JP6824654B2 - Cell information acquisition method and cell information acquisition device - Google Patents

Description

The present invention relates to a cell information acquisition method and a cell information acquisition device.

Various biological phenomena such as cell proliferation and differentiation involve the localization of various molecules such as proteins, mRNAs, and microRNAs in cells. Analyzing the localization of various molecules in cells is expected to lead to the elucidation of many biological phenomena such as molecular function analysis, protein-protein interaction analysis, and signal transduction pathway analysis.

The following Patent Document 1 discloses a method for analyzing the localization of a molecule in a cell by a fluorescence microscope and an imaging flow cytometer.

International Publication No. 2005/098430

Even if the cells are of the same origin, the individual cells are diverse. Therefore, for example, as shown in FIG. 23 (a), some cells have a specific molecule localized at a specific site. , As shown in FIG. 23 (b), some cells have a specific molecule localized at another site. Further, for example, as shown in FIG. 23 (c), the amount of molecules may not be uniform among cells due to various factors. Then, the inventor has found that when trying to acquire information from molecules having various distributions and amounts in cells, the acquisition results vary. Therefore, a method for accurately analyzing molecules having various distributions and amounts in cells is desired.

The first aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a plurality of fluorescent substances having different fluorescence wavelengths are bound to a test substance contained in the cell, and the cell is bound to the first light and a second light having a weaker intensity than the first light. by irradiating light to cause a plurality of fluorescent wavelength and intensity are different from each other by a plurality of fluorescent materials, based on the fluorescence produced by acquiring a plurality of fluorescent information, based on a plurality of fluorescent information, test substance Determines whether is localized in the nucleus or in the cytoplasm .

In the cell information acquisition method according to this aspect, the "plurality of fluorescent substances having different fluorescence wavelengths" means that when light is irradiated, the plurality of fluorescent substances emit fluorescence having different wavelengths from each other. In order to generate fluorescence having different wavelengths and intensities from a plurality of fluorescent substances, for example, when the wavelengths of the excitation lights are different from each other in the plurality of fluorescent substances, the plurality of lights having different wavelengths and intensities with respect to the cells. Irradiate. The fluorescence information is, for example, an image based on fluorescence. In addition, "binding a plurality of fluorescent substances to a test substance" does not necessarily mean that all the molecules of the same type of test substance contained in the cell are bound to the plurality of fluorescent substances, and at least a part thereof. It is shown that a plurality of fluorescent substances need be specifically bound to the molecules of the same type of test substance.

When the distribution and amount of the test substance in the cells are diverse, the intensity of fluorescence generated from the fluorescent substance may be too strong or too weak. Therefore, one fluorescence information is not always used to determine the distribution status of the test substance. It is not always possible to determine properly. However, according to the cell information acquisition method according to this aspect, fluorescence having different intensities can be generated from a plurality of fluorescent substances bound to a test substance, and a plurality of fluorescence information can be acquired based on the fluorescence having different intensities. As a result, even if the distribution status of the test substance cannot be properly determined in one fluorescence information because the fluorescence is too strong, the distribution status of the test substance can be properly discriminated by using other fluorescence information. become able to. Therefore, if any of the fluorescence information is used, the distribution status of the test substance can be appropriately determined. Therefore, even if the distribution and amount of the test substance in the cells are diverse, the test substance can be analyzed accurately. it can.

A second aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a substrate is brought into contact with a test substance contained in a cell to generate a plurality of fluorescent substances having different fluorescence wavelengths from each other, and the cell is subjected to the first light and the first light. It was irradiated with more intensity weak second light causing a plurality of fluorescent substance from the wavelength and intensity different from each other fluorescence to obtain a plurality of fluorescent information based on the fluorescence produced, based on a plurality of fluorescent information Therefore, it is determined whether the test substance is localized in the nucleus or the cytoplasm .

In the cell information acquisition method according to this embodiment, the test substance is labeled with the plurality of fluorescent substances by bringing the substrate into contact with the test substance contained in the cell to generate a plurality of fluorescent substances having different fluorescence wavelengths from each other. In the cell information acquisition method according to this aspect as well, as in the first aspect, if any of the fluorescence information is used, the distribution status of the test substance can be appropriately determined, so that the test substance can be analyzed accurately. it can.

A third aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a fluorescent substance is bound to a test substance contained in the cell, the cell is irradiated with light to generate fluorescence having a predetermined wavelength width from one type of fluorescent substance, and the generated predetermined wavelength. Multiple fluorescences having different wavelengths and intensities are acquired by dividing the width fluorescence according to the wavelength band , multiple fluorescence informations are acquired based on each acquired fluorescence, and the test substance in the cell is obtained based on the plurality of fluorescence informations. To determine the distribution status of.

In the cell information acquisition method according to this aspect, one kind of fluorescent substance is bound to the test substance. In order to obtain a plurality of fluorescence having different wavelengths and intensities from the generated fluorescence, for example, the fluorescence generated from the fluorescent substance is separated by passing through a plurality of filter members having different transmission wavelength bands. Also in the cell information acquisition method according to this aspect, the fluorescence information is, for example, an image based on fluorescence. In addition, not all of the molecules of the same type of test substance contained in the cell are necessarily bound to the fluorescent substance, and the fluorescent substance is specifically bound to at least some molecules of the same type of test substance. Just do it. In the cell information acquisition method according to this aspect as well, as in the first aspect, if any of the fluorescence information is used, the distribution status of the test substance can be appropriately determined, so that the test substance can be analyzed accurately. it can.

A fourth aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a substrate is brought into contact with a test substance contained in a cell to generate a fluorescent substance, and the cell is irradiated with light to generate fluorescence having a predetermined wavelength width from one type of fluorescent substance. , The generated fluorescence of a predetermined wavelength width is divided by a wavelength band to acquire a plurality of fluorescence having different wavelengths and intensities, a plurality of fluorescence information is acquired based on each acquired fluorescence, and a plurality of fluorescence information is acquired based on the plurality of fluorescence information. Determine the distribution of the test substance in the cells.

In the cell information acquisition method according to this embodiment, the test substance is labeled with the fluorescent substance by bringing the substrate into contact with the test substance contained in the cell to generate a fluorescent substance. In the cell information acquisition method according to this aspect as well, as in the third aspect, if any of the fluorescence information is used, the distribution status of the test substance can be appropriately determined, so that the test substance can be analyzed accurately. it can.

A fifth aspect of the present invention relates to a cell information acquisition device. The cell information acquisition device according to this embodiment irradiates a cell containing a test substance in which a plurality of fluorescent substances having different fluorescence wavelengths are bound to each other with a first light and a second light having a weaker intensity than the first light. Then, a light irradiation unit that generates a plurality of fluorescence having different wavelengths and intensities from a plurality of fluorescent substances, a light receiving unit that receives each fluorescence generated from the plurality of fluorescent substances, and a plurality of light receiving units based on fluorescence having different intensities. It includes an acquisition unit for acquiring fluorescence information and an analysis unit for determining whether the test substance is localized in the nucleus or the cytoplasm based on a plurality of fluorescence information .

According to the cell information acquisition device according to this aspect, the same effect as that of the first aspect can be achieved.

A sixth aspect of the present invention relates to a cell information acquisition device. The cell information acquisition device according to this embodiment includes a light irradiation unit that irradiates cells containing a test substance to which a fluorescent substance is bound with light to generate fluorescence of a predetermined wavelength width from one type of fluorescent substance, and one type of fluorescence. A light receiving unit that receives a plurality of fluorescences having different wavelengths and intensities by dividing fluorescence having a predetermined wavelength width generated from a substance by a wavelength band, and an acquisition unit that acquires a plurality of fluorescence information based on the plurality of received fluorescences. , It is provided with an analysis unit for discriminating the distribution status of a test substance in cells based on a plurality of fluorescence information.

According to the cell information acquisition device according to this aspect, the same effect as that of the third aspect can be achieved.

A seventh aspect of the present invention relates to a cell information acquisition device. The cell information acquisition device according to this embodiment irradiates cells containing a test substance that has generated a fluorescent substance by contact with a substrate with light to generate fluorescence having a predetermined wavelength width from one type of fluorescent substance. A unit, a light receiving part that receives a plurality of fluorescences having different wavelengths and intensities by dividing fluorescence having a predetermined wavelength width generated from one kind of fluorescent substance by a wavelength band , and a plurality of fluorescences based on the received multiple fluorescences. It includes an acquisition unit for acquiring information and an analysis unit for determining the distribution status of a test substance in cells based on a plurality of fluorescence information.

According to the cell information acquisition device according to this aspect, the same effect as that of the fourth aspect can be achieved.
An eighth aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a plurality of fluorescent substances having different wavelengths and intensities of fluorescence generated by irradiation with light of the same wavelength are bound to a test substance contained in the cell, and the cell is subjected to the first method. A plurality of fluorescences having different wavelengths and intensities are generated from a plurality of fluorescent substances by irradiating the first light and a second light having a weaker intensity than the first light, and a plurality of fluorescence informations are generated based on the generated fluorescences. To get.
A ninth aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a plurality of fluorescent substances having different fluorescence wavelengths are bound to a test substance contained in the cell, a sample containing the cell is flowed into a flow cell, and the cell flowing through the flow cell is first. By irradiating light and a second light having a weaker intensity than the first light, a plurality of fluorescence having different wavelengths and intensities are generated from a plurality of fluorescent substances, and a plurality of fluorescence information is acquired based on each of the generated fluorescence. To do.
A tenth aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a plurality of fluorescent substances having different fluorescence wavelengths are bound to a test substance contained in the cell, and the cell is bound to the first light and a second light having a weaker intensity than the first light. Is irradiated to generate multiple fluorescences having different wavelengths and intensities from a plurality of fluorescent substances, and multiple fluorescence information is acquired based on each of the generated fluorescences, and fluorescence obtained from a plurality of fluorescences having different intensities. Among the information, the localization status of the test substance in the cell is determined based on the fluorescence information obtained from the fluorescence whose intensity is within a predetermined range.
The eleventh aspect of the present invention relates to a method for acquiring cell information. In the cell information acquisition method according to this embodiment, a plurality of fluorescent substances having different fluorescence wavelengths are bound to a test substance contained in the cell, and the cell is bound to the first light and a first light having a weaker intensity than the first light. By irradiating 2 light, a plurality of fluorescences having different wavelengths and intensities were generated from a plurality of fluorescent substances, and a plurality of fluorescence informations were acquired based on each of the generated fluorescences, which were obtained from a plurality of fluorescences having different intensities. Of the fluorescence information, the localization status of the test substance in the cell is determined based on the fluorescence information obtained from the fluorescence whose intensity is within a predetermined range, and the localization status of the test substance is determined by the cell. Includes the calculation of the ratio of the localized amount of the test substance in the analysis target site to the amount of the test substance in the whole cell.

According to the present invention, molecules having various distributions and amounts in cells can be analyzed with high accuracy.

FIG. 1 is a flowchart showing a cell information acquisition method according to the first embodiment. FIG. 2 is a diagram showing an outline of fluorescence acquisition according to the first embodiment. FIG. 3 is a conceptual diagram showing an image acquired based on the high-intensity fluorescence and the low-intensity fluorescence according to the first embodiment. FIG. 4A is a diagram showing an image acquired in the verification according to the first embodiment. FIG. 4B is a diagram showing the numerical values acquired in the verification according to the first embodiment. FIG. 5 is a block diagram showing a configuration of the device according to the first embodiment. FIG. 6 is a diagram showing a configuration of an optical detection unit according to the first embodiment. FIG. 7 is a diagram showing a configuration of an optical detection unit according to a modified example of the first embodiment. FIG. 8 is a flowchart showing processing by the cell information acquisition device according to the first embodiment. FIG. 9 is a diagram showing a screen displayed on the display unit according to the first embodiment. FIG. 10 is a conceptual diagram showing a graph acquired based on high-intensity fluorescence and low-intensity fluorescence according to a modified example of the first embodiment. FIG. 11 is a diagram showing an outline of fluorescence acquisition according to the second embodiment. FIG. 12 is a diagram showing an outline of fluorescence acquisition according to the third embodiment. FIG. 13 is a diagram for explaining the transmission wavelength band and the acquired fluorescence wavelength band of the filter member according to the third embodiment. FIG. 14 is a diagram showing an image acquired in the verification according to the third embodiment. FIG. 15 is a diagram showing a configuration of an optical detection unit according to the third embodiment. FIG. 16 is a diagram showing an outline of fluorescence acquisition according to the fourth embodiment. FIG. 17 is a diagram showing a configuration of an optical detection unit according to the fourth embodiment. FIG. 18 is a diagram showing an outline of fluorescence acquisition according to the fifth embodiment. FIG. 19 is a diagram showing a configuration of an optical detection unit according to the fifth embodiment. FIG. 20 is a diagram showing an outline of fluorescence acquisition according to the sixth embodiment. FIG. 21 is a diagram showing an image acquired in the verification according to the sixth embodiment. FIG. 22 is a diagram showing an outline of fluorescence acquisition according to the seventh embodiment. FIG. 23 is a schematic diagram for explaining the problem to be solved by the invention.

<Embodiment 1>
The first embodiment is a cell information acquisition method in which a plurality of fluorescent substances having different fluorescence wavelengths are bound to a test substance contained in a cell, and the localization status of the test substance is determined based on a plurality of fluorescences generated from the fluorescent substances. This is an application of the present invention. In the first embodiment, the test substance is NF-κB. It is thought that NF-κB, which is a transcription factor, exists in the cytoplasm in a state of forming a complex with IκB, and is transferred to the inside of the nucleus by decomposition of IκB by various stimuli. In the first embodiment, NF-κB is specifically labeled with a fluorescent substance as a test substance, and an analysis is performed to determine whether NF-κB is present in the cytoplasm or the nucleus based on the fluorescence from the fluorescent substance. Be told. The test substance may be a protein or molecule other than NF-κB. For example, the test substance may be a transcription factor other than NF-κB, for example, STAT (Signal Transducer and Activator of Transcription), NFAT (nuclear factor of activated T cells), HIF (hypoxia-inducible factor). There may be. Further, the test substance may be mRNA or microRNA. Further, "binding a plurality of fluorescent substances to a test substance" does not necessarily mean that all the molecules of the same type of test substance contained in the cell are bound to the plurality of fluorescent substances, and at least a part thereof. It suffices if a plurality of fluorescent substances are specifically bound to the molecules of the same type of test substance. Further, the determination of the localization status is not limited to the determination of whether the test substance is localized in the nucleus or the cytoplasm. For example, in the case of having a protrusion shape like a nerve cell, it may be determined whether or not the test substance is localized at the tip of the protrusion.

As shown in FIG. 1, the cell information acquisition method includes steps S1 to S4. The case where the operator executes the cell information acquisition method of FIG. 1 by using a flow cytometer capable of capturing a fluorescent image and a processing device capable of analyzing the captured image will be described below. Each step of FIG. 1 may be executed by processing in the cell information acquisition device. The configuration and processing when the cell information acquisition device performs each step of FIG. 1 will be described later with reference to FIGS. 5 and later.

In step S1, the operator labels NF-κB contained in the cells collected from the subject with fluorescent substances 11 and 12 having different fluorescence wavelengths from each other. For example, as shown in FIG. 2, fluorescent substances 11 and 12 are bound to NF-κB contained in cells via a primary antibody and a secondary antibody. Fluorescent substances 11 and 12 may be bound to NF-κB by a plurality of primary antibodies, or may be bound to NF-κB by a part of the antibody or all of the antibodies. Further, in step S1, the operator labels the nucleus contained in the cell with a fluorescent substance 13 having a fluorescent wavelength different from that of the fluorescent substances 11 and 12.

Fluorescent substances 11, 12, and 13 are fluorescent dyes. The fluorescent substances 11, 12, and 13 are configured to excite fluorescence in wavelength bands different from each other when irradiated with light having wavelengths λ1, λ2, and λ3, respectively. That is, the wavelengths of light for exciting fluorescence from the fluorescent substances 11 to 13 are set to be different from each other. Thus, the sample is prepared in step S1. When the test substance is mRNA or microRNA, a fluorescent substance is bound to the test substance via a nucleic acid probe.

In step S2, the operator drives the flow cytometer to flow a sample containing cells labeled with fluorescent substances 11 to 13 into the flow cell, irradiates the cells flowing through the flow cell with light having wavelengths λ1 to λ3, and fluoresces. Fluorescence is generated from substances 11 to 13.

As shown in FIG. 2, when the cells are irradiated with laser light having wavelengths λ1 to λ3, fluorescence in different wavelength bands is generated from the fluorescent substances 11 to 13. The filter member 21 passes the fluorescence of the wavelength band B1 generated from the fluorescent substance 11 and blocks the light other than the wavelength band B1. The filter member 21 separates the fluorescence of the wavelength band B1 generated from the fluorescent substance 11. The filter member 22 passes the fluorescence of the wavelength band B2 generated from the fluorescent substance 12 and blocks the light other than the wavelength band B2. The filter member 22 separates the fluorescence of the wavelength band B2 generated from the fluorescent substance 12. The filter member 23 passes the fluorescence of the wavelength band B3 generated from the fluorescent substance 13 and blocks the light other than the wavelength band B3. The filter member 23 separates the fluorescence of the wavelength band B3 generated from the fluorescent substance 13.

Here, the laser beam having a wavelength of λ1 irradiates the cells with high power, and the laser beam having a wavelength λ2 irradiates the cells with low power. By irradiating the cells with a laser beam having a wavelength of λ1 with high power, the fluorescence in the wavelength band B1 passing through the filter member 21 becomes high intensity. By irradiating the cells with a laser beam having a wavelength of λ2 with low power, the fluorescence in the wavelength band B2 passing through the filter member 22 becomes low intensity.

In step S3, the processing apparatus acquires three fluorescence information for each cell based on the fluorescence generated from the fluorescent substances 11 to 13. The flow cytometer has a configuration for acquiring an image based on each fluorescence by forming an image of fluorescence in wavelength bands B1 to B3 separated by filter members 21 to 23 on a light receiving unit configured by an image sensor. It has. Based on the imaging signal output by the light receiving unit of the flow cytometer, the processing device provides fluorescence information such as an image based on high-intensity fluorescence in wavelength band B1, an image based on low-intensity fluorescence in wavelength band B2, and wavelength. An image based on the fluorescence of band B3 is acquired.

The fluorescence in the wavelength bands B1 to B3 may be individually imaged by the three light receiving units, or may be imaged by one light receiving unit. When the fluorescence of the wavelength bands B1 to B3 is imaged by one light receiving unit, the optical system is configured so that the fluorescence of the wavelength bands B1 to B3 is imaged in different regions on the light receiving surface of the light receiving unit.

As shown in FIG. 3, in the case of cells in which NF-κB is localized in the nucleus, for example, images 31 and 32 are acquired in step S3. Image 31 is an image based on high-intensity fluorescence in wavelength band B1, and image 32 is an image based on low-intensity fluorescence in wavelength band B2. In the images 31 and 32, a region 33 in which the nucleus exists is set. The region 33 is acquired from the fluorescence of the wavelength band B3 generated at the same time as the images 31 and 32, that is, the image based on the fluorescence generated from the nucleus.

In the case of cells where NF-κB is localized in the cytoplasm, for example, images 41 and 42 are acquired in step S3. Image 41 is an image based on high-intensity fluorescence in wavelength band B1, and image 42 is an image based on low-intensity fluorescence in wavelength band B2. The region 43 in which the nucleus exists is also set in the images 41 and 42. The region 43 is acquired from the fluorescence-based image of the wavelength band B3 generated at the same time as the images 41 and 42.

In the case of cells in which the expression level of NF-κB is low, according to image 31, the fluorescence intensity is appropriate and there is a difference in fluorescence intensity between the nucleus and the cytoplasm, so that NF-κB is located in the nucleus region 33. It can be determined that it is localized. On the other hand, according to the image 32, it cannot be determined that NF-κB is localized in the nuclear region 33 because the fluorescence intensity is too low. On the other hand, in the case of cells having a high expression level of NF-κB, according to image 41, the fluorescence intensity is too high, so that there is no difference in fluorescence intensity between the nucleus and the cytoplasm. Therefore, it is not possible to determine whether NF-κB is localized in the nuclear region 43 or the cytoplasmic region wider than the nuclear region 43. On the other hand, according to image 42, since the fluorescence intensity is appropriate and there is a difference in intensity between the nucleus and the cytoplasm, NF-κB is localized in the cytoplasmic region wider than the nucleus region 43. Can be identified.

When NF-κB is localized in the cytoplasm, NF-κB is distributed intracellularly so as to surround the nucleus. That is, when the cells are viewed in the imaging direction, NF-κB is also distributed in front of the nucleus. Therefore, in the image 41 based on high-intensity fluorescence, strong fluorescence is also generated in the nucleus region due to NF-κB distributed in front of the nucleus. Therefore, in the image 41, it tends to be difficult to properly determine whether NF-κB is localized in the nucleus or the cytoplasm. On the other hand, in the image 42 based on low-intensity fluorescence, NF-κB distributed in front of the nucleus causes fluorescence in the nucleus region as well, but the intensity of this fluorescence is low. Therefore, in the image 41, even when NF-κB is localized in the cytoplasm, the localization state can be appropriately determined.

When NF-κB is localized in the nucleus, most of NF-κB is distributed in the cytoplasm, although part of it is distributed in the cytoplasm. Therefore, in the image 31 based on high-intensity fluorescence, strong fluorescence is generated from NF-κB distributed in the nucleus, and it can be properly determined that NF-κB is localized in the nucleus. On the other hand, in the image 32 based on low-intensity fluorescence, since the fluorescence from NF-κB distributed in the nucleus is too weak, it is properly determined whether NF-κB is localized in the nucleus or the cytoplasm. I tend not to.

As described above, the appropriate intensity for determining the localization of NF-κB differs depending on the amount and distribution of NF-κB, which is a test substance in cells.

Therefore, the power of the laser beam having a wavelength of λ1 is set so that in a cell in which NF-κB is localized in the nucleus, it can be appropriately determined that NF-κB is localized in the nucleus as shown in image 31. The power of the laser beam having a wavelength of λ2 is set so that in a cell in which NF-κB is localized in the cytoplasm, it can be appropriately determined that NF-κB is localized in the cytoplasm as shown in image 42. As a result, regardless of whether NF-κB is localized in the nucleus or cytoplasm in the cell to be discriminated, the localization of NF-κB can be discriminated using at least one of the two images. Become.

Returning to FIG. 1, in step S4, the operator refers to the image based on the high intensity fluorescence in the wavelength band B1 and the image based on the low intensity fluorescence in the wavelength band B2, and refers to the image of the test substance NF-κB. Determine the distribution status. Specifically, the operator selects an image based on high-intensity fluorescence in wavelength band B1 and an image based on low-intensity fluorescence in wavelength band B2, in which the localized position of NF-κB can be discriminated. Based on the image, whether NF-κB is localized in the nucleus or cytoplasm in the cell, that is, the localization status is determined. Instead of the localization status, the position where NF-κB is distributed, for example, the distribution range in the cell, etc. may be determined.

As described above, in the first embodiment, among the images based on high-intensity fluorescence and the images based on low-intensity fluorescence, by adjusting the two fluorescences generated from the fluorescent substances 11 and 12 labeled with NF-κB, One of them is in a state where the localization of NF-κB can be appropriately determined. Therefore, the operator can accurately analyze NF-κB having various distributions in cells based on the two images. Specifically, the localization status of NF-κB in cells, that is, whether NF-κB is localized in the nucleus or cytoplasm can be accurately determined.

Further, in the first embodiment, even when NF-κB is localized in the nucleus, the fluorescence intensity of the image 31 is too high due to the large amount of NF-κB, and the fluorescence intensity of the image 32 is appropriate. It is possible that it will be in a state. Even in this case, it can be determined that NF-κB is localized in the nucleus by using the image 32 having an appropriate fluorescence intensity. Further, even when NF-κB is localized in the cytoplasm, since the amount of NF-κB is small, the fluorescence intensity of image 41 may be in an appropriate state, and the fluorescence intensity of image 42 may be too low. Conceivable. Even in this case, it can be determined that NF-κB is localized in the cytoplasm by using the image 41 having an appropriate fluorescence intensity. As described above, according to the first embodiment, the localization state of NF-κB in the cell can be accurately determined based on the two images regardless of the amount of NF-κB.

Here, the vascular endothelial cells are detached from the inner wall of the blood vessel and flow into the blood. Detachment of vascular endothelial cells is caused not only by irritation due to inflammation but also by changes in pressure due to compression or the like. In vascular endothelial cells exfoliated by inflammation stimulation, NF-κB tends to be localized in the nucleus, and in vascular endothelial cells exfoliated by stimulation other than inflammation stimulation, NF-κB tends not to be localized in the nucleus. .. According to the first embodiment, since the localization of NF-κB can be accurately determined as described above, NF-κB, which is a signal molecule, is localized in the nucleus of the exfoliation caused by inflammation stimulation among these exfoliation causes. It can be determined by whether or not it is present, and the presence or absence of activation of vascular endothelial cells can be determined. This makes it possible to determine, for example, whether the detachment of vascular endothelial cells is caused by pressure at the time of blood collection or is caused by a disease or the like, and has clinical significance.

An image of medium-intensity fluorescence may be acquired by a medium-power laser beam having a wavelength different from the wavelengths λ1 and λ2. That is, NF-κB is labeled with three fluorescent substances having different fluorescence wavelengths from each other, and the cells are irradiated with three laser beams to generate fluorescence having different intensities from the three fluorescent substances, and an image based on each fluorescence is acquired. You may. By doing so, the localization of NF-κB can be determined more accurately by using the most appropriate image from the three images having different fluorescence intensities. The intensity of fluorescence generated from the test material may be four or more steps, or four or more images based on fluorescence of four or more intensities may be acquired for one cell.

Further, in step S4, the operator acquires the proportion of cells in which NF-κB is localized in a specific site among the cells contained in the sample, based on the localization status of each cell determined as described above. Specifically, assuming that the number of cells determined to localize NF-κB to the nucleus is N1 and the number of cells determined to localize NF-κB to the cytoplasm is N2, the operator can use the following formula. Obtain the nuclear localization rate and cytoplasmic localization rate. In step S4, the operator may acquire the nuclear localization number and the cytoplasmic localization number instead of the nuclear localization rate and the cytoplasmic localization rate.

Nuclear localization = {N1 / (N1 + N2)} x 100
Cytoplasmic localization = {N2 / (N1 + N2)} x 100

In step S2, the image of each fluorescence was acquired by using the flow cytometer as described above, but the present invention is not limited to this, and the image of each fluorescence may be acquired as fluorescence information by using a microscope. That is, a high-intensity fluorescent image generated from the fluorescent substance 11, a low-intensity fluorescent image generated from the fluorescent substance 12, and an image corresponding to the nucleus generated from the fluorescent substance 13 may be acquired by a microscope. ..

<Verification of Embodiment 1>
Next, the verification of the first embodiment performed by the inventor will be described.

1. 1. Human cardiac microvascular endothelial cells (HMVEC-C) (Lonza Cat No. CC-7030, Lot No. 0000296500 (P4)) were prepared as preparatory cells. As the primary antibody, NF-κB p65 (D14E12) XP Rabbit mAb (Cell Signaling Technologies # 8242S) was prepared. As secondary antibodies, Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor 647 conjugate (Life technologies A-21245), Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor 488 conjugate (Life technologies) A-11008) was prepared. Alexa Fluor 647 and Alexa Fluor 488 are bound to the secondary antibody as fluorescent dyes. Cellstain Hoechst 33342 solution (DOjinDO H342) was prepared as a nuclear stain. In addition, EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (Lonza Cat No. CC-3202), PBS pH7.4 (GIBCO Cat No.10010-023), BSA (LAMPIRE Cat) No. 7500805), PFA (WAKO Cat No.160-16061), TritonX100 (Nakarai Tesk Cat No.35501-15) were prepared.

2. 2. Reagent Preparation Reagents other than FBS of EGM-2MV Single Quots Kit were added to 500 mL of EGM-2MV Medium, and 100 mL was transferred to a sterile bottle to prepare a serum-free medium. A culture medium was prepared by adding 20 mL of FBS of Single Quots Kit to the remaining amount (400 mL) of the serum-free medium. Paraformaldehyde was dissolved in PBS at pH 12 to a final concentration of 8% w / v, and then adjusted to pH 7.4. 1.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 3% BSA / PBS. 0.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 1% BSA / PBS. Triton X100 was prepared with PBS to a final concentration of 0.1% w / v.

3. 3. procedure
HMVEC-C was cultured in EGM-2MV medium according to the manufacturer's recommended protocol. Cells within 6 passages after purchase were used for this verification. The culture medium had an expiration date of 3 weeks after opening. For TNF-α stimulation culture, remove the culture supernatant of HMV EC-C cells with approximately 70% confluence, add EGM-2MV medium supplemented with Recombinant Human TNF-alpha to a final concentration of 25 ng / mL, and add 37 ° C. CO ₂ It was allowed to stand in the incubator for 1 hour. The medium was removed with an electric pipettor leaving about 3 mL, and the cells were detached with a scraper. Equivalent 8% PFA / PBS was added to the recovered suspension and reacted at room temperature for 15 minutes. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. The cell pellet was washed twice with 1 mL PBS. The supernatant was removed, 1 mL of 0.1% Triton X-100 / PBS was added, and the mixture was reacted at room temperature for 15 minutes. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. Washed twice with 1 mL of 1% BSA / PBS. The supernatant was removed, 1 mL of 3% BSA / PBS was added, and the mixture was allowed to stand at room temperature for 30 minutes. 400 μL of primary antibody to 1/1600 with 3% BSA / PBS was added. The reaction was carried out at room temperature for 1 hour. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. Washed with 1 mL of 1% BSA / PBS. 400 μL of secondary antibody reduced to 1/1000 with 3% BSA / PBS was added. The reaction was carried out at room temperature for 30 minutes. Washed twice with 1 mL of 1% BSA / PBS. The supernatant was removed and 50 μL of 1% BSA / PBS was added.

4. Detection by flow cytometer ImageStreamX Mark II Imaging Flow Cytometer (Merck Millipore) was used as a flow cytometer capable of acquiring fluorescence images. A sample prepared according to 3 above was flowed through the flow cell of this flow cytometer, and the sample flowing through the flow cell was irradiated with laser light having wavelengths of 488 nm, 647 nm, and 405 nm. The laser beams having wavelengths of 488 nm, 647 nm, and 405 nm correspond to the laser beams having the wavelengths λ1, λ2, and λ3. The emission powers of the laser beams having wavelengths of 488 nm, 647 nm, and 405 nm were 55 mW, 10 mW, and 120 mW, respectively. By irradiating the two types of fluorescent dyes labeling NF-κB with laser light having wavelengths of 488 nm and 647 nm, high-intensity fluorescence and low-intensity fluorescence were generated, respectively. Fluorescence was generated by irradiating the nuclear dye with a laser beam having a wavelength of 405 nm.

In the flow cytometer, the fluorescence generated by the laser beam having a wavelength of 488 nm was imaged through a filter member having a transmission wavelength band of 505 nm to 560 nm, and a high-intensity fluorescence image was acquired. The fluorescence generated by the laser beam having a wavelength of 647 nm was imaged through a filter member having a transmission wavelength band of 642 nm to 740 nm, and a low-intensity fluorescence image was obtained. The fluorescence generated by the laser beam having a wavelength of 405 nm was imaged through a filter member having a transmission wavelength band of 430 nm to 505 nm, and a fluorescence image corresponding to the nucleus was acquired. Further, the sample flowing through the flow cell was irradiated with a laser beam having a wavelength set between 430 nm and 480 nm. The light transmitted through the cells by the laser light was imaged through a filter member having a transmission wavelength band of 430 nm to 480 nm, and a bright field image was acquired. In the flow cytometer, unnecessary wavelength band light is removed by a filter member or the like so that light in the target wavelength band is properly incident on the light receiving portion. In this verification, a bright-field image was acquired, but the present invention is not limited to this, and a dark-field image may be acquired.

The image acquired by the above detection will be described with reference to FIG. 4A.

"Bright field" refers to a bright field image of a cell. "High-intensity fluorescence" and "low-intensity fluorescence" are images based on high-intensity fluorescence generated from a fluorescent dye labeled with NF-κB and low-intensity generated from a fluorescent dye labeled with NF-κB, respectively. It is an image based on the fluorescence of. "Fluorescence from the nucleus" is an image based on the fluorescence generated from the dye for nuclear staining that stained the nucleus. "Composite" is an image obtained by synthesizing the four images on the left side. The five images arranged side by side are images obtained from one cell. The images indicated by "high intensity fluorescence", "fluorescence from nucleus", "low intensity fluorescence", and "synthesis" are grayscaled versions of the acquired color image for convenience. In the images indicated by "high intensity fluorescence", "fluorescence from the nucleus", and "low intensity fluorescence", the white part indicates that the fluorescence intensity is strong.

In the case of the cells shown in the upper row, it is difficult to determine the localization of NF-κB because the image based on low-intensity fluorescence is too low in intensity. On the other hand, since the intensity of the image based on high-intensity fluorescence is appropriate, it can be determined that NF-κB is localized in the nucleus. In the case of the cells shown in the lower row, it is difficult to determine the localization of NF-κB because the image based on high intensity fluorescence is too intense. On the other hand, since the intensity of the image based on low-intensity fluorescence is appropriate, it can be determined that NF-κB is localized in the cytoplasm.

5. Calculation of nuclear localization rate The localization of NF-κB was determined for each cell by visually observing the acquired image. This determination was performed in the same manner as in step S4. That is, the nuclear region was set based on the fluorescence image from the nucleus, and the region other than the nuclear region was defined as the cytoplasmic region. When the fluorescence intensity of the nucleus is considered to be about twice or more the fluorescence intensity of the cytoplasm, it is determined that NF-κB is localized in the nucleus in this cell, and the fluorescence intensity of the nucleus is about about the fluorescence intensity of the cytoplasm. If it was considered to be less than twice, it was determined that NF-κB was localized in the cytoplasm in this cell.

The result of discriminating the localization of NF-κB in 131 cells identified by the flow cytometer will be described with reference to FIG. 4 (b).

The number of cells that could be determined to be localized in the nucleus was 44, and the number of cells that could be determined to be localized in the cytoplasm was 87. The number of cells whose localization could not be determined was 0. The nuclear localization rate at this time was 44/131 = 34%.

Here, Comparative Example 1 in which localization is determined based only on a high-intensity fluorescence image and Comparative Example 2 in which localization is determined based only on a low-intensity fluorescence image will be described. In the case of Comparative Example 1, the number of cells that could be determined to be localized in the nucleus was 42, and the number of cells that could be determined to be localized in the cytoplasm was 10. The number of cells whose localization could not be determined because the fluorescence intensity was too high was 79. The nuclear localization rate of Comparative Example 1 was 81%. In the case of Comparative Example 2, the number of cells that could be determined to be localized in the nucleus was 16, and the number of cells that could be determined to be localized in the cytoplasm was 84. The number of cells whose localization could not be determined because the fluorescence intensity was too low was 31. The nuclear localization rate of Comparative Example 2 was 16%.

As described above, according to this verification, when the localization is discriminated based on two fluorescent images having different intensities as in the first embodiment, the cells that cannot be discriminated in the cases of Comparative Examples 1 and 2 are also discriminated. It can be seen that the localization of NF-κB can be discriminated. Further, when the localization is discriminated as in the first embodiment, it can be seen that the localization of NF-κB in the cells can be accurately discriminated because there are few cells that cannot be discriminated. Therefore, according to the first embodiment, the number of indistinguishable cells can be suppressed to a low level, and the localization of NF-κB can be accurately discriminated. Thereby, for example, even when the number of cells collected from the subject is small, the localization of NF-κB can be accurately determined while securing the cells that can be identified.

In the first embodiment, as an example of discriminating the distribution status of the test substance, an example of discriminating the localization status of NF-κB has been shown, but when the amount of the molecule of the test substance changes, the amount of this molecule is used. You may discriminate. In this case, the operator labels the molecules with fluorescent substances 11 and 12, and the processing apparatus acquires an image based on the two fluorescences of different intensities generated from the fluorescent substances 11 and 12. When the amount of molecules is large, the amount is determined by using a low-intensity fluorescence image, and when the amount of molecules is small, the amount is determined by using a high-intensity fluorescence image. This makes it possible to accurately determine the amount of molecules.

<Device configuration of the first embodiment>
A configuration of a cell information acquisition device for capturing a cell image and determining the localization of a test substance in cells will be described based on the cell information acquisition method of the first embodiment.

As shown in FIG. 5, the cell information acquisition device 100 includes a processing unit 110, a sample preparation unit 120, an optical detection unit 130, a drive unit 140, a display unit 150, an input unit 160, and a storage unit 170. , Equipped with.

The processing unit 110 is composed of a microcomputer, a CPU, and the like. The storage unit 170 is composed of a RAM, a ROM, a hard disk, and the like. The storage unit 170 stores various data such as a processing program executed by the processing unit 110 and an image. The processing unit 110 transmits and receives signals to and from each unit of the cell information acquisition device 100, and controls each unit. The processing unit 110 is provided with the functions of the acquisition unit 111 and the analysis unit 112 by the program stored in the storage unit 170.

The sample preparation unit 120 prepares a sample by mixing the cells and the reagent according to step S1 of FIG. Sample preparation may be done by the operator. In this case, the sample preparation unit 120 is omitted from the cell information acquisition device 100. The optical detection unit 130 is a flow cytometer. The optical detection unit 130 irradiates the cells contained in the sample with light and images the generated fluorescence. The configuration of the optical detection unit 130 will be described later with reference to FIG. The drive unit 140 drives the light sources 301 to 304 of the optical detection unit 130, which will be described later.

The display unit 150 is composed of a display. The display unit 150 shows the image acquired for each cell, the localization of NF-κB determined for each cell, the number of cells in which NF-κB is localized in the nucleus, and the localization of NF-κB in the cytoplasm. The number of cells, nuclear localization rate, cytoplasmic localization rate, etc. are displayed. The input unit 160 includes a mouse and a keyboard. The operator inputs an instruction to the cell information acquisition device 100 via the input unit 160.

As shown in FIG. 6, the optical detection unit 130 includes a flow cell 200, a light irradiation unit 300, a light collection unit 400, and light receiving units 501 to 504. A flow path 210 is formed in the flow cell 200, and a sample prepared by the sample preparation unit 120 is flowed through the flow path 210. For convenience, FIG. 6 shows XYZ axes that are orthogonal to each other.

The light irradiation unit 300 irradiates the cells contained in the sample flowing through the flow cell 200 with light to generate fluorescence having different intensities from the fluorescent substances 11 and 12 shown in FIG. Further, the light irradiation unit 300 irradiates the cells with light to generate fluorescence from the fluorescent substance 13 shown in FIG. 2, and further irradiates the cells with light for a bright field. The light irradiation unit 300 includes light sources 301 to 304, condenser lenses 31 to 314, and dichroic mirrors 321 and 322.

The light sources 301 to 304 are composed of a semiconductor laser light source. The light emitted from the light sources 301 to 304 is laser light having wavelengths λ1 to λ4, respectively. The wavelengths λ1 to λ4 are, for example, 488 nm, 647 nm, 405 nm, and 785 nm, respectively. Wavelengths λ1 to λ3 are light for exciting fluorescence from fluorescent substances 11 to 13, as shown in FIG. The condensing lenses 31 to 314 respectively condense the light emitted from the light sources 301 to 304. The dichroic mirror 321 transmits light having a wavelength of λ1 and reflects light having a wavelength of λ2. The dichroic mirror 322 transmits light having wavelengths λ1 and λ2 and reflects light having wavelengths λ3.

In this way, the light irradiating unit 300 irradiates the cells contained in the sample flowing through the flow path 210 with the lights of wavelengths λ1 to λ3 emitted from the light sources 301 to 303 superposed on each other. Further, the light irradiation unit 300 irradiates the position of the flow path 210 to which the wavelengths λ1 to λ3 are irradiated with the light having the wavelength λ4. When the sample flowing through the flow cell 200 is irradiated with light having wavelengths λ1 to λ3, fluorescence in different wavelength bands is generated from the fluorescent substances 11 to 13 as described with reference to FIG. When the sample flowing through the flow cell 200 is irradiated with light having a wavelength of λ4, this light passes through the cells. The light of wavelength λ4 transmitted through the cells is used to acquire a bright-field image.

Here, the light source 301 emits light having a wavelength λ1 with high power, and the light source 302 emits light having a wavelength λ2 with low power. The emission power of the light sources 301 and 302 is controlled by the drive unit 140 shown in FIG. As a result, as described with reference to FIG. 2, the fluorescence generated from the fluorescent substance 11 becomes high intensity, and the fluorescence generated from the fluorescent substance 12 becomes low intensity. In the first embodiment and the second and third embodiments described later, it is not necessary to adjust the emission powers of the light sources 301 and 302. By selecting fluorescent labels having different fluorescence intensities even with the same emission power, the fluorescence generated from the fluorescent substance 11 becomes high intensity, and the fluorescence generated from the fluorescent substance 12 becomes low intensity.

The light collecting unit 400 collects the fluorescence generated from the flow cell 200 by irradiation with light having wavelengths λ1 to λ3. The light collecting unit 400 collects the fluorescence generated from the fluorescent substances 11 to 13 on the light receiving units 501 to 503, respectively. Further, the light collecting unit 400 collects the light having a wavelength λ4 generated from the flow cell 200 on the light receiving unit 504. The condensing unit 400 includes a condensing lens 401, filter members 411 to 413, 421 to 424, and condensing lenses 431 to 434.

The condenser lens 401 collects the fluorescence generated from the sample flowing through the flow cell 200 and the light having a wavelength λ4 transmitted through the sample flowing through the flow cell 200. The filter members 411 to 413 are composed of a dichroic mirror.

The filter member 411 reflects the light in the wavelength band B1 among the light collected by the condenser lens 401, and transmits the light other than the wavelength band B1. Of the light reflected by the filter member 411, the filter member 421 transmits only the light in the wavelength band B1 and blocks the light other than the wavelength band B1. In this way, the filter members 411 and 421 are configured to be able to separate only the fluorescence of the wavelength band B1 from the light generated from the flow cell 200. Similarly, the filter members 412 and 422 are configured so that only the fluorescence of the wavelength band B2 can be separated from the light generated from the flow cell 200, and the filter members 413 and 423 have the wavelength band of the light generated from the flow cell 200. Only the fluorescence of B3 can be separated. The filter member 424 transmits light having a wavelength of λ4 among the light transmitted through the filter members 411 to 413, and blocks light other than the wavelength λ4.

The light receiving unit 501 receives the light of the wavelength band B1 condensed by the condensing lens 431 and outputs the image information based on the received light as an imaging signal. The light receiving unit 502 receives the light in the wavelength band B2 condensed by the condenser lens 432 and outputs the image information based on the received light as an imaging signal. The light receiving unit 503 receives light in the wavelength band B3 focused by the condenser lens 433, and outputs image information based on the received light as an imaging signal. The light receiving unit 504 receives light having a wavelength λ4 collected by the condenser lens 434 and outputs image information based on the received light as an imaging signal. The light receiving units 501 to 504 are composed of an image pickup element such as a color CCD.

The light condensing unit 400 condenses the light of the wavelength bands B1 to B3 and the light of the wavelength λ4 on the light receiving units 501 to 504, respectively, but an image may be formed on one light receiving unit. In this case, the optical detection unit 130 is configured so that the light in the wavelength bands B1 to B3 and the light in the wavelength λ4 are imaged in different regions on the light receiving surface of the light receiving unit.

In the configuration shown in FIG. 6, a plurality of filter members were used to separate the light in the wavelength band B1, but as shown in FIG. 7, the light generated from the flow cell 200 is separated by one filter member. Is also good. As shown in FIG. 7, the condensing unit 400 includes condensing lenses 441 to 444, filter members 451 to 454, and condensing lenses 461 to 464. The light in the wavelength bands B1 to B3 is separated by the filter members 451 to 453, respectively, and the light in the wavelength λ4 is separated by the filter member 454.

Next, a case where the process of step S4 of FIG. 1 is performed by the cell information acquisition device 100 will be described with reference to the flowchart of FIG.

As shown in FIG. 8, in step S11, the processing unit 110 drives the sample preparation unit 120 to label NF-κB contained in the cells with fluorescent substances 11 and 12 as in step S1 of FIG. A sample is prepared by labeling the nucleus contained in the cell with the fluorescent substance 13. In step S12, the processing unit 110 causes the sample to flow through the flow cell 200, drives the light sources 301 to 304 by the driving unit 140, and irradiates the cells flowing through the flow cell 200 with light. In step S13, the processing unit 110 images the fluorescence of the wavelength bands B1 to B3 by the light receiving units 501 to 503, and the light of the wavelength λ4 by the light receiving unit 504. Then, the acquisition unit 111 of the processing unit 110 acquires an image based on the image pickup signal output by the light receiving units 501 to 504.

In step S14, the analysis unit 112 of the processing unit 110 selects an image whose localization can be discriminated from the high-intensity and low-intensity fluorescence images. Specifically, the analysis unit 112 selects an image in which the fluorescence intensity obtained from the image, for example, the brightness of the entire image is within a predetermined range, from the high-intensity fluorescence image and the low-intensity fluorescence image. .. As a result, the image with extremely high fluorescence intensity and the image with extremely low fluorescence intensity cannot discriminate the localization of NF-κB as in the image 32 and image 41 of FIG. Be excluded.

Further, the analysis unit 112 selects an image in which the difference between the fluorescence intensity at the cell analysis target site and the fluorescence intensity at the cells other than the analysis target site is larger than a predetermined threshold value among the high-intensity and low-intensity fluorescence images. To do. In the first embodiment, the analysis target site is the nucleus. That is, an image in which the difference between the fluorescence intensity in the nucleus and the fluorescence intensity of the cells outside the nucleus is larger than a predetermined threshold value is selected. As a result, an image in which the difference in fluorescence intensity between the inside and outside the nucleus is small cannot be discriminated from the localization of NF-κB as in the images 32 and 41 of FIG. 3, and is therefore excluded from the images used for discrimination.

Next, in step S15, the analysis unit 112 determines the localization of NF-κB in the cells for each cell using the image selected in step S14. That is, the analysis unit 112 calculates the ratio of the localized amount of NF-κB in the analysis target site of the cell to the amount of NF-κB in the whole cell. For example, in the image selected in step S14, the analyzer 112 divides the fluorescence intensity in the nuclear region by the fluorescence intensity in the entire cell region. The analysis unit 112 determines that NF-κB is localized in the nucleus when the division result is 2 or more, and NF-κB is localized in the cytoplasm when the division result is smaller than 2. To determine. The value for determining the division result is not limited to 2, and other values may be used.

When two images are selected in step S14, in step S15, the division results are acquired and the localization is determined for both images as described above. Further, when the image is not selected in step S14, the localization of this cell is regarded as "indistinguishable" in step S15.

In step S16, the analysis unit 112 calculates the above-mentioned nuclear localization number, cytoplasmic localization number, nuclear localization rate, and cytoplasmic localization rate based on the discrimination results of all the treated cells. In step S17, the processing unit 110 displays the numerical value calculated in step S16, the image acquired for each cell, the discrimination result for each cell, and the like on the display unit 150. Specifically, the processing unit 110 displays the screen 161 including the above contents on the display unit 150.

As shown in FIG. 9, the screen 161 includes regions 161a and 161b. Region 161a displays the number of nuclear localizations, the number of cytoplasmic localizations, the nuclear localization rate, and the cytoplasmic localization rate. The region 161b displays an image and a determination result of localization of NF-κB. In region 161b, the image used for localization determination in step S15 is surrounded by a solid line so that it can be seen that it was used for localization determination.

The fluorescence information acquired in step S13 may be a waveform signal indicating the fluorescence intensity that changes with time. In this case, in the optical detection unit 130, photodetectors such as photomultipliers are arranged as light receiving units 501 to 503, respectively. The three photodetectors receive high-intensity fluorescence in wavelength band B1, low-intensity fluorescence in wavelength band B2, and fluorescence in wavelength band B3, and output waveform signals indicating the intensity of each fluorescence, respectively.

As shown in FIG. 10, the analysis unit 112 acquires graphs 51 and 52, for example, in the case of cells having a low expression level of NF-κB based on the waveform signal output by the photodetector, and expresses NF-κB. For cells with a large amount, for example, graphs 61 and 62 are obtained. Further, the analysis unit 112 acquires a graph corresponding to the nucleus based on the waveform signal output by the photodetector. The analysis unit 112 sets the width W1 of the waveform corresponding to the nucleus in the graphs 51 and 52 from the graph of the nucleus acquired at the same time as the graphs 51 and 52, and from the graph of the nucleus acquired at the same time as the graphs 61 and 62, In the graphs 61 and 62, the width W2 of the waveform corresponding to the nucleus is set.

According to the graph 51, the peak value of fluorescence is between the threshold values Sh1 and Sh2, and the peak of the waveform exists in the width W1 corresponding to the nucleus. Therefore, the analysis unit 112 determines that NF-κB is localized in the nucleus. it can. On the other hand, according to the graph 52, since the peak value of fluorescence is smaller than the threshold value Sh1, the analysis unit 112 cannot determine the localization of NF-κB. According to the graph 61, since the peak value of fluorescence is larger than the threshold value Sh2, the analysis unit 112 cannot determine the localization of NF-κB. On the other hand, according to the graph 62, since the peak value of fluorescence is between the threshold values Sh1 and Sh2 and the corrugated depression exists in the width W2 corresponding to the nucleus, the analysis unit 112 indicates that NF-κB is localized in the cytoplasm. Then it can be determined. Therefore, also in this case, as in the case of using an image as shown in FIG. 3, the localization of NF-κB can be accurately discriminated by the two fluorescences having different intensities.

<Embodiment 2>
In the second embodiment, instead of using two lights, only light having a wavelength of λ1 is used to obtain fluorescence of different intensities. In the second embodiment, only a part of the steps of the cell information acquisition method shown in FIG. 1 in steps S1 and S2 is different from that in the first embodiment. Hereinafter, a procedure different from that of the first embodiment will be described.

In step S1, as shown in FIG. 11, NF-κB contained in the cell is labeled with fluorescent substances 14 and 15 having different fluorescence wavelengths from each other. Fluorescent substances 14 and 15 are fluorescent dyes. When the fluorescent substance 14 is irradiated with light having a wavelength of λ1, it excites fluorescence in the same wavelength band as that of the fluorescent substance 11 of the first embodiment. When the fluorescent substance 15 is irradiated with light having a wavelength of λ1, it excites fluorescence in the same wavelength band as that of the fluorescent substance 12 in FIG. That is, the fluorescent substances 14 and 15 have substantially the same wavelength of light for excitation.

In step S2, a sample containing cells labeled with fluorescent substances 14, 15 and 13 is flowed into the flow cell, the cells flowing through the flow cell are irradiated with light having wavelengths λ1 and λ3, and fluorescence is emitted from the fluorescent substances 14, 15 and 13. It is caused. The fluorescence generated from the fluorescent substances 14 and 15 is passed through the filter members 21 and 22, respectively, to become fluorescence in the wavelength bands B1 and B2. At this time, the fluorescent substances 14 and 15 are configured so that the fluorescence in the wavelength band B1 becomes high intensity and the fluorescence in the wavelength band B2 becomes low intensity.

In the apparatus configuration of the second embodiment, the light source 302, the condenser lens 312, and the dichroic mirror 321 are omitted from the optical detection unit 130 shown in FIG. 6 as compared with the first embodiment.

Also in the second embodiment, as in the first embodiment, high-intensity fluorescence in the wavelength band B1 and low-intensity fluorescence in the wavelength band B2 are generated, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, as in the first embodiment, NF-κB having various distributions and amounts in cells can be accurately analyzed based on a high-intensity fluorescence image and a low-intensity fluorescence image.

<Embodiment 3>
In the third embodiment, instead of using two lights and two fluorescent substances, light having one wavelength λ1 and one fluorescent substance 11 are used to obtain fluorescence having different intensities from each other. In the third embodiment, only a part of the steps of the cell information acquisition method shown in FIG. 1 in steps S1 and S2 is different from that in the first embodiment. Hereinafter, a procedure different from that of the first embodiment will be described.

In step S1, as shown in FIG. 12, NF-κB contained in the cells is labeled only with the same fluorescent substance 11 as in the first embodiment. At this time, the fluorescent substance 11 may be bound to NF-κB via one antibody. In step S2, a sample containing cells labeled with fluorescent substances 11 and 13 is flowed into a flow cell, the cells flowing through the flow cell are irradiated with light having wavelengths λ1 and λ3, and fluorescence is generated from the fluorescent substances 11 and 13.

As shown in FIG. 12, the fluorescence generated from the fluorescent substance 11 is divided into two, one is passed through the same filter member 21 as in the first embodiment, and the other is passed through the same filter member 22 as in the first embodiment. The filter member 21 transmits only the light in the wavelength band B1, and the filter member 21 transmits only the light in the wavelength band B4. As shown in FIG. 13, the wavelength band B1 includes, for example, a wavelength at which the intensity of fluorescence generated from the fluorescent substance 11 peaks. The wavelength band B4 is set to, for example, a wavelength band larger than the wavelength band B1 and not overlapping the wavelength band B1. As a result, as shown in FIG. 12, the fluorescence of the wavelength band B1 passing through the filter member 21 becomes high intensity, and the fluorescence of the wavelength band B4 passing through the filter member 22 becomes low intensity.

The wavelength band B1 does not necessarily have to include a wavelength at which the intensity of fluorescence generated from the fluorescent substance 11 peaks. The wavelength band B4 may be set to a wavelength band smaller than the wavelength band B1, or may partially overlap with the wavelength band B1.

<Verification of Embodiment 3>
Next, the verification of the third embodiment performed by the inventor will be described.

1. 1. Human cardiac microvascular endothelial cells (HMVEC-C) (Lonza Cat No. CC-7030, Lot No. 0000296500 (P4)) were prepared as preparatory cells. As the primary antibody, NF-κB p65 (D14E12) XP Rabbit mAb (Cell Signaling Technologies # 8242S) was prepared. As a secondary antibody, Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor 647 conjugate (Life technologies A-21245) was prepared. Alexa Fluor 647 is bound to the secondary antibody as a fluorescent dye. In addition, EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (Lonza Cat No. CC-3202), PBS pH7.4 (GIBCO Cat No.10010-023), BSA (LAMPIRE Cat) No. 7500805), PFA (WAKO Cat No.160-16061), TritonX100 (Nakarai Tesk Cat No.35501-15) were prepared.

2. 2. Reagent Preparation EGM-2MV Single Quots Kit was added to 500 mL of EGM-2MV Medium to prepare a culture medium. Paraformaldehyde was dissolved in PBS at pH 12 to a final concentration of 8% w / v, and then adjusted to pH 7.4. 1.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 3% BSA / PBS. 0.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 1% BSA / PBS. Triton X100 was prepared with PBS to a final concentration of 0.1% w / v.

3. 3. procedure
HMVEC-C was cultured in EGM-2MV medium according to the manufacturer's recommended protocol. Cells within 6 passages after purchase were used for this verification. The culture medium had an expiration date of 3 weeks after opening. For TNF-α stimulation culture, remove the culture supernatant of HMV EC-C cells with approximately 70% confluence, add EGM-2MV medium supplemented with Recombinant Human TNF-alpha to a final concentration of 25 ng / mL, and add 37 ° C. CO ₂ It was allowed to stand in the incubator for 1 hour. The medium was removed with an electric pipettor leaving about 3 mL, and the cells were detached with a scraper. Equivalent 8% PFA / PBS was added to the recovered suspension and reacted at room temperature for 15 minutes. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. The cell pellet was washed twice with 1 mL PBS. The supernatant was removed, 1 mL of 0.1% Triton X-100 / PBS was added, and the mixture was reacted at room temperature for 15 minutes. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. Washed twice with 1 mL of 1% BSA / PBS. The supernatant was removed, 1 mL of 3% BSA / PBS was added, and the mixture was allowed to stand at room temperature for 30 minutes. 400 μL of primary antibody to 1/1600 with 3% BSA / PBS was added. The reaction was carried out at room temperature for 1 hour. Centrifugation was performed at 1000 rpm for 3 minutes at room temperature. Washed with 1 mL of 1% BSA / PBS. 400 μL of secondary antibody reduced to 1/1000 with 3% BSA / PBS was added. The reaction was carried out at room temperature for 30 minutes. Washed twice with 1 mL of 1% BSA / PBS. The supernatant was removed and 50 μL of 1% BSA / PBS was added.

4. Detection by flow cytometer ImageStreamX Mark II Imaging Flow Cytometer (Merck Millipore) was used as a flow cytometer capable of acquiring fluorescence images. A sample prepared according to 3 above was flowed through the flow cell of this flow cytometer, and the sample flowing through the flow cell was irradiated with a laser beam having a wavelength of 647 nm. The laser beam having a wavelength of 647 nm corresponds to the laser beam having a wavelength λ1 shown in FIG. The emission power of the laser beam having a wavelength of 647 nm was set to 10 mW. Fluorescence was generated by irradiating the fluorescent dye labeling NF-κB with a laser beam having a wavelength of 647 nm.

In the flow cytometer, the fluorescence generated by the laser beam having a wavelength of 647 nm was imaged through a filter member having a transmission wavelength band of 642 nm to 740 nm, and a high-intensity fluorescence image was obtained. Further, the fluorescence generated by the laser beam having a wavelength of 647 nm was imaged through a filter member having a transmission wavelength band of 740 nm to 800 nm, and a low-intensity fluorescence image was obtained. In the flow cytometer, unnecessary wavelength band light is removed by a filter member or the like so that light in the target wavelength band is properly incident on the light receiving portion.

The image acquired by the above detection will be described with reference to FIGS. 14 (a) and 14 (b).

"High-intensity fluorescence" and "low-intensity fluorescence" are images based on high-intensity fluorescence generated from a fluorescent dye labeled with NF-κB and low-intensity generated from a fluorescent dye labeled with NF-κB, respectively. It is an image based on the fluorescence of. In FIGS. 14A and 14B, the two images arranged side by side are images obtained from one cell. Each image is a grayscale version of the acquired color image for convenience. In each image, the white part indicates that the fluorescence intensity is strong.

In the case of the three cells shown in FIG. 14 (a), it is difficult to determine the localization of NF-κB because the image based on low intensity fluorescence is too low in intensity. On the other hand, since the intensity of the image based on high-intensity fluorescence is appropriate, it can be determined that NF-κB is localized in the cytoplasm. In the case of the three cells shown in FIG. 14 (b), it is difficult to determine the localization of NF-κB because the image based on high intensity fluorescence is too intense. On the other hand, since the intensity of the image based on low-intensity fluorescence is appropriate, it can be determined that NF-κB is localized in the cytoplasm.

As described above, according to this verification, it can be seen that the localization of NF-κB can be discriminated even when the localization is discriminated based on two fluorescent images having different intensities as in the third embodiment. As described above, according to the third embodiment, one cell is obtained by passing the fluorescence generated from one kind of fluorescent dye through the filter members 21 and 22 that pass the fluorescence of different wavelength bands and acquiring two fluorescence images. The localization of NF-κB can be correctly obtained with a single measurement.

<Device configuration of the third embodiment>
As shown in FIG. 15, in the apparatus configuration of the third embodiment, the light source 302, the condenser lens 312, and the dichroic mirror 321 are omitted from the optical detection unit 130 shown in FIG. 6 as compared with the first embodiment. , Filter members 414 and 425 are added in place of the filter members 412 and 422, respectively. The filter member 414 reflects the light in the wavelength band B4 among the light transmitted through the filter member 411, and transmits the light other than the wavelength band B4. Of the light reflected by the filter member 414, the filter member 425 transmits only the light in the wavelength band B4 and blocks the light other than the wavelength band B4. In this way, the filter members 414 and 425 are configured to be able to separate only the fluorescence of the wavelength band B4 from the light generated from the flow cell 200. The light receiving unit 502 images low-intensity fluorescence in the wavelength band B4.

Also in the third embodiment, as in the first embodiment, high-intensity fluorescence and low-intensity fluorescence can be generated separately, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, as in the first embodiment, NF-κB having various distributions and amounts in cells can be accurately analyzed based on a high-intensity fluorescence image and a low-intensity fluorescence image.

<Embodiment 4>
In the fourth embodiment, only a part of the steps of the cell information acquisition method shown in FIG. 1 in steps S1 and S2 is different from that in the first embodiment. Hereinafter, a procedure different from that of the first embodiment will be described.

In step S1, as shown in FIG. 16, NF-κB contained in the cells is labeled only with the same fluorescent substance 11 as in the first embodiment. In step S2, a sample containing cells labeled with fluorescent substances 11 and 13 is flowed into a flow cell, the cells flowing through the flow cell are irradiated with light having wavelengths λ1 and λ3, and fluorescence is generated from the fluorescent substances 11 and 13. At this time, since the light having the wavelength λ1 is irradiated to the cells with high power, the fluorescence in the wavelength band B1 transmitted through the filter member 21 has high intensity as in the first embodiment. Further, the cells are moved in the flow cell, and the moved cells are irradiated with light having a wavelength of λ1 to generate fluorescence from the fluorescent substance 11. At this time, the light having a wavelength of λ1 is irradiated to the cells with low power. Therefore, the fluorescence of the wavelength band B1 transmitted through the filter member 21 has low intensity.

In the apparatus configuration of the fourth embodiment, as compared with the first embodiment, among the optical detection units 130 shown in FIG. 6, the light source 302, the condenser lens 312, the dichroic mirror 321 and the filter members 412 and 422 are collected. The optical lens 432 and the light receiving unit 502 are omitted. Further, as shown in FIG. 17, in the apparatus configuration of the third embodiment, the light source 305 and the condensing lens 315 are added to the light irradiation unit 300, and the condensing unit 400 collects light as compared with the first embodiment. A lens 471, a filter member 472, and a condensing lens 473 are added. Further, in the device configuration of the third embodiment, the light receiving unit 505 is added as compared with the first embodiment.

Note that FIG. 17 is a view of the optical detection unit 130 viewed in a direction parallel to the XY plane. In FIG. 17, for convenience, the light irradiation unit 300 is shown in a positive direction on the Y axis, and the light condensing unit 400 and the light receiving unit 505 are shown in a negative direction on the X axis.

The light of wavelength λ1 emitted from the light source 301 is applied to the position 211 in the flow path 210 of the flow cell 200. The light source 305 is configured in the same manner as the light source 301, and emits light with a lower power than the light source 301. The condensing lens 315 condenses the light emitted from the light source 305 at the position 212 located on the positive side of the Z axis of the position 211 in the flow path 210. The condenser lens 471 collects the fluorescence generated from the position 212. The filter member 472 transmits only the light in the wavelength band B1 out of the light collected by the condenser lens 471. The light receiving unit 505 receives low-intensity light in the wavelength band B1 focused by the condenser lens 473, and outputs image information based on the received light as an imaging signal.

The time T from position 211 to position 212 of the cell is obtained in advance. As a result, after the time T elapses after the light based on a certain cell is received by the light receiving unit 501, the light based on the same cell is received by the light receiving unit 505. Therefore, the image acquired based on the light receiving unit 501 and the image acquired based on the light receiving unit 505 can be associated with each other as if they were acquired from the same cell.

Also in the fourth embodiment, similarly to the first embodiment, high-intensity fluorescence and low-intensity fluorescence can be generated separately, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, as in the first embodiment, NF-κB having various distributions and amounts in cells can be accurately analyzed based on a high-intensity fluorescence image and a low-intensity fluorescence image.

<Embodiment 5>
In the fifth embodiment, only a part of the steps of the cell information acquisition method shown in FIG. 1 is different from that in the first embodiment as compared with the first embodiment. Hereinafter, a procedure different from that of the first embodiment will be described.

In step S2, as shown in FIG. 18, a sample containing cells labeled with fluorescent substances 11 to 13 is flowed into a flow cell, and the cells flowing through the flow cell are irradiated with light having wavelengths λ1 to λ3, and fluorescent substances 11 to 13 are irradiated. Fluorescence can be generated from. The fluorescence generated from the fluorescent substances 11 and 12 is incident on the filter member 24 together. The filter member 24 is composed of a prism. The fluorescence generated from the fluorescent substances 11 and 12 is divided into the fluorescence of the wavelength band B1 and the fluorescence of the wavelength band B2 by the filter member 24 due to the difference in the wavelength band. Here, as in the first embodiment, the light having a wavelength λ1 is irradiated to the cells with high power, and the light having a wavelength λ is irradiated to the cells with low power. As a result, as in the first embodiment, the fluorescence of the wavelength band B1 transmitted through the filter member 21 becomes high intensity, and the fluorescence of the wavelength band B2 transmitted through the filter member 22 becomes low intensity.

In addition, the fluorescence generated from the fluorescent substances 11 to 13 may be incidentally incident on the filter member 24, and may be divided into the fluorescence of the wavelength bands B1 to B3 by the filter member 24 due to the difference in the wavelength band. Although an example used for the configuration of the first embodiment is shown here, the filter member 24 may be used in the configurations of the second to fourth embodiments.

As shown in FIG. 19, in the apparatus configuration of the fifth embodiment, the filter members 411, 421, 421, and 422 are omitted from the optical detection unit 130 shown in FIG. 6, as compared with the first embodiment, and the light condensing unit is used. A filter member 481 is added to the 400. The filter member 481 is composed of a prism.

The fluorescence generated from the sample flowing through the flow cell 200 is incident on the filter member 481. The fluorescence incident on the filter member 481 is emitted from the filter member 481 at different angles depending on the wavelength of the fluorescence. The condenser lens 431 and the light receiving unit 501 are arranged in a direction corresponding to the fluorescence of the wavelength band B1 among the fluorescence emitted from the filter member 481. As a result, the light receiving unit 501 can image high-intensity fluorescence in the wavelength band B1. The condenser lens 432 and the light receiving unit 502 are arranged in a direction corresponding to the fluorescence in the wavelength band B2 among the fluorescence emitted from the filter member 481. As a result, the light receiving unit 502 can image low-intensity fluorescence in the wavelength band B2.

Also in the fifth embodiment, similarly to the first embodiment, high-intensity fluorescence and low-intensity fluorescence can be generated separately, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, as in the first embodiment, NF-κB having various distributions and amounts in cells can be accurately analyzed based on a high-intensity fluorescence image and a low-intensity fluorescence image.

<Embodiment 6>
In the sixth embodiment, the substrate is brought into contact with the test substance contained in the cell to generate a fluorescent substance, the generated fluorescent substance is irradiated with light, and the test substance is based on the fluorescence generated from the fluorescent substance by the irradiation of light. Determine the localization status of. In the sixth embodiment, the test substance is cytoplasm. The substrate contains a cleavage site that is cleaved upon contact with the test substance and produces a fluorescent substance when the cleavage site is cleaved. More specifically, the substrate is cleaved by an enzyme present in the cytoplasm upon contact with the cytoplasm, which is the test substance. In the sixth embodiment, the localization of the cytoplasm is determined based on the fluorescence from the fluorescent substance that labels the cytoplasm. The test substance may be, for example, a substance other than the cytoplasm contained in the cell, such as a protein in the cytoplasm, an organelle, or a cell membrane.

In the sixth embodiment, some of the steps in the cell information acquisition method shown in FIG. 1 are different from those in the third embodiment. Hereinafter, a procedure different from that of the third embodiment will be described.

In step S1, the cells and substrate 16a are mixed, as shown in FIG. Substrate 16a is a substance that produces fluorescent substance 16b when hydrolyzed by esterase contained in the cytoplasm. When the cell and the substrate 16a are mixed, the substrate 16a that has permeated the cell membrane is hydrolyzed by the esterase contained in the cytoplasm by contacting with the cytoplasm to produce a fluorescent substance 16b. Thus, the cytoplasm is labeled with the fluorescent material 16b.

Subsequently, as in the third embodiment, the processes after step S2 are performed. That is, in step S2, a sample containing cells labeled with the fluorescent substance 16b was flowed into the flow cell, and as shown in FIG. 20, the cells flowing through the flow cell were irradiated with light having wavelengths λ1 and λ3, respectively. , 13 gives rise to fluorescence. Then, as shown in FIG. 20, the fluorescence generated from the fluorescent substance 16b is divided into two, one is passed through the filter member 21, and the other is passed through the filter member 22. As a result, as in the third embodiment, the fluorescence of the wavelength band B1 transmitted through the filter member 21 becomes high intensity, and the fluorescence of the wavelength band B4 transmitted through the filter member 22 becomes low intensity. The device based on the sixth embodiment is configured in the same manner as the third embodiment.

Also in the sixth embodiment, as in the third embodiment, two fluorescences having different intensities are generated, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, the localization of the cytoplasm can be accurately discriminated based on the high-intensity fluorescence image and the low-intensity fluorescence image. If the localization of the cytoplasm can be determined accurately in this way, the range of the cytoplasm can be defined with high accuracy. This allows, for example, to combine the cytoplasmic range with other analyses for more detailed analysis. Further, for example, the range of cytoplasm can be used for research on cells and the like.

<Verification of Embodiment 6>
Next, the verification of the sixth embodiment performed by the inventor will be described.

1. 1. Human cardiac microvascular endothelial cells (HMVEC-C) (Lonza Cat No. CC-7030, Lot No. 0000296500 (P4)) were prepared as preparatory cells. Cell Explorer Fixable Live Cell Tracking Kit * Green Fluorescence * (Cosmo Bio 22621) was prepared as a cytoplasmic labeling reagent. The cytoplasmic labeling reagent contains a substance corresponding to the substrate 16a shown in FIG. The cytoplasmic labeling reagent has hydrophobicity. The cytoplasmic labeling reagent passes through the cell membrane and is hydrolyzed by intracellular esterase to produce a fluorescent substance. The fluorescent substance generated here corresponds to the fluorescent substance 16b shown in FIG. Cellstain Hoechst 33342 solution (DOjinDO H342) was prepared as a nuclear stain. In addition, EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (Lonza Cat No. CC-3202), PBS pH7.4 (GIBCO Cat No.10010-023), BSA (LAMPIRE Cat) No. 7500805), PFA (WAKO Cat No.160-16061), TritonX100 (Nakarai Tesk Cat No.35501-15) were prepared.

2. 2. Reagent Preparation EGM-2MV Single Quots Kit was added to 500 mL of EGM-2MV Medium to prepare a culture medium. Paraformaldehyde was dissolved in PBS at pH 12 to a final concentration of 8% w / v, and then adjusted to pH 7.4. 1.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 3% BSA / PBS. 0.5 g of BSA was added to PBS to dissolve it, and the mixture was scalpel-up to 50 mL to prepare 1% BSA / PBS. Triton X100 was prepared with PBS to a final concentration of 0.1% w / v. The Track kit working solution was adjusted by adding 100 μL of DMSO to the Vial of Track kit Green to prepare 1000 × Track kit Green stock solution, and adding 1/1000 amount to the Assay buffer attached to the Kit.

3. 3. procedure
HMVEC-C was cultured in EGM-2MV medium according to the manufacturer's recommended protocol. Cells within 6 passages after purchase were used for this verification. The culture medium had an expiration date of 3 weeks after opening. After the cells were cultured to 70% confluent, the medium was removed with an electric pipettor leaving about 3 mL of the medium, and the cells were detached with a scraper. 3 μL of Track kit working solution was added to the recovered 3 mL suspension, and the mixture was allowed to stand in a 37 ° C. CO ₂ incubator for 30 minutes. After allowing to stand for 30 minutes, the mixture was centrifuged at 1000 rpm for 3 minutes at room temperature. The cell pellet was washed 3 times with 5 mL PBS. The supernatant was removed and 50 μL of 1% BSA / PBS was added.

4. Detection by flow cytometer ImageStreamX Mark II Imaging Flow Cytometer (Merck Millipore) was used as a flow cytometer capable of acquiring fluorescence images. A sample prepared according to 3 above was flowed through the flow cell of this flow cytometer, and the sample flowing through the flow cell was irradiated with laser light having a wavelength of 488 nm and 405 nm. The laser beams having wavelengths of 488 nm and 405 nm correspond to the laser beams having wavelengths λ1 and λ3 shown in FIG. The emission powers of the laser beams having wavelengths of 488 nm and 405 nm were set to 50 mW and 20 mW, respectively. Fluorescence was generated by irradiating the fluorescent dye that labels the cytoplasm with a laser beam having a wavelength of 488 nm. Fluorescence was generated by irradiating the nuclear dye with a laser beam having a wavelength of 405 nm.

In the flow cytometer, the fluorescence generated by the laser beam having a wavelength of 488 nm was imaged through a filter member having a transmission wavelength band of 480 nm to 560 nm, and a high-intensity fluorescence image was acquired. Further, the fluorescence generated by the laser beam having a wavelength of 488 nm was imaged through a filter member having a transmission wavelength band of 560 nm to 595 nm, and a low-intensity fluorescence image was acquired. The fluorescence generated by the laser beam having a wavelength of 405 nm was imaged through a filter member having a transmission wavelength band of 420 nm to 505 nm, and a fluorescence image corresponding to the nucleus was acquired. Further, the sample flowing through the flow cell was irradiated with a laser beam having a wavelength set between 420 nm and 480 nm. The light transmitted through the cells by the laser light was imaged through a filter member having a transmission wavelength band of 420 nm to 480 nm, and a bright field image was acquired. In the flow cytometer, unnecessary wavelength band light is removed by a filter member or the like so that light in the target wavelength band is properly incident on the light receiving portion.

The image acquired by the above detection will be described with reference to FIG.

"Bright field" refers to a bright field image of a cell. "High intensity fluorescence" and "low intensity fluorescence" are based on an image based on high intensity fluorescence generated from a fluorescent dye labeled with a cytoplasm and a low intensity fluorescence generated from a fluorescent dye labeled with a cytoplasm, respectively. It is an image. "Fluorescence from the nucleus" is an image based on the fluorescence generated from the dye for nuclear staining that stained the nucleus. The four images arranged side by side are images obtained from one cell. For convenience, the images other than the bright-field image are grayscale images of the acquired color image. In images other than the bright field image, the white part indicates that the fluorescence intensity is strong.

In the case of the cells shown at the top and the cells one below the top, the cytoplasmic localization is unclear because the low-intensity fluorescence-based images are too low-intensity. On the other hand, since the intensity of the image based on high-intensity fluorescence is appropriate, the localization of the cytoplasm can be accurately discriminated. In the case of the cells shown at the bottom and the cells shown one above the bottom, it is difficult to determine the localization of the cytoplasm because the image based on high intensity fluorescence is too strong. On the other hand, since the intensity of the image based on low-intensity fluorescence is appropriate, the localization of the cytoplasm can be accurately determined.

As described above, according to this verification, it can be seen that the localization of the cytoplasm can be discriminated based on two fluorescence images having different intensities as in the sixth embodiment. As described above, according to the sixth embodiment, the fluorescence generated from one kind of fluorescent dye that labels the cytoplasm is passed through the filter members 21 and 22 that pass the fluorescence in different wavelength bands to acquire two fluorescence images. It can be seen that the localization of the cytoplasm can be correctly obtained by one measurement for one cell.

<Embodiment 7>
In the seventh embodiment, two kinds of substrates are brought into contact with a test substance contained in a cell to generate two kinds of fluorescent substances, and the two kinds of fluorescent substances generated are irradiated with light, and two kinds of fluorescent substances are irradiated with light. The localization status of the test substance is determined based on the fluorescence generated from the fluorescent substance. That is, in the seventh embodiment, instead of using one light as in the sixth embodiment, different wavelengths of light are used to obtain fluorescence of different intensities. In addition, one kind of substrate may be brought into contact with a test substance to generate two kinds of fluorescent substances.

In the seventh embodiment, only a part of the steps of the cell information acquisition method shown in FIG. 1 in steps S1 and S2 is different from that in the sixth embodiment. Hereinafter, a procedure different from that of the sixth embodiment will be described.

In step S1, as shown in FIG. 22, the cells and the substrates 17a and 18a are mixed. Substrate 17a and 18a are substances that produce fluorescent substances 17b and 18b, respectively, when hydrolyzed by esterase contained in the cytoplasm. The fluorescent substances 17b and 18b are configured to excite fluorescence in wavelength bands different from each other when irradiated with light having wavelengths λ1 and λ2, respectively. When the cells and the substrates 17a and 18a are mixed, the substrates 17a and 18a that have permeated the cell membrane are hydrolyzed by the esterase contained in the cytoplasm by contact with the cytoplasm to produce fluorescent substances 17b and 18b. Thus, the cytoplasm is labeled with fluorescent substances 17b, 18b.

In step S2, a sample containing cells labeled with fluorescent substances 17b and 18b is flowed into the flow cell, and as shown in FIG. 22, the cells flowing through the flow cell are irradiated with light having wavelengths λ1, λ2, and λ3, and fluorescent. Fluorescence is generated from the substances 17b, 18b, 13. At this time, the laser beam having a wavelength of λ1 irradiates the cells with high power, and the laser beam having a wavelength λ2 irradiates the cells with low power. The fluorescence generated from the fluorescent substance 17b becomes fluorescence in the wavelength band B1 by being passed through the filter member 21. The fluorescence generated from the fluorescent substance 18b becomes fluorescence in the wavelength band B2 by being passed through the filter member 22. In this way, the fluorescence of the wavelength band B1 that has passed through the filter member 21 becomes high intensity, and the fluorescence of the wavelength band B2 that has passed through the filter member 22 becomes low intensity. The device based on the seventh embodiment is configured in the same manner as the first embodiment.

Also in the seventh embodiment, as in the sixth embodiment, two fluorescences having different intensities are generated, and a high-intensity fluorescence image and a low-intensity fluorescence image can be obtained. Therefore, as in the sixth embodiment, the localization of the cytoplasm can be accurately discriminated based on the high-intensity fluorescence image and the low-intensity fluorescence image.

11,12 Fluorescent substance 16a, 17a, 18a Substrate 16b, 17b, 18b Fluorescent substance 21, 22, 24 Filter member 100 Cell information acquisition device 111 Acquisition unit 112 Analysis unit 200 Flow cell 300 Light irradiation unit 301, 302 Light source 411, 412, 414, 421, 422, 425, 451, 452, 481 Filter members 501, 502, 505 Light receiving part

Claims (28)

Multiple fluorescent substances having different fluorescence wavelengths are bound to the test substance contained in the cell,
The cells are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescence having different wavelengths and intensities from the plurality of fluorescent substances.
A plurality of fluorescence information is acquired based on each of the generated fluorescence, and the fluorescence information is acquired .
A cell information acquisition method for determining whether the test substance is localized in the nucleus or the cytoplasm based on the plurality of fluorescence information . The substrate is brought into contact with the test substance contained in the cell to generate a plurality of fluorescent substances having different fluorescence wavelengths from each other.
The cells are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescence having different wavelengths and intensities from the plurality of fluorescent substances.
A plurality of fluorescence information is acquired based on each of the generated fluorescence, and the fluorescence information is acquired .
A cell information acquisition method for determining whether the test substance is localized in the nucleus or the cytoplasm based on the plurality of fluorescence information . The cell information acquisition method according to claim 1 or 2, wherein the distribution status of the test substance in the cells is determined based on the plurality of fluorescence information. The cell information acquisition method according to any one of claims 1 to 3 , wherein the plurality of fluorescent substances have different wavelengths of light for excitation. The cell information acquisition method according to any one of claims 1 to 3 , wherein the plurality of fluorescent substances have different wavelengths and intensities of fluorescence generated by irradiation with light of the same wavelength. The invention according to any one of claims 1 to 5 , wherein each fluorescence generated from the plurality of fluorescent substances is separated into a first fluorescence and a second fluorescence having a weaker intensity than the first fluorescence. Cell information acquisition method. Fluorescent substance is bound to the test substance contained in the cell,
The cells are irradiated with light to generate fluorescence having a predetermined wavelength width from one type of the fluorescent substance.
The generated fluorescence of the predetermined wavelength width is divided by the wavelength band to obtain a plurality of fluorescence having different wavelengths and intensities.
A plurality of fluorescence information is acquired based on each of the acquired fluorescence, and
A cell information acquisition method for determining the distribution status of the test substance in the cells based on the plurality of fluorescence information. The substrate is brought into contact with the test substance contained in the cell to generate a fluorescent substance.
The cells are irradiated with light to generate fluorescence having a predetermined wavelength width from one type of the fluorescent substance.
The generated fluorescence of the predetermined wavelength width is divided by the wavelength band to obtain a plurality of fluorescence having different wavelengths and intensities.
A plurality of fluorescence information is acquired based on each of the acquired fluorescence, and
A cell information acquisition method for determining the distribution status of the test substance in the cells based on the plurality of fluorescence information. The cell information acquisition method according to claim 7 or 8 , wherein the localization status of the test substance in the cells is determined based on the plurality of fluorescence information. The cell information according to any one of claims 7 to 9 , wherein the fluorescence generated from the fluorescent substance is separated into a first fluorescence and a second fluorescence having a weaker intensity than the first fluorescence. Acquisition method. A first fluorescence image of a first fluorescence wavelength generated from the cell irradiated with the first light was acquired.
The invention according to any one of claims 1 to 10, wherein a second fluorescence image having a second fluorescence wavelength generated from the cell irradiated with a second light having a weaker intensity than the first light is acquired. Cell information acquisition method. A sample containing the cells was poured into a flow cell and
The cell information acquisition method according to any one of claims 1 to 11 , wherein the cells flowing through the flow cell are irradiated with light to generate the fluorescence. Determination of the localization conditions of the test substance, the localization of the analyte in the analyzed region of the cell, including calculating the percentage of relative amount of the analyte in the whole cells, claim 9 The cell information acquisition method described in. The determination of the localization state of the test substance is based on the fluorescence information obtained from the fluorescence whose intensity is within a predetermined range among the fluorescence information obtained from the plurality of fluorescences having different intensities. The cell information acquisition method according to claim 9 or 13 . The determination of the localization state of the test substance is performed by determining the fluorescence intensity at the analysis target site of the cell and the cell portion other than the analysis target site among the fluorescence information obtained from the plurality of fluorescences having different intensities. The cell information acquisition method according to any one of claims 9 , 13 , or 14 , which is performed based on the fluorescence information whose difference from the fluorescence intensity in the above is larger than a predetermined threshold value. The cell information acquisition method according to any one of claims 1 to 15 , wherein the test substance is a protein, mRNA, microRNA, cytoplasm, organelle, or cell membrane. The invention according to any one of claims 1 to 16 , wherein the ratio or number of cells in which the test substance is localized in a specific site among the cells contained in the sample is calculated based on the plurality of fluorescence information. Cell information acquisition method. A cell containing a test substance to which a plurality of fluorescent substances having different fluorescence wavelengths are bound to each other is irradiated with a first light and a second light having a weaker intensity than the first light, and wavelengths from the plurality of fluorescent substances are obtained. And a light-irradiating section that produces multiple fluorescences of different intensities,
A light receiving unit that receives each of the fluorescence generated from the plurality of fluorescent substances,
An acquisition unit that acquires a plurality of fluorescence information based on the fluorescence having different intensities, and
A cell information acquisition device including an analysis unit for determining whether the test substance is localized in the nucleus or the cytoplasm based on the plurality of fluorescence information . The cell information acquisition device according to claim 18 , further comprising a filter member for separating each fluorescence generated from the plurality of fluorescent substances. 18. The claim 18 includes a first light receiving unit that receives the first fluorescence and a second light receiving unit that receives the second fluorescence that receives the second fluorescence that is weaker than the first fluorescence. Alternatively, the cell information acquisition device according to any one of 19 . The acquisition unit includes a first fluorescence image having a first fluorescence wavelength generated from the cell irradiated with the first light, and the cell irradiated with a second light having a weaker intensity than the first light. The cell information acquisition device according to any one of claims 18 to 20 , which acquires a second fluorescence image having a second fluorescence wavelength generated from the above. A light irradiation unit that irradiates cells containing a test substance to which a fluorescent substance is bound with light to generate fluorescence having a predetermined wavelength width from one type of the fluorescent substance.
A light receiving unit that receives a plurality of fluorescence having different wavelengths and intensities by dividing the fluorescence having the predetermined wavelength width generated from the one type of fluorescent substance by a wavelength band.
An acquisition unit that acquires a plurality of fluorescence information based on the plurality of received fluorescence.
A cell information acquisition device including an analysis unit that determines the distribution status of the test substance in the cells based on the plurality of fluorescence information. A light irradiation unit that irradiates cells containing a test substance that has generated a fluorescent substance by contact with a substrate with light to generate fluorescence having a predetermined wavelength width from one type of the fluorescent substance.
A light receiving unit that receives a plurality of fluorescence having different wavelengths and intensities by dividing the fluorescence having the predetermined wavelength width generated from the one type of fluorescent substance by a wavelength band.
An acquisition unit that acquires a plurality of fluorescence information based on the plurality of received fluorescence.
A cell information acquisition device including an analysis unit that determines the distribution status of the test substance in the cells based on the plurality of fluorescence information. The cell information acquisition device according to claim 22 or 23 , comprising a filter member for separating the fluorescence generated from the fluorescent substance into a first fluorescence and a second fluorescence having a weak intensity. A plurality of fluorescent substances having different wavelengths and intensities of fluorescence generated by irradiation with light of the same wavelength are bound to a test substance contained in a cell.
The cells are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescence having different wavelengths and intensities from the plurality of fluorescent substances.
A cell information acquisition method for acquiring a plurality of fluorescence information based on each of the generated fluorescence. Multiple fluorescent substances having different fluorescence wavelengths are bound to the test substance contained in the cell,
A sample containing the cells was poured into a flow cell and
The cells flowing through the flow cell are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescences having different wavelengths and intensities from the plurality of fluorescent substances.
A cell information acquisition method for acquiring a plurality of fluorescence information based on each of the generated fluorescence. Multiple fluorescent substances having different fluorescence wavelengths are bound to the test substance contained in the cell,
The cells are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescence having different wavelengths and intensities from the plurality of fluorescent substances.
A plurality of fluorescence information is acquired based on each of the generated fluorescence, and the fluorescence information is acquired.
Of the fluorescence information obtained from the plurality of fluorescences having different intensities, the localization status of the test substance in the cells based on the fluorescence information obtained from the fluorescences having the intensity within a predetermined range. Cell information acquisition method to determine. Multiple fluorescent substances having different fluorescence wavelengths are bound to the test substance contained in the cell,
The cells are irradiated with a first light and a second light having a weaker intensity than the first light to generate a plurality of fluorescence having different wavelengths and intensities from the plurality of fluorescent substances.
A plurality of fluorescence information is acquired based on each of the generated fluorescence, and the fluorescence information is acquired.
Of the fluorescence information obtained from the plurality of fluorescences having different intensities, the localization status of the test substance in the cells based on the fluorescence information obtained from the fluorescences having the intensity within a predetermined range. To determine
The determination of the localization status of the test substance includes the calculation of the ratio of the localized amount of the test substance in the analysis target site of the cell to the amount of the test substance in the whole cell, and obtains cell information. Method.