region identification unit 2a (that is, the fluorescence luminance of each pixel constituting the pixel group). The ratio of the total value) and the increase in the total amount of fluorescence luminance for each different color are collated with the data table 2t-1, and the cell cycle period of the living cell is detected.
The data table 2t-1 acquires, via the image acquisition device 1, an image of a cell of the same type as that of a living cell, in which a fluorescent protein is introduced in advance and stained with a predetermined fluorescent reagent different from the fluorescent protein. The total amount of fluorescence luminance emitted from a predetermined fluorescent reagent in the data table creation pixel group specified as the cell region via the means 2a (that is, the total value of the fluorescence brightness of each pixel constituting the data table creation pixel group) And the maximum value of the fluorescence brightness (that is, the fluorescence brightness of the pixel having the highest fluorescence brightness among the pixels constituting the pixel group for creating the data table), and classifying the period of the cell cycle, and the pixel for creating the data table The total amount of fluorescence luminance emitted from a given fluorescent reagent in the group, the maximum value of fluorescence luminance, and the fluorescence protein emitted from the data table creation pixel group The ratio of the total amount of fluorescent luminance between different colors emitted from fluorescent proteins, obtained by associating with the ratio of the total amount of fluorescent luminance between different colors and the increase amount of the total amount of fluorescent luminance for each different color In addition, the period of the cell cycle corresponding to the increase amount of the total amount of fluorescence luminance for each different color is stored.

  FIG. 2 is a diagram showing the correlation between the fluorescence intensity emitted from the cells and the cell cycle. (A) is emitted from the fluorescent reagent staining the cells in the pixel group specified as the cell region in the captured cell image. Explanatory diagram showing the correlation between the total amount of fluorescence brightness and the maximum value of fluorescence brightness and the cell cycle period, (b) is the fluorescence introduced into the cell in the pixel group specified as the cell region in the captured cell image Explanatory diagram showing the time course characteristics of the total amount of fluorescent luminance for each different color emitted from protein, (c) is identified as a cell region in the captured cell image classified by the correspondence between (a) and (b) It is a figure which shows the correlation with the total amount of the fluorescence luminance for every different color emitted from the fluorescent protein introduce | transduced into the cell in the pixel group, and the period of the cell cycle of the cell.

In FIG. 2 (a), the total amount of fluorescence luminance reflects the amount of DNA in the cell. In addition, the maximum value of intracellular fluorescence luminance reflects the degree of chromatin aggregation.
The cell cycle is divided into an M phase, which is a phase in which cell division occurs, and an I phase, which is a phase between M and M phases. Furthermore, the I phase is a G1 phase which is a preparation phase for DNA synthesis, an S phase where DNA is synthesized and chromosomes are replicated, and a cell division preparation phase between the S phase and the M phase. It is divided into a certain G2 period. Further, there is a G0 phase as a phase out of the cell cycle.

In the M phase, the replicated chromosomes are condensed by the synthesis of intracellular DNA during cell division. For this reason, in the M period, as shown in FIG. 2 (a), the total amount of fluorescence luminance is large and the maximum value of fluorescence luminance is large.
After cell division (post M phase), there is less DNA in each divided cell. In the G1 phase, enzymes necessary for DNA synthesis are activated. For this reason, in the G1 period, as shown in FIG. 2A, the total amount of fluorescence luminance is small.
Note that cells normally shift to S phase due to enzyme activation in G1 phase, but cells that do not continue cell division shift to G0 phase.
In the S phase, DNA is synthesized in the cell and the chromosome is replicated. For this reason, as shown in FIG. 2 (a), the total amount of fluorescence luminance is increased.
In the G2 phase, protein synthesis increases in the cell as the final step in preparation for cell division. However, at this stage, the chromosomes are not aggregated. For this reason, as shown in FIG. 2A, the total amount of fluorescence luminance is large and the maximum value of fluorescence luminance is small.

Thus, there is a correlation between the total amount of fluorescence luminance in the pixel group of living cells and the maximum value of fluorescence luminance and the period of the cell cycle of living cells.
Such correlation is the maximum amount of fluorescence brightness in the pixel group specified as each cell region and the maximum fluorescence brightness in the pixel group specified as the cell region, stained with a fluorescent reagent such as DAPI. Can be determined using a relative value in the scatter diagram. However, the method using a fluorescent reagent such as DAPI cannot keep the cells alive for a long time.

  In addition, a fluorescent probe using two fluorescent proteins having different colors that emit fluorescence specifically in a specific phase of the cell cycle, such as FUCCI, or one fluorescent protein that emits different colors depending on the phase of the cell cycle When a specific protein is expressed depending on the phase of the cell cycle, the fluorescent protein is simultaneously expressed and emits different fluorescence, or one fluorescent protein emits a different color depending on the phase of the cell cycle. For this reason, the temporal change in the total amount of fluorescent luminance for each different color emitted from the fluorescent protein in the cell (for example, the change in the luminance value of each of red and green light in FUCCI as shown in FIG. 2 (b) For example, it is considered that the reference luminance is set as a threshold value for each of red and green light, and the period of the cell cycle can be determined based on the relative magnitude relationship with the reference luminance. However, when FUCCI is used, the brightness values of red and green vary greatly due to factors such as the nature of each cell and the surrounding noise of the cell being observed, and the green is stronger than red. It is difficult to distinguish each phase (S phase, G2 phase, M phase) of the cell cycle. Further, in order to increase the accuracy of the reference luminance, the average of the luminance values of a large number of cells must be used as the reference luminance. However, in that case, it is accurate for cells that emit light having a luminance value far from the reference luminance. It becomes difficult to specify the period.

  Therefore, in the present invention, by combining the method using a fluorescent reagent such as DAPI described above and the method using FUCCI in advance, the ratio of the total amount of fluorescent luminance between different colors emitted from the fluorescent protein and the different colors A data table 2t-1 in which the period of the cell cycle corresponding to the amount of increase in fluorescence brightness for each cell is stored. Then, the cell cycle period detection means 2b is configured to calculate the ratio of the total amount of fluorescent luminance between different colors emitted from the fluorescent protein in the pixel group specified as the cell region by the cell region specifying means 2a and the total amount of fluorescent luminance for each different color. By comparing the increase amount with this data table 2t-1, the period of the cell cycle of the living cell is detected.

Here, a specific example of creating the data table 2t-1 will be described.
For example, cell nuclei are stained using Hoechst and DRAQ5 as a DNA intercalator that is a predetermined fluorescent reagent. As will be described later, this cell has two fluorescent proteins with different colors that emit fluorescence specifically in a specific phase of the cell cycle, or one fluorescent protein that emits different colors depending on the phase of the cell cycle. FUCCI has also been introduced.

First, the cell cycle phase is detected using a fluorescent reagent.
A cell image is acquired via the image acquisition device 1. Next, the region of the cell nucleus in the cell is specified from the acquired cell image via the cell region specifying means 2a of the cell image analyzer 2.
Next, a scatter diagram is created using the total amount of fluorescence luminance and the maximum value of fluorescence luminance as parameters in the data table creation pixel group identified by the cell region identification means 2a as the cell nucleus region in the cell (FIG. 2 (a)). Thereby, each cell which comprises a cell population is represented as a point on a scatter diagram. Next, the G1 phase, S phase, G2 phase, M phase, and post M phase (after division) are stratified from the relative luminance values of the respective cells constituting the cell population in the scatter diagram.

In general, in the G2 period and the M period, the total value of luminance is about twice that in the G1 period and the post M period (after division). Therefore, in the scatter diagram, the range in which the total luminance value is distributed twice is determined. Of these ranges, the range with the smaller total luminance is the G1 period and post M period (after division) range, and the larger range is the G2 period and M period range. Next, the average value (μ) and standard deviation (sigma: σ) of the total luminance values in the respective ranges corresponding to the G1 period and the post M period (after division), the G2 period and the M period are calculated. And the average value μ of the total value in the range corresponding to the G1 period and the post M period (after division) exceeds + 3σ, and the average value of the total value in the range corresponding to the G2 period and the M period The range below μ to −3σ is the range of S period.
Next, the average value (μ) and standard deviation (sigma: σ) of the maximum luminance values in the range of G1 period and post M period (after division) are calculated. Then, the range of ± 3σ from the average value μ of the maximum luminance value is set as the range of the G1 period, and the other range is set as the range of the post M period (after division).
Similarly, the average value (μ) and standard deviation (sigma: σ) of the maximum luminance values in the G2 period and M period ranges are calculated. Then, the range of ± 3σ from the average value μ of the maximum luminance values is set as the G2 period range, and the other ranges are set as the M period range.
The G1 period, G2 period, S period, M period, and post M period (after division) corresponding to the maximum luminance value and the total luminance value thus determined are stored in the storage area of the computer.

Next, the period of the cell cycle obtained using the fluorescent reagent is associated with the total amount of fluorescence luminance for each different color emitted from the fluorescent protein.
For example, a fluorescent probe called FUCCI, which is composed of two types of fluorescent proteins that can be fused to two types of proteins, Geminin and Cdt1, which are present only at different specific times in the cell cycle, is introduced into the nucleus of the cell. Yes.
Of the fluorescence in the data table creation pixel group specified by the cell region specifying means 2a as the cell nucleus region in the cell, green light emitted from mAG1, which is a fluorescent protein fused with Geminin, and mkO2, which is a protein fused with Cdt1 The red light is detected from each image acquired at a predetermined pitch in the time corresponding to one cycle of the cell cycle. Here, the time for which the change in the characteristics of the green light and the red light makes one turn is one cycle. As a result, a graph showing the characteristics of the total amount of the luminance value with respect to time in each of the green light and the red light as shown in FIG.
At this time, in the pixel group specified by the cell region specifying means 2a as the cell nucleus region in the cell, as shown in FIG. 2 (a), the total amount of fluorescent luminance and the maximum fluorescent luminance are obtained by the method using the fluorescent reagent described above. As the values are obtained, the phases of the cell cycle are stratified.

Therefore, next, the total amount of fluorescence brightness and the maximum value of fluorescence brightness in the cell group in the scatter diagram shown in FIG. 2 (a) and the brightness values for the respective times of green light and red light shown in FIG. 2 (b). And the associated data are stored in the storage area of the computer. As a result, the characteristics of the luminance values with respect to time of the green light and the red light obtained in FIG. 2 (b) and the G1, S, G2, and M phases of the cell cycle are shown in FIG. ).
Next, in FIG. 2 (c), the ratio of the total amount of luminance values of green light and red light in each period divided into the G1, S, G2, and M phases of the cell cycle, and the green light And the amount of increase in the total amount of luminance values of red light is calculated, and the calculated value is stored in a storage area of the computer.
As a result, the ratio of the total amount of fluorescent luminance between different colors emitted from fluorescent proteins in the data table pixel group and the period of the cell cycle corresponding to the increased amount of fluorescent luminance for each different color are stored in the storage area of the computer. A data table 2t-1 is created.

  When the data table 2t-1 of the above creation example is used, the cell cycle period detection means 2b is fused to Cdt out of the total amount of fluorescence luminance values in the pixel group specified as the cell area by the cell area specifying means 2a. When the red light emitted from mKO2 which is the fluorescent protein is larger than the green light emitted from mAG1 which is the fluorescent protein fused with Geminin, the G1 phase is detected by collating with the data table 2t-1. Further, when the green light emitted from mAG1 is larger than the red light emitted from mKO2, the ratio of the total amount of green fluorescence emitted from mAG1 to the total amount of fluorescence of red light emitted from mKO2, and the respective fluorescence luminances The cell cycle period (any one of the S period, the G2 period, and the M period) of the living cell is detected by collating with the data table 2t-1 in which the period of the cell cycle corresponding to each increase amount is stored.

  By using the data table 2t-1 created in this way, the cells actually observed need not be stained with a highly toxic fluorescent reagent, so that the cells remain alive for a long time. Can do. Further, when the data table 2t-1 is created, the ratio of the total amount of fluorescence brightness emitted from the fluorescent protein to the total amount of fluorescence brightness emitted from the fluorescent reagent and the maximum value of the fluorescence brightness, and the fluorescence brightness for each different color. Since the increase amount of the total amount is associated, it is possible to correct the variation in the luminance value of the fluorescence for each cell when the fluorescent protein is used, and to accurately discriminate the S phase, the G2 phase, and the M phase. As a result, the period of the cell cycle can be accurately detected while observing living cells using the fluorescent protein.

The cell cycle period time measurement means 2c measures the generation time until the cell cycle period of the living cell detected by the cell cycle period detection means 2b changes to the cell cycle period in the next generation.
As described above, in the cell image analysis system of the present embodiment, the image acquisition device 1 acquires live cell images in time series at regular time intervals via the imaging time control means 1b1 and the like. Therefore, the desired time can be calculated by multiplying the fixed time interval controlled by the imaging time control means 1b1 by the number of images analyzed by the cell image analysis device 2.
Specifically, the time measurement means 2c in the cell cycle period is an analysis target until the cell cycle period of the living cell detected by the cell cycle period detection means 2b changes to the cell cycle period in the next generation. The number of images obtained is counted, and the number of images is multiplied by a predetermined time interval determined in the imaging time control means 1b1. Thereby, the generation period is calculated.

In this embodiment, the time measuring means 2c in the cell cycle period measures the period time until the cell cycle period of the living cell detected by the cell cycle period detecting means 2b changes to the next period. It also has a function to do.
That is, the time measuring means 2c in the cell cycle period counts the number of images to be analyzed until the cell cycle period of the living cell detected by the cell cycle period detecting means 2b changes to the next period. Then, the number of images is multiplied by a predetermined time interval determined in the imaging time control means 1b1. Thereby, the time of the cell cycle period is calculated.
That is, the “time of the cell cycle period” here is the generation time until the cell cycle period of the living cell changes to the cell cycle period of the next generation, and the cell cycle period of the living cell. Any period of time until the next period can be reached.

  The detailed phase detection means 2d in the M phase, when the cell cycle phase of the living cell detected by the cell cycle phase detection means 2b is the M phase, displays the morphological characteristic element of the living cell in the M phase. The detailed period in the M phase of the living cell is detected by collating with the data table 2t-2 in which the detailed period in the M phase corresponding to the morphological characteristic element of the living cell is stored.

FIG. 3 is an explanatory diagram schematically showing the state of cells in each detailed period within M phase, (a) is a diagram showing the state of cells in the previous period, (b) is a diagram showing the state of cells in the previous mid-stage, (c) is a diagram showing the state of the cell in the middle phase, (d) is a diagram showing the state of the cell in the late phase, (e) is a diagram showing the state of the cell in the final phase, (f) is a diagram showing the state of cytokinesis It is.
In the M phase, mitosis and cytokinesis occur. The M stage is divided into the first, middle, middle, late, and final stages according to the stage of mitosis.
In the first period, as shown in FIG. 3 (a), the chromosome 3a is condensed, two centrosomes (central bodies) 3b are formed, and the microtubule 3c is extended. In the previous middle period, as shown in FIG. 3 (b), the nuclear membrane 3d disappears and the chromosome 3a begins to move to the equator plane. In the middle period, as shown in FIG. 3 (c), chromosomes 3a are arranged on the equator plane. In the latter period, as shown in FIG. 3 (d), the chromosome 3a starts to separate and starts moving in the polar direction. At the final stage, as shown in FIG. 3 (e), the chromosome 3a completes its movement to both poles, and no aggregation occurs, and a new nuclear membrane 3d is formed. Thereafter, cytokinesis begins during the terminal phase. And as shown in FIG.3 (f), a cell divides into two by cytokinesis.

Thus, in each detailed phase of the M phase, morphological features appear in the cells.
Therefore, in the cell image analysis system of the present embodiment, the data table 2t in which the detailed period in the M phase corresponding to the morphological feature element of the living cell in the M phase is stored in the storage area of the computer provided in the cell image analyzing apparatus 2. -2. Then, when the detailed period detecting means 2d in the M period is in the M period, the morphological characteristic element of the living cell is expressed as M, when the cell cycle period of the living cell detected by the cell cycle period detecting means 2b is M phase. The detailed period in the M phase of the living cell is detected by collating with the data table 2t-2 in which the detailed period in the M phase corresponding to the morphological characteristic element of the living cell in the phase is stored. .

The detailed period time measuring means 2e in the M phase changes the detailed period in the M phase of the living cell detected by the detailed period detecting means 2d in the M phase to the next detailed period or the next cell cycle period. Measure the time until the detailed period.
That is, the time measuring means 2e for the detailed period in the M period is used until the detailed period in the M phase of the living cell detected by the detailed period detecting means 2d in the M period changes to the next detailed period or The number of cell images to be analyzed until the end of the period changes to the G1 period which is the period of the next cell cycle is counted, and the number of images is counted at a predetermined time interval determined by the imaging time control means 1b1. Multiply. Thereby, the time of the detailed period within the M period is calculated.

The time comparison means 2f in the cell cycle period includes the first period time from the first period of the living cell measured by the time measurement means 2c in the cell cycle period to the next period, and the cell cycle. The time of the second period until the change from the second period to the next period of the living cells measured by the time measuring means 2c of the second period is compared. Then, for example, numerical data indicating an objective comparison value such as a time difference between the two periods is created.
In addition, as “the time of the first period” and “the time of the second period” here, for example, the time between two different cell cycle periods within the same generation, The time between periods of one cell cycle can correspond.

The detailed period time comparison means 2g in the M phase is changed until the first detailed period in the M phase of the living cell measured by the detailed time measurement means 2e in the M period changes to the next detailed period. Alternatively, the time of the first detailed period until the end of the M phase changes to the G1 phase, which is the next cell cycle period, and the M phase of the living cell measured by the detailed time measuring means 2e within the M phase. The time of the second detailed period is compared until the second detailed period changes to the next detailed period, or until the end of M phase changes to G1, which is the period of the next cell cycle. Then, for example, numerical data indicating an objective comparison value such as a difference in time between detailed periods within the M periods is created.
The “first detailed period time” and the “second detailed period time” are, for example, two different detailed periods in the same generation M period or two different detailed periods. It can correspond to the time of a detailed period within one M period in a generation.

The identification means 2h is the time in the cell cycle of the living cell measured by the time measurement means 2c in the cell cycle period, the time measurement means 2e in the detailed period in the M phase, and the time in the M phase of the living cell is measured. Detailed period time, cell cycle period time comparison means 2f compared with the cell cycle period time comparison value, and M period detailed period time comparison means 2g compared with the living cell M At least one of cell types is identified using at least one of the comparison values of the detailed periods of different detailed periods within the period.
The identification means 2h further includes the time of the cell cycle period measured by the time measurement means 2c in the cell cycle period, and the M phase of the living cells measured by the detailed time measurement means 2e within the M phase. The time of the detailed period in the cell, the comparison value of the time of the cell cycle of the living cell compared with the time comparison means 2f of the period of the cell cycle, and the living cell of the detailed period time comparison means 2g within the M phase Using at least one of the comparison values of the detailed periods between different detailed periods in the M phase, cell division, cell growth rate, cell population stratification, cell population growth rate, cell property at least Identify either.

Next, an overall processing procedure from live cell labeling / staining to analysis of live cell images using the thus configured cell image analysis system of the present embodiment will be described.
FIG. 4 is an explanatory diagram showing an outline of the entire processing procedure from introduction of fluorescent protein into living cells to analysis of images of living cells as a previous stage processing using the cell image analysis system of the present embodiment.
As shown in FIG. 4, the entire process includes introduction of fluorescent protein into live cells as a previous process (step S1), acquisition of live cell images by the image acquisition device 1 (step S2), and cell image analysis. This is performed in the order of analysis of live cell images by the apparatus 2 (step S3). After introducing the fluorescent protein into the living cell (step S1), time-lapse observation is performed, and the acquisition of the live cell image (step 2) and the analysis of the live cell image (step 3) are repeated.

Fluorescent protein introduction processing stage (step S1)
In the fluorescent protein introduction treatment step, FUCCI (as two fluorescent proteins having different colors that emit fluorescence specifically in a specific phase of the cell cycle, or one fluorescent protein that emits different colors depending on the phase of the cell cycle, is used. Fluorescent Ubiqutination-based Cell Cycle Indicator (Foot) is introduced into the cell. Then, a plurality of cells into which the fluorescent protein is introduced are seeded in, for example, a multiwell plate, and time-lapse observation can be performed with a microscope.

Cell image acquisition stage (step S2)
In the imaging processing stage, the microscope observation optical system 1a of the cell image acquisition device 1 configured using a microscope forms an image of a living cell in the sample, and the imaging element 1b2 is connected via the microscope observation optical system 1a. The imaged cell image is captured at a predetermined time interval. Thereby, the image of the living cell which introduce | transduced fluorescent protein is acquired in time series.

Analysis processing stage of live cell image (step S3)
FIG. 5 is a flowchart showing a processing procedure of the cell image analysis apparatus in the cell image analysis system of the present embodiment.
In the live cell image analysis processing stage, first, in the image acquired by the image acquisition device 1, the cell region specifying means 2a of the cell image analysis device 2 is a pixel group in which the fluorescence luminance exceeds a predetermined luminance threshold or adjacent to the pixel group. A pixel group whose luminance difference from the luminance exceeds a predetermined luminance difference threshold is specified as a cell region (step S31).

  Next, the cell cycle period detection means 2b determines the ratio of the total amount of fluorescent luminances of different colors emitted from the fluorescent protein in the pixel group specified as the cell region by the cell region specifying unit 2a and the total amount of fluorescent luminance for each different color. Data table 2t-1 in which the increase amount is stored as described above, the ratio of the total amount of fluorescence luminance between different colors emitted from the fluorescent protein, and the period of the cell cycle corresponding to the increase amount of the total amount of fluorescence luminance for each different color And the period of the cell cycle of the living cell is detected (step S32).

  Here, when the cell cycle phase shifts to the next phase, the time measurement means 2c in the cell cycle period causes the cell cycle period of the living cell detected by the cell cycle period detection means 2b to change to the next period. The number of images to be analyzed until they change is counted, and the number of images is multiplied by a predetermined time interval determined in the imaging time control means 1b1 to calculate the time of the cell cycle period (step) S33, S34).

  Further, when the cell cycle period shifts to the next generation of the next generation of the present generation, the time measurement means 2c of the cell cycle period detects the cell cycle period of the living cell detected by the cell cycle period detection means 2b. The number of images to be analyzed until they change to the period of the cell cycle in the next generation is counted, and the number of images is multiplied by a predetermined time interval determined in the imaging time control means 1b1 to generate a generation period. Is calculated (steps S35 and S36).

  Further, when the cell cycle period of the living cell detected by the cell cycle period detection means 2b is the M period, the detailed period detection means 2d in the M phase may determine the morphological characteristic element of the living cell, The detailed period in the M phase of the living cell is detected by collating with the data table 2t-2 in which the detailed period in the M phase corresponding to the morphological characteristic element of the living cell in the M phase is stored (step S37). , S38).

  Further, when the detailed period in the M phase shifts to the next period, the time measuring means 2e in the detailed period in the M period is detected by the detailed period detecting means 2d in the M period in the M phase of the living cell. The number of images to be analyzed until the detailed period changes to the next detailed period or the next cell cycle period is counted, and the number of images is counted at a predetermined time interval determined in the imaging time control means 1b1. By multiplying, the time of the detailed period within the M period is calculated (steps S39 and S40).

  Then, the time comparison means 2g of the cell cycle period is the time of the first period until the living cell changes from the first period to the next period measured by the time measurement means 2c of the cell cycle period, The time of the period of the cell cycle is compared with the time of the second period from the second period to the next period of the living cell measured by the time measurement means 2c, for example, the difference between the times of both periods, etc. Create numerical data showing objective comparison values. (Step S41).

  Further, the detailed period time comparison means 2g in the M phase is the next detailed period or the next detailed period or the next detailed period in the M phase of the living cell measured by the detailed time measurement means 2e in the M period. The second detailed period in the M phase of the living cell measured by the time measuring means 2e in the detailed period in the M period and the time period measuring means 2e in the detailed period in the M period is the next detailed period. Or, compare with the time of the second detailed period until it changes to the next cell cycle period, for example, create numerical data showing objective comparison values such as the difference of the time of the detailed period in both M phases (Step S42).

  Thus, in the cell image analysis system of the present embodiment, various information related to cell division is acquired using the image acquired via the image acquisition device 1. Therefore, the significance will be described.

The cell is provided with a control mechanism (cell cycle checkpoint) that monitors the progress of the cell cycle and decelerates or stops the progress of the cell cycle when the state of the cell is abnormal.
The cell cycle checkpoint is a complex composed of two types of proteins, protein phosphorylase (cyclin-dependent kinase: Cdk) and cyclin, which are called checkpoint regulators, when the next point is monitored and an abnormality is detected. The cell cycle progression is slowed or stopped by controlling the activity of.
DNA replication check Monitors whether DNA replication is complete and preparations are made to proceed to the M phase.
-Spindle assembly check Monitors whether spindle formation is normal and can shift to late M phase.
Chromosome segregation check Monitors whether normal chromosome segregation was made at the end of M phase.
DNA damage check Functions during G1, G1, and S phases, S phase, G2 and M phases, and monitors whether repair of DNA damage is completed.

The cell cycle checkpoint is, for example, a checkpoint when transitioning from the G1 phase to the S phase, an S phase checkpoint, a checkpoint when shifting from the G2 phase to the M phase, M phase, depending on the progress stage of the cell cycle Classified as checkpoints.
At the check point when transitioning from the G1 phase to the S phase, it is monitored whether the repair of DNA damage in the G1 phase has been completed, the nutrition necessary for DNA replication, the presence of growth factors, and the like.
At the S phase checkpoint, it is monitored whether there is any defect in DNA replication.
At the check point when transitioning from the G2 phase to the M phase, whether or not DNA replication is completed, whether or not DNA damage has been repaired, whether or not DNA distribution is possible is monitored.
In the M-phase checkpoint, it is monitored whether or not the spindle formation is completed in the middle M-phase.

  The G1 phase passage is cyclin D-Cdk4 complex, the S phase start is cyclin E-Cdk2 complex, the S phase passage is cyclin A-Cdk2 complex, and the G2 phase passage is cyclin A-Cdk1 (Cdc2). The start of the complex, M phase, is controlled by the cyclin B-Cdk1 (Cdc2) complex, respectively.

  Thus, when a cell does not satisfy the conditions for advancing normal cell division, such as DNA damage, due to a cell cycle checkpoint, the cell stops the cell cycle and repairs DNA damage etc. Prevents cell mutation such as canceration due to accumulation of genetic information.

By the way, it is considered that the failure of the control of the cell cycle progression by the cell cycle checkpoint causes the occurrence and progression of cancer (uncontrolled abnormal growth of cells).
For example, the tumor suppressor gene products p53, Rb, and BRCA1 are involved in cell cycle checkpoint control, and many tumor suppressor gene products are frequently inactivated in human cancers, causing many cancers. Often becomes. Abnormal cell cycle checkpoints result in genetic instability, which is a major feature in many cancer cells.
For this reason, changes in the time of each phase in the cell cycle and the time of each detailed phase in the M phase indicate a change in some state of the cell, and can also be a signal indicating a sign of canceration of the cell. It is considered a thing.

  Therefore, in the cell image analysis system of this embodiment, the cells are classified and stratified according to the length of time in the cell cycle period of the living cells or the detailed period in the M phase, and the ratio of the time lengths. ing. Specifically, the identification means 2h measures the time of the cell cycle period measured by the time measurement means 2c in the cell cycle period, and the living cells measured by the detailed time measurement means 2e within the M period. The comparison of the time of the detailed period in the M phase, the time comparison means 2f of the cell cycle period compared with the time of the cell cycle period of the living cell, and the time comparison means 2g of the detailed period in the M phase At least one of the comparison values of the detailed period within the M phase of the living cell is used to identify at least the cell type. Further, the identification unit 2h further includes the time of the cell cycle period measured by the time measurement unit 2c of the cell cycle period, the time period of the living cell measured by the time measurement unit 2e of the detailed period within the M phase. Time of detailed period within M phase, time comparison means 2f of cell cycle period compared with time comparison value of cell cycle period of living cell, time comparison means 2g of detailed period within M phase compared At least one of the comparison values of the detailed period within the M phase of the living cell is used to identify cell division, cell growth, cell population stratification, and / or cell properties. .

  For this reason, according to the cell image analysis system of the present embodiment, the cell type can be identified based on temporal information related to cell division, and further, for example, changes in cell characteristics, cell types, cell Differentiation stability, identification of iPS cell established cells and other cells, identification of differentiated cells after iPS cell establishment, identification of each differentiated cell, etc. New analysis data considered useful in research is obtained.

  In the cell image analysis system of this embodiment, the cell image analysis apparatus 2 can be connected to display means (not shown) or includes display means. Therefore, the data identified by the identification unit 2h can be displayed via the display unit.

Hereinafter, examples using the cell image analysis system of the present embodiment will be described.
Example 1
FIG. 6 is a block diagram showing the overall configuration of the cell image analysis system of Example 1, and FIG. 7 is a graph schematically showing generation times for different types of cells.
For example, established iPS cells, cells differentiated from iPS cells, cancer cells, egg cells, and the like have different generation times for each type.
Therefore, in the cell image analysis system of Example 1, the cell image analysis device 2 includes a data table 2t-3 that stores generation times for different types of cells. Further, the identification unit 2h compares the generation time of each living cell measured by the time measurement unit 2c in the cell cycle period with the data table 2t-3 in which the generation time for each different type of cell is stored. It is configured to identify the cell type.
According to the cell image analysis system of the first embodiment, the cell type can be specified from the generation time.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 2
FIG. 8 is a block diagram showing the overall configuration of the cell image analysis system of Example 2, and FIG. 9 schematically shows the correlation between the number of generations for different types of cells and the total value of generation times according to the number of generations. It is a graph.
For example, established iPS cells, cells differentiated from iPS cells, cancer cells, egg cells, etc. have different correlations between the number of generations and the total generation time according to the number of generations. The difference in relationships becomes clear with each generation.
Therefore, in the cell image analysis system according to the second embodiment, the cell image analysis apparatus 2 includes a data table 2t-4 that stores the generation number for each different type of cell and the generation time integrated value corresponding to the generation number. . In addition, the image analysis software provided in the cell image analysis device 2 is a computer, and when the cell cycle period of the living cell detected by the cell cycle period detection means 2b reaches the cell cycle period in the next generation. That is, it is configured to function as the generation number measuring means 2i that counts the number of generations every time the generation of each live cell specified by the cell region specifying means 2a changes. The time measuring means 2c ′ in the cell cycle period is configured to calculate the generation period of the generation and to integrate the generation periods for each generation. In addition, the identification unit 2h determines the integrated value of the generation time of each living cell measured by the time measuring unit 2c ′ in the cell cycle period and the generation number of each living cell measured by the generation number measuring unit 2i. The type of cell is specified by collating with the data table 2t-4 in which the number of generations for each different cell and the integrated value of the generation time according to the number of generations are stored.
According to the cell image analysis system of the second embodiment, the type of cell can be specified even when it is difficult to specify from the generation time of one generation.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 3
FIG. 10 is a block diagram showing the overall configuration of the cell image analysis system of Example 3, and FIG. 11 shows the ideal value and the measured value in the correlation between the number of generations of a specific cell and the total value of generation times according to the number of generations. It is a graph typically shown.
In general, a cell changes into a cell having a specific shape and function as it repeats division. Differentiation refers to this morphological and functional cell change. In general, cells have a shorter cell cycle and a tendency to repeat cell division vigorously as undifferentiated cells. In addition, normal tissue cells are unidirectionally differentiated. In contrast, cancer cells have the property of blastogenesis / dedifferentiation in which the direction of differentiation is reversed. In addition, cancer cells tend to proliferate faster and have higher malignancy as the degree of differentiation becomes lower, and cancer cells tend to have a lower malignancy as the degree of differentiation becomes higher. For this reason, the degree of differentiation of cells and the stability of differentiation are considered to be an index for detecting canceration of cells and the state of cancer cells.

Therefore, in the cell image analysis system of Example 3, the cell image analysis device 2 uses the generation value for each different type of cell and the correlation between the generation value and the degree of deviation from the ideal value according to the generation number. Is provided with a data table 2t-5 that stores the stability of cell differentiation according to the above. Further, the identification unit 2h further uses an integrated value of the generation time of each living cell measured by the time measuring unit 2c ′ in the cell cycle period and the number of generations of each living cell measured by the generation number measuring unit 2i. The number of generations in each living cell and the correlation of the generation time integration value according to the number of generations are calculated, and the calculated correlation value is used to calculate the generation time according to the number of generations and the number of generations for different types of cells. The configuration is such that the stability of cell differentiation is detected for each cell by comparing with the data table 2t-5 in which the ideal value of the correlation of values and the stability of cell differentiation corresponding to the degree of deviation from the ideal value are stored. Has been.
According to the cell image analysis system of Example 3, the stability of cell differentiation can be detected from the generation times of multiple generation cells, such as canceration of cells and malignancy of cells that have become cancerous. Can contribute to diagnosis.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIGS.

Example 4
FIG. 12 is a block diagram showing the overall configuration of the cell image analysis system of Example 4, and FIG. 13 is a graph schematically showing the time of the detailed period within the M period for each different type of cell.
For example, established iPS cells, cells differentiated from iPS cells, cancer cells, egg cells, and the like have different time periods in the M phase and the entire M phase for each type.
Therefore, in the cell image analysis system of Example 4, the cell image analysis apparatus 2 includes a data table 2t-6 in which the time in the M period for each different type of cell is stored in the detailed period. In addition, the identification unit 2h determines the time of the detailed period in the M phase and the time of the entire M period of each living cell measured by the time measuring unit 2e of the detailed period in the M phase for each cell of different types. The cell type is identified by collating it with the data table 2t-6 which stores the time in the detailed period.
According to the cell image analysis system of Example 4, the cell type can be specified from the time of the detailed period within the M period and the time of the entire M period.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 5
FIG. 14 is a block diagram showing the overall configuration of the cell image analysis system of Example 5, and FIG. 15 is a graph schematically showing the time in each phase of the cell cycle in one generation for each different type of cell.
For example, established iPS cells, cells differentiated from iPS cells, cancer cells, egg cells, and the like have different times and generation times in each phase of the cell cycle in each generation.
Therefore, in the cell image analysis system of Example 5, the cell image analysis apparatus 2 includes a data table 2t-7 that stores the time of the cell cycle in one generation for each different type of cell divided into the time of each period. Yes. In addition, the identification unit 2h uses the time and generation time of each cell cycle of each live cell in one generation measured by the time measurement unit 2c in the cell cycle period as the cell cycle in one generation for each different type of cell. The cell type is identified by collating it with the data table 2t-7 that stores the time in each period divided and stored.
According to the cell image analysis system of Example 5, the type of cell can be specified from the time and generation time of each phase of the cell cycle in one generation.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 6
FIG. 16 is a block diagram illustrating the overall configuration of the cell image analysis system according to the sixth embodiment.
It is considered that the generation time changes with cell changes such as canceration.
Therefore, in the cell image analysis system of Example 6, the cell image analysis apparatus 2 uses a living cell corresponding to a comparison value of generation times of different generations (for example, a difference value of both generation times) for different types of cells. Is provided with a data table 2t-8 storing the change information. In addition, the time measuring means 2c in the cell cycle period is configured to measure generation times of a plurality of generations in one living cell. Further, the image analysis software provided in the cell image analysis apparatus 2 is a generation time comparison means for comparing the generation times of different generations of the one living cell measured by the computer and further by the time measurement means 2c in the cell cycle period. 2j is also configured to function. Further, the identification unit 2h further compares the generation time comparison value between the different generations of the one living cell compared by the generation time comparison unit 2j with the comparison value of the generation time between the different generations of the living cells. By comparing with the data table 2t-8 in which the cell change information is stored, the change of the one living cell is detected.
According to the cell image analysis apparatus of the sixth embodiment, it is possible to detect a change in cells from a comparison value of generation times between different generations of one living cell.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 7
FIG. 17 is a block diagram illustrating the overall configuration of the cell image analysis system according to the seventh embodiment.
For cancer cells and the like, stratification is considered to be important in order to increase the subsequent therapeutic effect.
Therefore, in the cell image analysis system of Example 7, the time measuring means 2c ″ in the cell cycle period is configured to measure generation times of a plurality of generations in each of two or more living cells. The image analysis software provided in the cell image analysis apparatus 2 is a generation for comparing a generation time of different generations in each of the two or more living cells measured by the time measurement means 2c ″ of the cell cycle period with a computer. It is configured to function as the time comparison means 2j ′. Further, the identification unit 2h further compares the generation times of different generations in the two or more living cells compared by the generation time comparison unit 2j ′, and compares the generation times of different generations of living cells. It is configured to stratify the two or more living cells by collating with the data table 2-9 storing the stratification information of the living cells corresponding to the values.
Other configurations are substantially the same as those of the cell image analysis system shown in FIG.
According to the cell image analyzer of Example 7, the two or more living cells can be stratified from the comparison value of the generation times of different generations in each of the two or more living cells. .
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 8
FIG. 18 is a block diagram showing the overall configuration of the cell image analysis system of Example 18.
In the cell image analysis system according to the eighth embodiment, the cell image analysis apparatus 2 changes the living cells corresponding to the comparison value of the generation times between different generations (for example, the difference value between the two generation times) for different types of cells. A data table 2t-10 storing information and stratification information is provided. Further, the time measuring means 2c ″ at the cell cycle period is configured to measure generation times of a plurality of generations in each of two or more living cells. Further, the image analysis software provided in the cell image analysis apparatus 2 Is configured to cause the computer to function as generation time comparison means 2j ′ for comparing generation times of different generations in each of the two or more living cells measured by the time measurement means 2c ″ in the cell cycle period. ing. Further, the identification unit 2h further compares the generation times of different generations in the two or more living cells compared by the generation time comparison unit 2j ′, and compares the generation times of different generations of living cells. By comparing with the data table 2t-10 in which the change information and stratification information of the live cell corresponding to the value are stored, the change in each of the two or more live cells is detected and the two or more live cells are detected. It is configured to stratify cells.
According to the cell image analysis apparatus of Example 8, two changes in each of the two or more living cells are detected simultaneously from the comparison values of the generation times of the different generations in each of the two or more living cells. The above living cells can be stratified.
Other configurations and operational effects are substantially the same as those of the cell image analysis system shown in FIG.

Example 9
In the cell image analysis system of Example 9, the image acquisition device 1 varied at least one of chemicals to be administered, light stimulation, genetic manipulation, environment such as temperature / humidity / pressure / PH, nutrient of medium, and the like. It is configured to acquire live cell images under two measurement conditions in time series.
The generation time comparison means 2j (2j ′) compares the generation time of the living cell measured by the time measurement means 2c (2c ′, 2c ″) in the cell cycle period under each of the two measurement conditions. .
Other configurations are substantially the same as those of any of the cell image analysis systems of Examples 1-8.

In the cell image analysis system of each of the above embodiments, it is preferable that the living cell to be analyzed is at least one of an ES cell, an iPS cell, and a cancer stem cell. Alternatively, the living cells may be at least one of ES cells, iPS cells, and cells differentiated from cancer stem cells.
Furthermore, in the cell image analysis system of each of the above embodiments, the fluorescent dye is preferably FUCCI.

According to the cell image analysis system of each of the above embodiments, cells that are likely to continue cell division due to canceration or the like can be recognized in advance, and changes in cells in the cell cycle can be accurately captured.
Note that the cell image analysis system of the present invention is not limited to the configuration described in the above-described embodiments, and for example, may be configured by arbitrarily combining the configurations specific to each of the above-described embodiments.

In the cell image analysis system of the present invention, the image acquisition device 1 is configured to acquire a detailed image for a specific cell according to the analysis result analyzed through the cell image analysis device 2. The image acquisition operation may be controlled.
For example, when a specific cell is detected through the cell image analysis device 2, the magnification of the microscope observation optical system 1a in the image acquisition device 1 is increased, or the imaging time interval of the imaging time control means 1 is shortened. A detailed image at the time of division of this specific cell may be acquired by controlling such as.

In addition, it is preferable that the time for acquiring the cell image is short in order to accurately measure the time of the cell cycle period. On the other hand, when the microscope apparatus used as the image acquisition apparatus 1 is a laser microscope, it is preferable that the imaging interval is long in consideration of damage to cells by the laser during imaging. Therefore, from the data obtained via the cell image analyzer 2, the start time and end time of the period in which attention is desired in the cell cycle (a predetermined period in the cell cycle or a predetermined detailed period in the M period) are predicted, The imaging interval may be shortened only during the time period before and after the desired period.
For example, when the living cells are stratified by the ratio of the lengths of the G1 phase and the non-G1 phase, the image acquisition device 1 initially captures the live cells, for example, at an interval of one hour, and the cell image analysis device 2 Analyze the image. Here, when the cell image analysis device 2 detects a living cell that is likely to shift from the G1 phase to the S phase, the image acquisition device 1 images the living cell by shortening the interval to, for example, an interval of 10 minutes. Then, the cell image analysis device 2 measures the time in the period when the cell phase has shifted to the S phase, and then the image acquisition device 1 captures images at the original interval (for example, one hour interval).
Similarly, for example, when stratifying by the time from the M phase to the transition to the PostM phase, when the cell image analyzer 2 detects a living cell that is likely to shift from the M phase to the PostM phase, the image acquisition device 1 Live cells are imaged with the interval shortened to, for example, 5 minutes. Then, the cell image analysis device 2 measures the time when the cell transitions to the PostM phase (the cell is completely divided), and then the image acquisition device 1 captures images at the original interval (for example, one hour interval).
In this way, it is possible to accurately measure the desired period of time in the cell cycle while minimizing damage to the cells.

  The cell image analysis system of the present invention is useful in the field of diagnosis and cytogenetics research using iPS cells, ES cells, and cancer cells for medical applications.

DESCRIPTION OF SYMBOLS 1 Cell image acquisition apparatus 1a Microscope observation optical system 1b Imaging means 1b1 Imaging time control means 1b2 Imaging element 2 Cell image analysis apparatus 2a Cell area specifying means 2b Cell cycle period detection means 2c, 2c ′, 2c ″ Cell cycle period Time measurement means 2d Detailed period detection means 2e within M period Detailed measurement time measurement means 2f within M period Time comparison means 2g during cell cycle period Detailed comparison time period within M period 2h Identification means 2i Generation number measurement Means 2j, 2j 'Generation time comparison means 2t-1, 2t-2, 2t-3, 2t-4, 2t-5, 2t-6, 2t-7, 2t-8, 2t-9 Data table 3a Chromosome 3b Centro Zome (central body)
3c microtubule 3d nuclear membrane

Claims (18)

An image acquisition device equipped with a microscope for acquiring, in time series, images of live cells into which fluorescent proteins that specifically emit fluorescence in a specific phase of the cell cycle are introduced, and images acquired by the image acquisition device in time series A cell image analysis system having a computer for performing a predetermined analysis on living cells using a cell image analysis system,
The cell image analysis apparatus includes the computer,
In the image acquired by the image acquisition device, a cell region specification that identifies, as a cell region, a pixel group whose fluorescence luminance exceeds a predetermined luminance threshold value or a pixel group whose luminance difference from adjacent luminance exceeds a predetermined luminance difference threshold value means,
A cell cycle period detecting means for detecting a cell cycle period of the living cell by comparing with a data table storing a cell cycle period corresponding to fluorescence emitted from the fluorescent protein,
A time measuring means for measuring the period of the cell cycle until the period of the cell cycle of the living cell detected by the detecting means of the cell cycle changes to another period,
An identification means for identifying at least the cell type using the time of the phase of the cell cycle;
A cell image analysis system comprising image analysis software that functions as a computer. The data table is
The cell cycle period is classified using fluorescence emitted from the predetermined fluorescent reagent in cells of the same type as the living cells previously introduced with the fluorescent protein and stained with a predetermined fluorescent reagent different from the fluorescent protein. The cell image analysis system according to claim 1, wherein the cell image analysis system is obtained by associating fluorescence emitted from the predetermined fluorescent reagent with fluorescence emitted from the fluorescent protein. . The fluorescent protein is two fluorescent proteins having different colors, or one fluorescent protein that emits different colors depending on the phase of the cell cycle,
The cell cycle phase detection means comprises:
Introducing the fluorescent protein in advance, the ratio of the total amount of fluorescent luminance of different colors emitted from the fluorescent protein in the pixel group specified as the cell region by the cell region specifying means and the amount of increase in the total amount of fluorescent luminance for each different color In addition, an image of the same type of cells as the living cells stained with a predetermined fluorescent reagent different from the fluorescent protein is acquired through the image acquisition device, and is specified as a cell region through the cell region specifying means. The period of the cell cycle is classified using the total amount of fluorescence luminance emitted from the predetermined fluorescent reagent in the data table creation pixel group and the maximum value of the fluorescence luminance, and the predetermined fluorescent reagent in the data table creation pixel group The total amount of fluorescence brightness emitted from the light and the maximum value of the fluorescence brightness and whether the fluorescent protein in the data table creation pixel group The total amount of fluorescent luminances of different colors emitted from the fluorescent protein, obtained by associating with the ratio of the total amount of fluorescent luminances of different colors emitted and the increase amount of the total amount of fluorescent luminances of different colors The cell cycle period of the living cell is detected by comparing with the data table storing the period of the cell cycle corresponding to the increase ratio and the increase in the total amount of fluorescence luminance for each different color. The cell image analysis apparatus according to 1 or 2.   The time in the cell cycle period is a generation time until the cell cycle period of the living cell changes to the cell cycle period in the next generation. Cell image analysis system.   The cell image analysis according to any one of claims 1 to 3, wherein the time period of the cell cycle is a time period until the cell cycle period of the living cell changes to the next period. system.   When the cell cycle phase of the living cell detected by the cell cycle phase detection means is M phase, the morphological characteristic element of the living cell is represented by M corresponding to the morphological characteristic element of the living cell in M phase. The detailed period detecting means for detecting the detailed period in the M phase of the living cell by comparing with the data table in which the detailed period in the period is stored is further provided. The cell image analysis system according to any one of 5.   The time period of the detailed period until the detailed period in the M phase of the live cell detected by the detailed period detecting means in the M period changes to the next detailed period or the next cell cycle period is measured. The cell image analysis system according to claim 6, further comprising time measuring means for a detailed period.   The time of the first period from the first phase of the living cell measured by the time measurement means of the cell cycle period to the next period, and the time measurement means of the cell cycle period measured by the time measurement means The cell image according to any one of claims 1 to 7, further comprising time comparison means in a cell cycle phase that compares a time of a second phase from the second phase of the living cell to the next phase. Analysis system.   The first detailed period until the first detailed period in the M period of the living cell measured by the time measuring means in the detailed period in the M period changes to the next detailed period or the next cell cycle period. A second period until the second detailed period in the M phase of the living cell measured by the time measuring means of the detailed period in the M period changes to the next detailed period or the next cell cycle period. 9. The cell image analysis system according to claim 7, further comprising time comparison means for the detailed period within the M period for comparing the time of the detailed period. The identification means is
The time of the period of the cell cycle of the living cell measured by the time measuring means of the period of the cell cycle, the time of the detailed period within the M phase of the living cell measured by the time measuring means of the detailed period within the M phase, The comparison value of the time of the cell cycle period of the living cell compared with the time comparison means of the cell cycle period, the detailed period of the living cell within the M phase compared with the detailed time comparison means of the M phase 10. The cell image analysis system according to claim 9, which is dependent on claim 8, wherein at least one cell type is identified using at least one of the time comparison values.   The identification means further includes the time of the cell cycle period measured by the time measurement means of the cell cycle period, and the M phase of the living cells measured by the detailed time measurement means within the M phase. Of the detailed period of the cell, the comparison value of the time of the cell cycle period of the living cell compared by the time comparison means of the cell cycle, and the living cell of the detailed period of the M phase compared And / or identifying at least one of cell division, cell growth level, cell population stratification, and cell properties using at least one of the comparison values of the detailed period within the M phase. Item 11. The cell image analysis system according to Item 10. The time measuring means of the cell cycle period measures generation times of a plurality of generations in one living cell,
The image analysis software further causes the computer to function as a generation time comparison unit that compares generation times of different generations of the single living cell measured by the time measurement unit of the cell cycle period,
The identification means further includes a comparison value of generation times of different generations of the one living cell compared by the generation time comparison means of a living cell corresponding to a comparison value of generation times of different generations of living cells. The cell image analysis system according to claim 1, wherein a change in the one living cell is detected by collating with a data table in which change information is stored. The time measurement means of the cell cycle period measures generation times of multiple generations in each of two or more living cells,
The image analysis software further causes the computer to function as generation time comparison means for comparing generation times of different generations in each of the two or more living cells measured by the time measurement means of the cell cycle period. ,
The identification means further corresponds to a comparison value of generation times of different generations in each of the two or more living cells compared by the generation time comparison means to a comparison value of generation times of different generations of living cells. The cell image according to any one of claims 1 to 11, wherein the two or more living cells are stratified by collating with a data table in which stratification information of live cells to be stored is stored. Analysis system. The time measurement means of the cell cycle period measures generation times of multiple generations in each of two or more living cells,
The image analysis software further causes the computer to function as generation time comparison means for comparing generation times of different generations in each of the two or more living cells measured by the time measurement means of the cell cycle period. ,
The identification means further corresponds to a comparison value of generation times of different generations in each of the two or more living cells compared by the generation time comparison means to a comparison value of generation times of different generations of living cells. The change in each of the two or more live cells is detected and the two or more live cells are stratified by collating with the data table storing the change information and stratification information of the live cells The cell image analysis system according to claim 1, wherein: The image acquisition device is an image of living cells under two measurement conditions in which at least one of chemicals to be administered, light stimulation, genetic manipulation, temperature / humidity / pressure / PH environment, and nutrients of the medium are different. In chronological order,
The generation time comparison means compares the generation time of the living cells measured by the time measurement means in the cell cycle period under each of the two measurement conditions. A cell image analysis system according to claim 1.   The cell image analysis system according to any one of claims 1 to 15, wherein the living cells are at least one of ES cells, iPS cells, and cancer stem cells.   The cell image analysis system according to any one of claims 1 to 15, wherein the living cells are at least one of ES cells, iPS cells, and cells differentiated from cancer stem cells.   The cell image analysis system according to claim 1, wherein the fluorescent protein is FUCCI.

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