JP2010252648A - Method for analyzing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing cells by which the throughput of the analysis is improved by shortening an imaging time, and a management cost of data can be reduced by reducing the amount of the data. <P>SOLUTION: The method for analyzing the cells by taking the image of a cell group, recognizing each of the cells by recognizing a first component in each cell by using the taken image, and evaluating characteristic amounts of components except the first component by the cell includes the step of labeling the first component with a first dye, the step of labeling the components except the first component with a second dye, the step of simultaneously taking the image of the first component and the components except the first component by changing the brightness level of the light from a first dye from the brightness level of the light from the second dye in a degree of enabling a threshold value to be set, the step of recognizing the first component and the components except the first component from the obtained image via the brightness threshold value, and the evaluation step of evaluating by the cell the characteristic amounts of the components except the first component by evaluating the characteristics of the light from the second dye in the region of the components except the first component in the obtained image. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention captures a cell population with a fluorescence microscope, recognizes individual cells from the captured fluorescence microscope image, evaluates the characteristic amount of a cell component to be analyzed for each cell, The present invention relates to a cell analysis method for quantitatively analyzing the characteristics of a cell population by performing numerical evaluation as an average value.

  In the conventional cell analysis method, when the feature amount of the cell component to be analyzed is evaluated for each cell, the cell image is acquired by labeling with a fluorescent dye or a luminescent reporter molecule corresponding to each cell component, and corresponding to the fluorescent dye. The selected wavelength element is used to individually capture each cell component, and the captured images are superimposed. Individual cells are recognized based on the nuclear image.

  For example, when a cell image as shown in FIG. 7 is acquired, a microscope imaging apparatus having a configuration as shown in FIG. 8 is used. In FIG. 7, 7 is a nucleus and 8 is a cytoplasm. In FIG. 8, 51 is a light source, 52 is an excitation filter switching means such as a turret or slider, 52a and 52b are excitation filters, 53 is a dichroic mirror switching means such as a turret or slider, 53a and 53b are dichroic mirrors, and 54 is a culture vessel. Samples of cells and the like cultured in the above, 55 is an absorption filter switching means such as a turret and a slider, 55a and 55b are absorption filters, and 56 is a photodetector.

First, an excitation filter 52a, a dichroic mirror 53a, and an absorption filter 55a each having wavelength transmission characteristics as shown in FIG. 9 are combined as an excitation filter, a dichroic mirror, and an absorption filter in a microscope imaging apparatus, and DAPI (4 ′ , 6-diamidino-2-phenylindole) to obtain an image of the nucleus 7 labeled (see FIG. 10).
Next, the excitation filter, dichroic mirror, and absorption filter in the microscope imaging apparatus are switched to a combination of an excitation filter 52b, a dichroic mirror 53b, and an absorption filter 55b each having wavelength transmission characteristics as shown in FIG.
An image of the cytoplasm 8 labeled with (Fluorescein isothiocianate) is acquired (FIG. 12).
Next, an image of the nucleus 7 and an image of the cytoplasm 8 are synthesized. Thereby, a cell image as shown in FIG. 7 is obtained.

JP 2001-307066 A

  Evaluation of the response of a drug using cells is an extremely important step in the drug discovery process, and for accurate evaluation, it is necessary to statistically evaluate the cell population. For this purpose, it is necessary to recognize individual cells, analyze the characteristic amount of the cell component to be analyzed for each cell, and statistically evaluate it as an average value per cell.

  Although cells are cultured in a culture container such as a petri dish or a microwell plate, it is difficult in practice to scan and image the entire region of the culture container such as a well because of time and cost limitations. For this reason, in most cases, the culture vessel is partially scanned and imaged, cell recognition is performed using digital data from the obtained image, and the number of cells to be analyzed is evaluated. A method for statistically evaluating the feature amount of the image is taken.

  Furthermore, in order to accurately perform statistical evaluation of the feature amount per cell, at least 10,000 cells are required on the image, and images with 10,000 or more cells can be acquired. The number of images to be taken and the amount of image data stored are enormous.

  Further, the time required for imaging is substantially proportional to the number of images to be captured. However, in the conventional cell analysis method, each cell component such as nucleus, cytoplasm, mitochondria, endoplasmic reticulum, Golgi body, etc. to be evaluated is labeled with a dye corresponding to the cell component at the time of cell image acquisition. Since an optical element such as a wavelength selection element corresponding to the wavelength is selected and an image is captured each time, it takes a long imaging time and the throughput cannot be improved.

  The present invention has been made in view of such conventional problems, and can shorten the imaging time and improve the analysis throughput, and can reduce the data amount and reduce the data management cost. An object of the present invention is to provide a simple cell analysis method.

  In order to achieve the above object, the cell analysis method according to the present invention captures a cell population with a microscopic imaging device, recognizes a first cell component in each cell by using an image of the captured cell population, and individually The cell component is recognized, the feature amount of the cell component other than the first cell component is evaluated for each cell, and the average value per cell is calculated using the evaluated feature amount of the cell component other than the first cell component. A cell analysis method for statistically evaluating the first cell component, wherein the first cell component is labeled with a first fluorescent dye or a first luminescent reporter molecule; A second labeling step of labeling a cell component with a second fluorescent dye or a second luminescent reporter molecule; a luminance level of light from the first fluorescent dye or the first luminescent reporter molecule; and the second fluorescence. Dye or second luminescent reporter An imaging step of simultaneously imaging the first cell component and a cell component other than the first cell component by varying a luminance level of light from the child to an extent that a threshold can be set; and A recognition step for recognizing the first cell component and a cell component other than the first cell component at least via a threshold value for luminance from the obtained image, and among the images obtained in the imaging step , By evaluating the characteristics of light from the second fluorescent dye or the second luminescent reporter molecule in a region of cell components other than the first cell component recognized in the recognition step, An evaluation step of evaluating for each cell a feature amount of a cell component other than the cell component.

  Moreover, in the cell analysis method of the present invention, in the imaging step, the light in the first excitation wavelength band that excites the first fluorescent dye or the first luminescent reporter molecule and the second fluorescent dye or the second fluorescent dye. An excitation filter having a wavelength characteristic that transmits only light in the second excitation wavelength band for exciting the luminescent reporter molecule, and a first excitation wavelength band for exciting the first fluorescent dye or the first luminescent reporter molecule. Reflecting light and light in a second excitation wavelength band that excites the second fluorescent dye or the second luminescent reporter molecule, and the first fluorescence from the first fluorescent dye or the first luminescent reporter molecule A die having a characteristic of transmitting light in a first predetermined band including a wavelength band and light in a second predetermined band including a second fluorescent wavelength band from the second fluorescent dye or the second luminescent reporter molecule Of the light transmitted through the Loic mirror and the dichroic mirror, the first fluorescent wavelength band light from the first fluorescent dye or the first luminescent reporter molecule and the second fluorescent dye or the second luminescent reporter It is preferable to use an absorption filter having a characteristic of transmitting only light in the second fluorescence wavelength band from the molecule and absorbing light in other wavelength bands.

  Further, in the cell analysis method of the present invention, the transmittance for transmitting the light in the first excitation wavelength band for exciting the first fluorescent dye or the first luminescent reporter molecule in the excitation filter; It is preferable to make a difference from the transmittance for transmitting light in the second excitation wavelength band for exciting the fluorescent dye or the second luminescent reporter molecule.

  In the cell analysis method of the present invention, the absorption filter transmits the light in the first fluorescence wavelength band from the first fluorescent dye or the first luminescent reporter molecule, and the second filter. It is preferable to make a difference from the transmittance that transmits light in the second fluorescence wavelength band from the fluorescent dye or the second luminescent reporter molecule.

  In the cell analysis method of the present invention, the concentration of the first fluorescent dye or the first luminescent reporter molecule for labeling the first cell component and the cell component other than the first cell component are labeled. It is preferable to make a difference between the two fluorescent dyes or the concentration of the second luminescent reporter molecule.

  The cell analysis method of the present invention further includes a reference area setting step for setting a reference value for the area of the first cell component. In the recognition step, the threshold value for the luminance is set for the luminance. The area value of the first cell component obtained by setting the threshold value satisfies a predetermined condition with respect to the reference value of the area of the first cell component set in the reference area setting step. It is preferable to perform the adjustment.

  The cell analysis method of the present invention further includes a reference shape setting step for setting a reference characteristic of the shape of the first cell component. In the recognition step, the threshold value for the luminance is set for the luminance. The shape characteristic of the first cell component obtained by setting the threshold is adjusted so as to approach the reference characteristic of the shape of the first cell component set in the reference shape setting step. Is preferred.

  In the cell analysis method of the present invention, it is preferable that, in the reference shape setting step, the reference characteristic of the shape of the first cell component when the first cell component is a nucleus is set to a perfect circle. .

  In the cell analysis method of the present invention, the cell is a nerve cell, the cell component other than the first cell component is cytoplasm, and the feature value to be evaluated for each cell is the neurite length. preferable.

  In the cell analysis method of the present invention, the cell is a cancer cell, the cell component other than the first cell component is a cytoplasm, and the feature amount evaluated for each cell is apoptosis induction. preferable.

  ADVANTAGE OF THE INVENTION According to this invention, the imaging time can be shortened and the throughput of an analysis can be improved, and the cell analysis method which can reduce the amount of data and can reduce the management cost of data is obtained.

It is explanatory drawing which shows schematic structure of the microscope imaging device used for the cell analysis method of this invention. It is a graph which shows the wavelength characteristic of an excitation filter, a dichroic mirror, and an absorption filter used for the cell analysis method concerning Example 1 of this invention. It is explanatory drawing which shows an example of the cell image acquired simultaneously via the microscope imaging device shown in FIG.1 and FIG.2. It is a graph which shows the brightness | luminance cross section in line segment AA in the cell image shown in FIG. It is a graph which shows the wavelength characteristic of an excitation filter, a dichroic mirror, and an absorption filter used for the cell analysis method concerning Example 2 of this invention. It is a graph which shows an example of the brightness | luminance cross section of a cell image in case a sufficient difference does not arise in the brightness | luminance of cell components other than a 1st cell component and a 1st cell component. It is explanatory drawing which shows an example of a cell image. It is explanatory drawing which shows an example of schematic structure of the microscope imaging device used in the conventional cell analysis method. It is a graph which shows the wavelength characteristic of an excitation filter, a dichroic mirror, and an absorption filter combined when acquiring a 1st cell component using the microscope imaging device of FIG. It is explanatory drawing which shows the image of the nucleus which is the 1st cell component acquired combining the excitation filter with a wavelength characteristic shown in FIG. 9, a dichroic mirror, and an absorption filter. It is a graph which shows the wavelength characteristic of an excitation filter, a dichroic mirror, and an absorption filter combined when acquiring cell components other than a 1st cell component using the microscope imaging device of FIG. It is explanatory drawing which shows the image of the cytoplasm which is cell components other than the 1st cell component acquired combining the excitation filter with a wavelength characteristic shown in FIG. 11, a dichroic mirror, and an absorption filter.

The present invention is a method for evaluating the feature amount of a cell component for each cell, so that the brightness of light obtained from a dye labeled with each cell component is different so that a threshold can be set. Each cell component labeled with a combination of dyes can be imaged simultaneously using a common optical system, and each cell component can be separated and recognized from the difference in fluorescence brightness in the obtained image Thus, the imaging time is shortened.
According to the present invention, since it is not necessary to perform imaging for each cell component to be analyzed in the microscopic observation of a cell population, the imaging time can be shortened and the analysis throughput can be improved. In addition, since the amount of data can be reduced, the data management cost can be reduced.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
Example 1
FIG. 1 is an explanatory diagram showing a schematic configuration of a microscope imaging apparatus used in the cell analysis method of the present invention. FIG. 2 is a graph showing the wavelength characteristics of the excitation filter, dichroic mirror, and absorption filter used in the cell analysis method according to Example 1 of the present invention. In FIG. 1, 1 is a light source, 2 is an excitation filter, 3 is a dichroic mirror, 4 is a sample such as a cell cultured in a culture vessel, 5 is an absorption filter, and 6 is a photodetector.
In the cell analysis method of Example 1, DAPI is used as the first fluorescent dye or the first luminescent reporter molecule for labeling the cell component of the sample 4, and FITC is used as the second fluorescent dye or the second luminescent reporter molecule. Then, the nucleus as the first cell component in the present invention is labeled, and the cytoplasm as the cell component other than the first cell component in the present invention is labeled with FITC.

Further, in the cell analysis method of Example 1, in order to obtain a cell image as shown in FIG. 7, the excitation filter 2, the dichroic mirror 3, and the absorption filter 5 in the microscope imaging apparatus shown in FIG. As shown, a combination of an excitation filter, a dichroic mirror, and an absorption filter each having multiband wavelength characteristics corresponding to DAPI and FITC is used.
The excitation filter 2 has a characteristic of transmitting only light in an excitation wavelength band for exciting DAPI and light in an excitation wavelength band for exciting FITC. The dichroic mirror 3 reflects light in an excitation wavelength band for exciting DAPI and light in an excitation wavelength band for exciting FITC, and also reflects light in a predetermined band including a fluorescence wavelength band from DAPI and a fluorescence wavelength band from FITC. It has a characteristic of transmitting light in a predetermined band including. The absorption filter 5 transmits only light in the fluorescence wavelength band from DAPI and light in the fluorescence wavelength band from FITC, and absorbs light in the other wavelength bands among the light transmitted through the dichroic mirror 3. Have

  The light emitted from the light source 1 enters the excitation filter 2, and only the light in the wavelength band that excites DAPI and FITC passes through the excitation filter 2. The light transmitted through the excitation filter 2 is reflected by the dichroic mirror 3 and irradiates the sample 4. Then, the nucleus 7 labeled with DAPI and the cytoplasm 8 labeled with FITC are excited to emit fluorescence. After passing through the dichroic mirror 3, extraneous light is cut off by the absorption filter 5 and detected by the photodetector 6, and these fluorescences are captured as one image at the same time.

In addition, the microscope imaging apparatus using the cell analysis method of the first embodiment is provided with an image analysis apparatus (not shown) that analyzes the image captured by the photodetector 6. In the image analysis apparatus, threshold values for the luminance of these fluorescence are set.
For example, it is assumed that a cell image as shown in FIG. 3 is obtained through the microscope imaging apparatus shown in FIGS. In this cell image, the luminance cross section at line segment AA has a shape as shown in FIG. 4, for example. In the luminance cross section shown in FIG. 4, the image analysis apparatus has a core 7 labeled with DAPI in a region where the luminance is higher than the set threshold value 9 and is labeled with FITC in a region where the luminance is lower than the threshold value 9. Identified as cytoplasm 8. Then, the image analysis device executes the discrimination process between the nucleus 7 and the cytoplasm 8 using the threshold 9 for the entire region of the cell image obtained through the microscope imaging device. Using this discrimination processing result, colors of different strains can be given to the nucleus 7 and the cytoplasm 8, respectively, to form a cell image that can be clearly recognized for each cell.

  In the above example, the nucleus 7 is used as the first cell component in the cell. However, in the cell analysis method of Example 1, other cell components (mitochondria, endoplasmic reticulum, Golgi body, etc.) are used as the first cell component. Similarly, even when used as a cell component, the first cell component and a cell component other than the first cell component can be identified and imaged. In that case, as the excitation filter 2, dichroic mirror 3, and absorption filter 5 in the microscope imaging apparatus, instead of the combination of the excitation filter, dichroic mirror, and absorption filter having multiband wavelength characteristics as shown in FIG. A combination of an excitation filter, a dichroic mirror, and an absorption filter each having multiband wavelength characteristics reflecting the excitation absorption characteristics of a fluorescent dye or luminescent reporter molecule that labels the corresponding cell component may be used.

Example 2
FIG. 5 is a graph showing the wavelength characteristics of the excitation filter, dichroic mirror, and absorption filter used in the cell analysis method according to Example 2 of the present invention.

  When the cell analysis method of Example 1 is used to capture an image of a cell through the microscope imaging apparatus shown in FIGS. 1 and 2, and the captured image is analyzed by an image analysis apparatus (not shown), FIG. As shown in FIG. 6, there is no sufficient luminance difference between DAPI-labeled nucleus 7 (or other cell components such as mitochondria, endoplasmic reticulum, Golgi body) and FITC-labeled cytoplasm 8, It may be difficult to set a threshold value.

  In such a case, the difference between the wavelength characteristic of the DAPI wavelength band and the wavelength characteristic of the FITC wavelength band in a filter having multiband wavelength characteristics such as the excitation filter 2 and the absorption filter 5 Can provide a sufficient luminance difference between DAPI-labeled nucleus 7 (or other cellular components such as mitochondria, endoplasmic reticulum, Golgi body) and FITC-labeled cytoplasm 8, and setting a threshold for luminance Is possible.

  In the cell analysis method of Example 2, in the microscope imaging device shown in FIG. 1, the excitation filter, dichroic mirror, and absorption filter having the wavelength characteristics shown in FIG. 5 are used as the excitation filter 2, dichroic mirror 3, and absorption filter 5, respectively. Use. Other configurations are substantially the same as those of the cell analysis method of Example 1.

As shown in FIG. 5, when an excitation filter having a wavelength characteristic in which the transmittance of light in the excitation wavelength band for FITC is lower than the transmittance of light in the excitation wavelength band for DAPI is used as the excitation filter 2, FITC Excitation can be suppressed. In addition, when an absorption filter having a wavelength characteristic in which the transmittance of light in the fluorescence wavelength band for FITC is lower than the transmittance of light in the fluorescence wavelength band for DAPI is used as the absorption filter 5, fluorescence excited by FITC Can be reduced. In the example of FIG. 5, the wavelength transmission characteristics for FITC in the excitation filter and absorption filter are both 30% or less compared to the wavelength transmission characteristics for FITC in the excitation filter and absorption filter shown in FIG. , It is possible to suppress the transmittance of fluorescence from FITC to 10% or less comprehensively. As a result, it is possible to give a sufficient difference in luminance between the DAPI-labeled nucleus 7 (or other cell components such as mitochondria, endoplasmic reticulum, and Golgi apparatus) and the cytoplasm 8 labeled with FITC.
In the example of FIG. 5, the wavelength characteristics of the DAPI wavelength band and the wavelength characteristics of the FITC wavelength band are different from those of both the excitation filter 2 and the absorption filter 5. 2 and the absorption filter 5 may be provided with such a difference in wavelength characteristics.

Example 3
In the cell analysis method of Example 3, when an image of a cell is captured through the microscope imaging apparatus shown in FIGS. 1 and 2 and the captured image is analyzed by an image analysis apparatus (not shown), for example, the nucleus 7 Increase the concentration of the first fluorescent dye or luminescent reporter molecule such as DAPI for labeling, or decrease the concentration of the second fluorescent dye or luminescent reporter molecule such as the second FITC for labeling the cytoplasm 8. Differentiate the concentration of the dye or luminescent reporter molecule.

  If the imaging analysis method of Example 2 is used, the cell analysis method of Example 1 is used to capture an image of a cell via the microscope imaging device shown in FIGS. 1 and 2, and the captured image is converted into an image analysis device. When analyzing with (not shown), as shown in FIG. 6, the nucleus 7 (or other cell components such as mitochondria, endoplasmic reticulum, Golgi body etc.) labeled with DAPI and cytoplasm 8 labeled with FITC DAPI-labeled nucleus 7 (or other cell components such as mitochondria, endoplasmic reticulum, and Golgi apparatus) even when there is no sufficient difference in luminance and it is difficult to set a threshold for luminance. A sufficient difference can be given to the brightness of the cytoplasm 8 labeled with FITC, and a threshold value for the brightness can be set.

  As the cell analysis method of Example 2, the method for differentiating the concentration of the fluorescent dye or the luminescent reporter molecule to be labeled has been described. Further, as another method, a combination of a fluorescent dye or a luminescent reporter molecule such as DAPI or FITC is used. May be combined with each other having a large difference in fluorescence characteristics such as molar extinction coefficient and quantum yield.

Example 4
The cell analysis method of Example 4 is a method for improving the accuracy of analysis by resetting the threshold value 9 in the cell analysis methods of Examples 1, 2, and 3.
In the cell analysis method of Example 4, a reference value for the area of the first cell component is set in advance, and the threshold value 9 for luminance is set to the first cell obtained by setting the threshold value for luminance. The area value of the component is adjusted so as to satisfy a predetermined condition (for example, within the range of the reference value) with respect to the reference value of the area of the first cell component.

Specifically, in the cell analysis method of Example 4, in the cell analysis method of Examples 1, 2, and 3, the cell image as shown in FIG. 3 is obtained through the microscope imaging device shown in FIG. The constituent elements of the first cell component such as the nucleus 7 are recognized once by the default threshold value 9 through the omitted image analysis apparatus.
Further, the area of the first cell component such as the nucleus 7 is calculated from the image acquired by the microscope imaging device via the image analysis device. In the image analysis device, a reference value for the area of the first cell component such as the nucleus 7 is set in advance. The reference value of the area is usually set by a combination of a minimum value and a maximum value having a certain width.
Then, the area of the first cell component such as the nucleus 7 calculated from the acquired image is compared with a reference value of this area.

When the area of the first cell component such as the nucleus 7 calculated from the acquired image is less than the minimum value in the reference value of this area, the actual first cell component and other cell components in the luminance cross section Since the threshold value 9 for distinguishing between the first cell component and the other cell components is set with a brightness higher than the brightness of the boundary portion, the threshold value 9 is reset with a lower brightness.
On the other hand, when the area of the first cell component such as the nucleus 7 calculated from the acquired image exceeds the maximum value in the reference value of this area, the actual first cell component and other cell components in the luminance cross section Is set to a threshold value 9 for discriminating between the first cell component and the other cell components with a luminance smaller than the luminance of the boundary portion, that is, in a portion closer to the cytoplasm 8 than the boundary portion. Since 9 is set, the threshold value 9 is reset to a larger luminance. The optimum threshold value 9 can be set by sequentially repeating these threshold value adjustment processes.

  It should be noted that the reference value of the area of the first cell component such as the nucleus 7 set in advance in the image analysis apparatus may be a constant value without having the minimum value and the maximum value width. In this case, the size relationship between the area of the nucleus 7 calculated from the acquired image and the reference value is determined. For example, the area of the first cell component such as the nucleus 7 calculated on the acquired image is the reference of the area. When the value exceeds the value, the optimum threshold value 9 is set. When the value is less than the value, a message indicating that the threshold value 9 is insufficiently set is displayed to the measurer.

Further, as a modification of the cell analysis method of Example 4, the following method may be used.
A cell image as shown in FIG. 3 is acquired, and the first cell component component such as the nucleus 7 is recognized by the threshold 9 set by default once through an image analysis device (not shown). The characteristic of the shape of the first cell component such as the nucleus 7 is recognized from the image acquired by the microscope imaging device via the analysis device. In the image analysis apparatus, a reference characteristic pattern of the shape of the first cell component such as the nucleus 7 is set in advance.
The shape characteristic of the first cell component such as the nucleus 7 calculated from the acquired image is compared with the reference characteristic pattern of this shape, and the threshold value 9 is reset so as to approach the reference characteristic pattern of the shape. The optimum threshold value 9 can be set by sequentially repeating this threshold value adjustment process. When the first cell component is the nucleus 7, the reference characteristic pattern of the shape is set to a perfect circle.

  According to the cell analysis method of Examples 1 to 4, the first cell component in the cell and the cell component other than the first cell component can be simultaneously imaged. Since it is not necessary to switch the optical system for each cell component and perform imaging, the imaging time can be shortened and the analysis throughput can be improved. Furthermore, since the amount of data can be reduced, the data management cost can be reduced.

  Note that the cell analysis methods of Examples 1 to 4 are suitable for evaluating the neurite length for each cell targeting nerve cells or for evaluating apoptosis induction for each cell targeting cancer cells. is there.

  The cell analysis method of the present invention acquires images of a large number of living cells using a microscopic observation apparatus, recognizes individual cells from the acquired images, and evaluates feature quantities of cell components to be analyzed for each cell. In addition, it is useful in the field of biotechnology for quantitatively analyzing the characteristics of a cell population by performing numerical evaluation as an average value per cell.

DESCRIPTION OF SYMBOLS 1 Light source 2 Excitation filter 3 Dichroic mirror 4 Sample 5 Absorption filter 6 Photo detector 7 Nucleus 8 Cytoplasm 51 Light source 52 Excitation filter switching means 52a, 52b Excitation filter 53 Dichroic mirror switching means 53a, 53b Dichroic mirror 54 Sample 55 Absorption filter switching means 55a, 55b Absorption filter 56 Photodetector

Claims (10)

A cell population is imaged with a microscope imaging device, and an individual cell is recognized by recognizing a first cell component in each cell using an image of the imaged cell population, and cell components other than the first cell component A cell analysis method that statistically evaluates an average value per cell using a feature amount of a cell component other than the evaluated first cell component.
A first labeling step of labeling the first cell component with a first fluorescent dye or a first luminescent reporter molecule;
A second labeling step of labeling a cell component other than the first cell component with a second fluorescent dye or a second luminescent reporter molecule;
The brightness level of light from the first fluorescent dye or first luminescent reporter molecule is different from the brightness level of light from the second fluorescent dye or second luminescent reporter molecule to such an extent that a threshold can be set. An imaging step of simultaneously imaging the first cell component and a cell component other than the first cell component;
A recognition step for recognizing the first cell component and a cell component other than the first cell component from the image obtained in the imaging step through at least a threshold value for luminance;
Characteristics of light from the second fluorescent dye or the second luminescent reporter molecule in a region of cell components other than the first cell component recognized in the recognition step in the image obtained in the imaging step An evaluation step for evaluating, for each cell, a characteristic amount of a cell component other than the first cell component by evaluating
A cell analysis method characterized by comprising at least In the imaging step,
Only light in the first excitation wavelength band that excites the first fluorescent dye or the first luminescent reporter molecule and light in the second excitation wavelength band that excites the second fluorescent dye or the second luminescent reporter molecule. An excitation filter having a wavelength characteristic that transmits light;
Light in a first excitation wavelength band that excites the first fluorescent dye or first luminescent reporter molecule and light in a second excitation wavelength band that excites the second fluorescent dye or second luminescent reporter molecule. A first predetermined band of light that is reflected and includes a first fluorescence wavelength band from the first fluorescent dye or first luminescent reporter molecule and from the second fluorescent dye or second luminescent reporter molecule A dichroic mirror having a characteristic of transmitting light in a second predetermined band including the second fluorescence wavelength band;
Of the light transmitted through the dichroic mirror, the first fluorescent wavelength band light from the first fluorescent dye or the first luminescent reporter molecule and the second fluorescent dye or the second luminescent reporter molecule from the second luminescent reporter molecule. An absorption filter having a characteristic of transmitting only light in the fluorescence wavelength band of 2, and absorbing light in other wavelength bands,
The cell analysis method according to claim 1, wherein:   A transmittance for transmitting light in a first excitation wavelength band for exciting the first fluorescent dye or the first luminescent reporter molecule in the excitation filter; and the second fluorescent dye or the second luminescent reporter molecule. The cell analysis method according to claim 2, wherein a difference is made between the transmittance for transmitting light in the second excitation wavelength band to be excited.   In the absorption filter, a transmittance for transmitting light in the first fluorescence wavelength band from the first fluorescent dye or the first luminescent reporter molecule, and from the second fluorescent dye or the second luminescent reporter molecule The cell analysis method according to claim 2 or 3, wherein a difference is made between the transmittance for transmitting light in the second fluorescence wavelength band.   The concentration of the first fluorescent dye or first luminescent reporter molecule for labeling the first cell component, and the second fluorescent dye or second luminescent reporter molecule for labeling cell components other than the first cell component The cell analysis method according to claim 1, wherein a difference is made from the concentration of the cell. A reference area setting step for setting a reference value of the area of the first cell component;
In the recognition step, the first cell in which the area value of the first cell component obtained by setting the threshold value for the luminance is set in the reference area setting step is set. The cell analysis method according to any one of claims 1 to 5, wherein the cell analysis method is carried out by adjusting so as to satisfy a predetermined condition with respect to a reference value of the component area. A reference shape setting step for setting a reference characteristic of the shape of the first cell component;
In the recognizing step, the threshold value for the luminance is set, and the characteristic of the shape of the first cell component obtained by setting the threshold value for the luminance is the first shape set in the reference shape setting step. The cell analysis method according to any one of claims 1 to 5, wherein the cell analysis method is performed by adjusting so as to approach the reference characteristic of the shape of the cell component.   8. The cell analysis method according to claim 7, wherein, in the reference shape setting step, a reference characteristic of the shape of the first cell component when the first cell component is a nucleus is set to a perfect circle. .   The cell is a nerve cell, a cell component other than the first cell component is a cytoplasm, and a feature amount evaluated for each cell is a neurite length. The cell analysis method described in 1.   8. The cell according to claim 1, wherein the cell is a cancer cell, the cell component other than the first cell component is a cytoplasm, and the characteristic amount evaluated for each cell is apoptosis induction. The cell analysis method described in 1.

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