JP2020060573A - Cell analysis device and program - Google Patents

Abstract

To provide a cell analysis device and program that make it possible to highly accurately perform diagnoses with no need for a cumbersome work.SOLUTION: A cell analysis device 10 comprises: a first light source 11c and second light source 11a that irradiate an examined cell containing first substances bonded to first fluorescent pigments with light rays mutually different in wavelength; an imaging unit 19 that captures the examined cell; and a processing unit 111. The processing unit 111 is configured to: for activating the first fluorescent pigment of the examined cell, cause the first light source 11c to irradiate the examined cell; for inactivating the first fluorescent pigment of the examined cell, cause the second light source 11a to irradiate the examined cell; for inactivating the first fluorescent pigment, cause the imaging unit 19 to capture a plurality of images while irradiating the examined cell by the second light source 11a; and count first substances contained in the examined cell on the basis of the plurality of images captured by the imaging unit 19.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell analyzer and a program executed by a computer of the cell analyzer.

  In a given disease, a specific gene, a specific protein, etc. are involved in the progress of the disease state. Confirming the presence and status of a specific substance in cells collected from a subject is extremely useful in diagnosing these diseases and determining a therapeutic course.

  For example, in the case of breast cancer, the prognostic factor HER2 gene is amplified as the disease progresses. In Patent Document 1, amplification of the HER2 gene is analyzed using the FISH method. Specifically, a nucleic acid probe that binds to the DNA of the HER2 gene (HER2 probe) and a nucleic acid probe that binds to the centromere region of chromosome 17 (CEP17) (CEP17 probe) are labeled with different fluorescent dyes, respectively, and one cell Fluorescence generated from each probe in is counted. The amplification of the HER2 gene is positive when the ratio of the fluorescence number of the HER2 gene to the fluorescence number of CEP17 is a predetermined value or more, and the amplification of the HER2 gene is performed when this ratio is less than the predetermined value. Is judged to be negative.

JP2012-103077A

  In the above-mentioned diagnostic method, for example, an image showing the distribution state of fluorescence is provided to a doctor or the like upon diagnosis. Doctors and the like have been required to perform complicated work such as determining a medical condition while referring to the provided image. Further, in the above-mentioned diagnostic method, since the provided image is visually checked to determine the medical condition, a doctor or the like is required to have a skilled technique in making the determination, and the diagnosis may vary from person to person. It was

  A cell analysis device according to a first aspect of the present invention includes a first light source and a second light source for irradiating test cells containing a first substance bound to a first fluorescent dye with lights having wavelengths different from each other; An imaging unit that images cells and a processing unit are provided. The processing unit irradiates the test cell with the first light source in order to activate the first fluorescent dye of the test cell, and inactivates the first fluorescent dye of the test cell in the second light source. While irradiating the cells and irradiating the test cells with the second light source to inactivate the first fluorescent dye, the imaging unit captures a plurality of images, and the plurality of images captured by the imaging units. Based on the above, the first substance contained in the test cells is counted.

  The program according to the second aspect of the present invention includes first and second light sources for irradiating test cells containing a first substance bound to a first fluorescent dye with light having different wavelengths, and imaging the test cells. And a step of irradiating the test cell with the first light source to activate the first fluorescent dye of the test cell in the computer of the cell analysis device including the image capturing section. The step of irradiating the test cells with the second light source for activation, the step of irradiating the test cells with the second light source for inactivating the first fluorescent dye, and The step of capturing an image and the step of counting the first substance contained in the test cell based on the plurality of images captured by the image capturing unit are executed.

  According to the present invention, it is possible to perform diagnosis with high accuracy without requiring complicated work.

FIG. 1 is a diagram showing the configuration of the cell analyzer according to the first embodiment. FIG. 2A is a diagram showing a state in which the first fluorescent dye according to the first embodiment is bound to the first substance, and FIG. 2B is a diagram showing that the second fluorescent dye according to the first embodiment is It is a figure which shows the state couple | bonded with two substances. FIG. 3A is a diagram showing that all the first fluorescent dyes according to the first embodiment are in an active state, and FIG. 3B is a diagram showing that all the first fluorescent dyes according to the first embodiment are quenched. It is a figure which shows that it is in a state, and FIG.3 (c), (d) is a figure which shows a part of 1st fluorescent dye which concerns on Embodiment 1 in an active state. 4A and 4B are flowcharts showing the image acquisition processing and the analysis processing in the first processing step according to the first embodiment, respectively. FIG. 5A is a diagram illustrating a procedure for acquiring the reactivated diffraction-limited image and the number of substances according to the first embodiment, and FIG. 5B is a diagram illustrating a diffraction-limited image according to the first embodiment. It is a figure explaining the procedure to do. 6A and 6B are flowcharts showing the image acquisition processing and the analysis processing in the second processing step according to the first embodiment. FIG. 7 is a diagram illustrating a procedure for acquiring the super-resolution image and the number of substances according to the first embodiment. FIG. 8A to FIG. 8D are diagrams showing positive reference images acquired in the first processing step according to the first embodiment. 9A to 9D are diagrams showing a positive reference image acquired in the second processing step according to the first embodiment, and FIGS. 9E to 9G are related to the first embodiment. It is a figure which shows the negative reference image acquired in the 2nd process process. 10A to 10D are diagrams showing positive reference images acquired in Comparative Example 1, and FIGS. 10E to 10G are positive reference images acquired in Comparative Example 2. FIG. FIG. 11 is a flowchart showing a display process according to the first embodiment. 12A and 12B are diagrams showing a configuration of a screen displayed on the display unit according to the first embodiment. FIG. 13 is a flowchart showing the image acquisition process in the first processing step according to the second embodiment. 14A and 14B are flowcharts showing the image acquisition processing and the analysis processing in the second processing step according to the second embodiment, respectively. 15A and 15B are flowcharts showing image acquisition processing in the first processing step and the second processing step according to the modification of the second embodiment, respectively. FIG. 16A is a diagram showing the configuration of the cell analyzer according to the third embodiment, and FIG. 16B is a diagram showing the two focal points according to the third embodiment at the position of the emission point of fluorescence in the optical axis direction. It is a figure which shows rotating according to a light-receiving surface according to it. 17A and 17B are diagrams showing a three-dimensional super-resolution image according to the third embodiment, and FIGS. 17C and 17D are three-dimensional super-resolution images according to the third embodiment. It is a figure which shows the image at the time of referring to Z axis direction and Y axis direction. FIG. 18 is a diagram showing a configuration of a screen displayed on the display unit according to the third embodiment.

  The embodiment described below is one in which the present invention is applied to a cell analyzer that acquires information about breast cancer.

  The “treatment index information” displayed by the cell analysis device is information that serves as an index when a doctor or the like decides a treatment policy such as medication, surgery, or follow-up observation. In the following embodiments, as an example of the therapeutic index information, the number of HER2 genes, which is one of the prognostic factors of breast cancer, is counted as the number of first substances, and the determination result of the ratio between the count value and the number of CEP17 is: It is acquired and displayed as treatment index information. Based on this treatment index information, a doctor or the like can confirm the degree of amplification of the HER2 gene. Thus, for example, it is possible to determine whether or not to use Herceptin (registered trademark), which is a molecular targeting drug specifically targeting the HER2 gene, or trastuzumab (generic name) for treating breast cancer of the patient. It should be noted that what the cell analyzer displays is not limited to the above-mentioned treatment index information. The count value of the disease marker which is the first substance, the ratio of the first substance and the second substance, etc. are output as "therapeutic index information" which is an index for judging the progress of the disease related to the disease marker. Good.

1. Embodiment 1
As shown in FIG. 1, the cell analysis device 10 includes a light source unit 11, a shutter 12, a quarter-wave plate 13, a beam expander 14, a condenser lens 15, a dichroic mirror 16, and an objective lens 17. The stage 18, the imaging unit 19, the controllers 20 and 21, and the information processing apparatus 100. The image pickup unit 19 includes a condenser lens 19a and an image pickup element 19b. The image sensor 19b is, for example, a CCD, EMCCD, CMOS, or a scientific CMOS image sensor.

  A slide glass 22 on which a sample is placed is installed on the stage 18. The sample contains a test cell, and the nucleus of the test cell contains a first substance and a second substance. The first substance and the second substance to be detected are biological substances such as genes, proteins or peptides that serve as disease markers. Specifically, in the first embodiment, the test cell is a cell collected from the diseased tissue. The first substance is the HER2 gene, and the second substance is the centromere region of chromosome 17 (CEP17). The HER2 gene is a disease marker for breast cancer. CEP17 is present in the same number as the HER2 gene in normal cells, and does not proliferate even if a patient suffers from breast cancer or the like. Therefore, CEP17 is used as an internal control serving as a standard when measuring the amplification of the HER2 gene.

  In the sample, the first fluorescent dye is bound to the first substance and the second fluorescent dye is bound to the second substance. The first fluorescent dye can be switched between an active state in which fluorescence is generated by irradiation with light from the light source 11a and an inactive state in which fluorescence is not generated even when irradiated with light from the light source 11a. The first fluorescent dye is inactivated when the light from the light source 11a is irradiated, and is activated when the light from the light source 11c is irradiated. Below, inactivating is called "quenching". The nucleus of the test cell is stained with the third fluorescent dye.

  In the first embodiment, the first fluorescent dye is activated from the extinguished state by the light from the light source 11c. The light from the light source 11c is also used when exciting the third fluorescent dye with fluorescence. In this way, since the light from the light source 11c can be shared by the activation of the first fluorescent dye and the excitation of the third fluorescent dye, the configuration of the cell analyzer 10 can be simplified. Instead of the action of light, the activation of the first fluorescent dye may be performed by the action of heat or a chemical agent.

  The light source unit 11 irradiates the test cells with light. The light source unit 11 includes light sources 11a, 11b, 11c and dichroic mirrors 11d, 11e. The light sources 11a, 11b, 11c respectively emit light of different wavelengths. Although it is preferable to use a laser light source as the light source unit 11, a mercury lamp, a xenon lamp, an LED, or the like may be used. The dichroic mirror 11d transmits the light emitted from the light source 11b and reflects the light emitted from the light source 11a. The dichroic mirror 11e transmits the light emitted from the light sources 11a and 11b and reflects the light emitted from the light source 11c. The optical axes of the lights emitted from the light sources 11a, 11b, and 11c are matched with each other by the dichroic mirrors 11d and 11e. Usually, it is preferable that the light sources in the light source unit 11 are arranged such that the wavelength of the light emitted from the light source 11b is the longest and the wavelength of the light emitted from the light source 11c is the shortest.

  The light source unit 11 irradiates the test cell with light to cause fluorescence from the test cell. Specifically, the light emitted from each of the light sources 11a, 11b, and 11c excites the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye contained in the test cell to generate fluorescence. Let

  The shutter 12 is driven by the controller 20 and switches between a state in which the light emitted from the light source unit 11 passes and a state in which the light emitted from the light source unit 11 is blocked. Thereby, the irradiation time of the light to the test cell is adjusted. The quarter-wave plate 13 converts the linearly polarized light emitted from the light source unit 11 into circularly polarized light. Fluorescent dyes respond to light of a given polarization direction. Therefore, by converting the excitation light into circularly polarized light, the polarization direction of the excitation light easily matches the polarization direction in which the fluorescent dye reacts. As a result, fluorescence can be efficiently excited by the fluorescent dye contained in the test cell. The beam expander 14 expands the light irradiation area on the slide glass 22. The condensing lens 15 condenses the light so that the slide glass 22 is irradiated with parallel light from the objective lens 17. The shutter 12 and the quarter-wave plate 13 may be arranged immediately after the light sources 11a, 11b, and 11c.

  The dichroic mirror 16 reflects the light emitted from the light source unit 11 and transmits the fluorescence generated from the test cell. The objective lens 17 guides the light reflected by the dichroic mirror 16 to the slide glass 22. The stage 18 is driven by the controller 21 and moves in a horizontal plane. As a result, the slide glass 22 is widely irradiated with light. The fluorescence generated from the test cell passes through the objective lens 17 and the dichroic mirror 16. The condenser lens 19a collects the fluorescence and guides it to the light receiving surface of the image sensor 19b. The image sensor 19b images fluorescence and outputs the imaged image.

  The information processing device 100 is a personal computer, and includes a main body 110, a display unit 120, and an input unit 130. The main body 110 includes a processing unit 111, a storage unit 112, and an interface 113.

  The processing unit 111 is, for example, a CPU. The storage unit 112 is a ROM, a RAM, a hard disk, or the like. The processing unit 111 executes various functions based on the programs stored in the storage unit 112. The processing unit 111 processes the image obtained by the image sensor 19b and performs various other processes. Further, the processing unit 111 controls the light sources 11 a, 11 b and 11 c of the light source unit 11, the image pickup device 19 b, and the controllers 20 and 21 via the interface 113. The display unit 120 is a display for displaying the processing result and the like by the processing unit 111. The input unit 130 is a keyboard and a mouse for receiving an instruction input by the user.

  Hereinafter, the first processing step and the second processing step executed by the processing unit 111 will be described. Hereinafter, the captured images of fluorescence excited by the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye are referred to as “first image”, “second image”, and “third image”, respectively. The wavelength, intensity, and irradiation time of each light described in the following description of the first treatment step and the second treatment step are the same as those described in “(3) Experiment described below” for the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye, respectively. Is applied when it is the dye used in ". The wavelength, intensity, and irradiation time of each light are appropriately changed as the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye are changed, and the labeling method and labeling density of the dye are changed. It

(1) First Treatment Step First, the binding form of the fluorescent dye will be described with reference to FIGS. 2 (a) and 2 (b). In the first treatment step, the first fluorescent dye is bound to the first substance when the sample is prepared. As shown in FIG. 2A, the first fluorescent dye is bound to the first substance via an intermediate substance that specifically binds to the first substance. When the sample is prepared, the second fluorescent dye is bound to the second substance. As shown in FIG. 2B, the second fluorescent dye is also bound to the second substance via the intermediate substance that specifically binds to the second substance. At the time of sample preparation, the nucleus of the test cell is specifically stained with the third fluorescent dye. Here, when the first substance or the second substance is a gene, a nucleic acid probe can be used as an intermediate substance. When the first substance or the second substance is a protein, an antibody specific to the protein can be used as the intermediate substance. The target and the number of the substance that binds the fluorescent dye may be changed according to the purpose of analysis.

  As schematically shown in FIG. 3A, many first fluorescent dyes are bound to one first substance. In FIG. 3A, the two first substances to which the first fluorescent dyes are respectively bound are schematically shown. Similarly, many second fluorescent dyes also bind to one second substance. As described above, the first fluorescent dye and the second fluorescent dye are fluorescent dyes that can be switched between the quenching state and the active state by the laser light having the predetermined wavelength.

  In the initial state, as shown schematically in FIG. 3A, all the first fluorescent dyes are in the active state. In FIG. 3A, the active state is indicated by a black circle. In this state, when the test cell is irradiated with the light from the light source 11a for a predetermined time, all the first fluorescent dyes are extinguished as shown in FIG. 3 (b). In FIG. 3B, the extinction state is indicated by a white circle.

  Then, when the test cell is irradiated with the light from the light source 11c for a predetermined time, a part of the first fluorescent dye is activated as shown in FIG. 3C, for example. By adjusting the irradiation time of the light from the light source 11c, the ratio of the activated first fluorescent dye changes. When the test cell is irradiated with the light from the light source 11a again for a predetermined time, all the first fluorescent dyes are extinguished as shown in FIG. 3 (b). After that, when the light from the light source 11c is again applied to the test cells for a predetermined time, some of the first fluorescent dyes are activated as shown in FIG. 3D, for example. As shown in FIGS. 3C and 3D, the distribution of the first fluorescent dye that is activated by each activation process is different each time.

  In the first processing step, the first fluorescent dye is once quenched, then reactivated, and then irradiated with excitation light to image fluorescence. Therefore, the first image is acquired in a state in which the first fluorescent dye emits sparse fluorescence, as shown in FIG. 3C, for example. The second fluorescent dye can be switched between a quenching state and an active state, but in the first processing step, an image is captured in the initial state without quenching. Therefore, the second image is acquired in a state in which all the second fluorescent dyes emit fluorescence, as shown in FIG.

  In the first processing step, the wavelengths of the light emitted from the light sources 11a, 11b, and 11c are 640 nm, 730 nm, and 405 nm, respectively.

  As shown in FIG. 4A, in step S101, the processing unit 111 irradiates the test cell with light from the light source 11b at 20 mW for 1.5 seconds to cause fluorescence from the second fluorescent dye, The generated fluorescence is imaged by the imaging unit 19. The processing unit 111 repeats imaging while the test cells are being irradiated with light, and acquires 100 second images. Although 100 second images are acquired in step S101, the number of images to be acquired is not limited to this, and for example, one image may be acquired.

  In step S102, the processing unit 111 irradiates the test cell with light from the light source 11c at 1 mW for 1.5 seconds to generate fluorescence from the third fluorescent dye, and the imaging unit 19 images the generated fluorescence. . The processing unit 111 repeats imaging while the test cells are being irradiated with light to acquire 100 third images. Although 100 third images are acquired in step S102, the number of images to be acquired is not limited to this, and for example, one image may be acquired.

  In step S103, the processing unit 111 extinguishes the first fluorescent dye by irradiating the test cell with light from the light source 11a at 80 mW. In step S104, the processing unit 111 activates the first fluorescent dye by irradiating the test cell with light from the light source 11c at 15 mW for 0.15 seconds. In step S105, the processing unit 111 irradiates the test cell with light from the light source 11a at 80 mW for 2 seconds to cause fluorescence to be generated from the first fluorescent dye, and the generated fluorescence is imaged by the imaging unit 19. The processing unit 111 repeats imaging while the test cells are being irradiated with light to acquire 100 first images. Since the same light as used in step S103 is used in step S105, the first fluorescent dye is extinguished while the light is irradiated in step S105. In this way, the image acquisition processing of the first processing step ends. Although 100 first images are acquired in step S105, the number of images to be acquired is not limited to this, and for example, one image may be acquired.

  After that, in step S111 shown in FIG. 4B, the processing unit 111 determines the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye based on the first image, the second image, and the third image, respectively. Create a diffraction limited image. Hereinafter, the diffraction-limited image of the fluorescent dye obtained through quenching and reactivation is particularly referred to as “reactivation diffraction-limited image”.

  As shown in FIG. 5A, the reactivation diffraction-limited image is obtained by averaging images obtained by performing quenching and activation only once as shown in steps S103 to S105 of FIG. 4A. It is created by converting. Therefore, the reactivated diffraction-limited image of the first fluorescent dye is created by averaging the 100 first images acquired in step S105.

  As shown in FIG. 5B, the diffraction limited image is created by averaging the images acquired without extinction and activation as shown in steps S101 and S102 of FIG. 4A. It Therefore, the diffraction limited image of the second fluorescent dye is created by averaging the plurality of second images acquired in step S101. The diffraction limited image of the third fluorescent dye is created by averaging the plurality of third images acquired in step S102.

  Returning to FIG. 4B, in step S112, the processing section 111 creates a reference image by superimposing the three images acquired in step S111. The reference image is displayed on a screen that displays a determination result described later. In step S113, the processing unit 111 identifies the nucleus region of the test cell based on the diffraction-limited image of the third fluorescent dye obtained in step S111.

  In step S114, the processing unit 111 acquires the total number of first substances. Specifically, as shown in FIG. 5A, the processing unit 111, in the reactivation diffraction limit image of the first fluorescent dye acquired in step S111, the region of the nucleus of the test cell acquired in step S113. The total area of the fluorescent region inside is calculated. Subsequently, the processing unit 111 divides the calculated total area of the fluorescent region by the area of the fluorescent region corresponding to one first substance stored in the storage unit 112 in advance, and the division result is the total number of the first substances. And The method for obtaining the total number of the first substances is not limited to the method of dividing the total area of the fluorescent region by the unit area. For example, the method of FIG. 7 used in the second processing step described below may be applied to 100 first images to obtain the total number of first substances.

  Returning to FIG. 4B, in step S115, the processing unit 111 acquires the total number of the second substances as in step S114. That is, the processing unit 111 calculates the total area of the fluorescent region in the nucleus region of the test cell acquired in step S113 in the diffraction limit image of the second fluorescent dye acquired in step S111. Subsequently, the processing unit 111 divides the calculated total area of the fluorescent region by the area of the fluorescent region corresponding to one second substance stored in the storage unit 112 in advance, and the division result is the total number of the second substances. And The process of step S114 and the process of step S115 may be interchanged so that the order is reversed.

  In step S116, the processing unit 111 calculates a ratio of the total number of the first substances and the total number of the second substances, that is, "the number of the first substances / the number of the second substances". For example, when the captured image includes 30 test cells, the processing unit 111 calculates the total number of the first substances in the 30 test cells as the second count in the 30 test cells. The ratio is calculated by dividing by the total number of substances. Thus, the analysis process of the first processing step is completed.

  In step S116, the processing unit 111 may calculate the ratio by dividing the number of first substances in one test cell by the number of second substances in one test cell. In this case, the processing unit 111 acquires the number of first substances in one test cell by averaging the number of first substances acquired for each test cell. In addition, the processing unit 111 acquires the number of second substances in one test cell by averaging the number of second substances acquired for each test cell.

(2) Second Processing Step In the first processing step, the quenching process and the reactivating process were performed only once for the first fluorescent dye at the time of imaging, but in the second processing step, the first fluorescent dye was used for the first fluorescent dye. The first image is acquired by repeating the quenching process, the reactivating process, and the imaging process a plurality of times. In the second processing step, the bright points are extracted from each of the acquired first images, the extracted bright points are classified into groups corresponding to the first substance, and the number of the first substances is acquired.

  Note that it is assumed that the second processing step is carried out by taking over these processing after the image acquisition processing and the analysis processing in the first processing step are performed. Therefore, in the second processing step, at the start of the image acquisition processing, the first fluorescent dye is in the extinguished state by the imaging processing in the first processing step, that is, step S105 in FIG. 4A. When performing the processing of the second processing step independently without taking over the first processing step, steps S101 to S103 of FIG. 4A are added before step S121 of FIG. 4B is added after step S132 of FIG. 6B, and step S115 of FIG. 4B is added after step S133 of FIG. 6B.

  As shown in FIG. 6A, in step S121, the processing unit 111 activates the first fluorescent dye by irradiating the test cell with light from the light source 11c at 15 mW for 0.15 seconds. In step S122, the processing unit 111 irradiates the test cell with light from the light source 11a at 80 mW for 2.25 seconds to cause fluorescence to be generated from the first fluorescent dye, and the generated fluorescence is imaged by the imaging unit 19. . The processing unit 111 repeats imaging while the test cells are being irradiated with light to acquire 100 first images. The first fluorescent dye is extinguished while being irradiated with light in step S122.

  In step S123, the processing unit 111 determines whether or not the acquisition of the first image is completed. The processing unit 111 repeats the processing of steps S121 and S122 a predetermined number of times. Here, the processing of steps S121 and S122 is repeated 29 times. In this way, the processing unit 111 combines the 100 first images acquired in step S105 of FIG. 4A with the 2900 first images acquired by repeating the processes of steps S121 and S122 29 times, A total of 3000 first images are acquired.

  As shown in FIG. 6B, in step S131, the processing unit 111 creates a super-resolution image of the first fluorescent dye.

  As shown in FIG. 7, the super-resolution image is created based on the first image acquired in steps S103 to S105 of FIG. 4A and steps S121 to S123 of FIG. 6A. Specifically, for each first image, a bright spot of fluorescence is extracted by Gaussian fitting. As a result, the coordinates of each bright point are acquired on the two-dimensional plane. Here, for each fluorescent region on the first image, when the matching with the reference waveform is obtained in a predetermined range by Gaussian fitting, a bright spot region having a width corresponding to this range is assigned to each bright spot. . For the bright spots in the fluorescent region that match the reference waveform at one point, the bright spot region with the smallest level is assigned. The super-resolution image is created by superimposing the bright spot regions of the respective bright spots thus obtained on all the first images.

  Therefore, in the super-resolution image of the first fluorescent dye, bright spots are extracted from the 3000 first images acquired in steps S103 to S105 of FIG. 4A and steps S121 and S122 of FIG. 6A. Is created by overlapping the bright spot regions of the extracted bright spots.

  Returning to FIG. 6B, in step S132, the processing unit 111 diffracts the super-resolution image of the first fluorescent dye acquired in step S131 and the second fluorescent dye acquired in step S111 of FIG. 4B. A reference image is created by superimposing the limit image and the diffraction limit image of the third fluorescent dye. The reference image is displayed on a screen that displays a determination result described later.

  In step S133, the processing unit 111 acquires the number of first substances. Specifically, as shown in FIG. 7, the processing unit 111 classifies the bright spots extracted at the time of creating the super-resolution image in step S131 into groups corresponding to one first substance. That is, the processing unit 111 first maps all the bright points extracted from the 3000 first images on the coordinate plane. Next, the processing unit 111 scans the coordinate plane in a reference area having a predetermined size and refers to the number of bright points included in the reference area. Furthermore, the processing unit 111 extracts the positions of the reference areas in which the number of bright points included in the reference area is larger than the threshold value and is larger than the surroundings, and the bright point groups included in the reference area are extracted at the extracted positions. Classify into the group corresponding to the first substance. The method of classifying the bright points into the group corresponding to the first substance is not limited to this, and the bright points may be classified into the group corresponding to the first substance by another clustering method.

  Here, as described with reference to FIGS. 3A to 3D, many first fluorescent dyes are bound to one first substance. In addition, as shown in FIGS. 3C and 3D, the first fluorescent dye bound to one first substance is sparsely activated and is activated by each quenching treatment and activation treatment. The distribution of the first fluorescent dye is different. Therefore, the bright spots corresponding to the first substance are slightly shifted for each first image. However, by grouping the bright spots as described above, the bright spots based on the plurality of first fluorescent dyes bound to one first substance, which are close to each other, are classified into one group.

  In step S133 of FIG. 6B, the processing unit 111 further specifies the nuclear region of the test cell acquired in step S113 on the coordinate plane, and determines the group of groups included in the nuclear region of the test cell. Count the number. In this way, the processing unit 111 acquires the counted number of groups as the number of first substances. Here, when a plurality of test cells are included in the first image, the number of the first substances is acquired by, for example, averaging the number of the first substances acquired for each of the test cells.

  As described above, since the nuclear region of the test cell is specified based on the captured image captured by the imaging unit 19 in step S113 of FIG. 4B, the nuclear region of the test cell is determined in step S133. The bright spots of the first fluorescent dye can be superimposed on the image, and the bright spots of the first fluorescent dye can be extracted smoothly for each nucleus region of the test cell. Thereby, the number of the first substances can be acquired smoothly.

  In step S134, the processing unit 111 sets the ratio of the number of the first substances obtained in step S133 to the number of the second substances in one test cell, that is, “the number of the first substances / the number of the second substances”. Calculate the number ". The number of second substances in one test cell is obtained by dividing the total number of second substances obtained in step S115 of FIG. 4B by the number of nuclei in the test cell. Thus, the analysis processing of the second processing step is completed. Note that the number of first images acquired in step S122 of FIG. 6A is not limited to 100, and may be another number.

  In the following description, the number of first substances and the number of second substances for each nucleus are acquired based on the total number of first substances and the total number of second substances acquired in steps S114 and S115 of the first processing step. In this case, as in step S134, a process of dividing the total number of the first substances and the total number of the second substances by the number of nuclei in the test cell is performed.

  As described above, when the inactivation treatment for quenching the first fluorescent dye is performed and then the activation treatment is performed, only a part of the first fluorescent dye bound to each first substance is activated. It In addition, the first fluorescent dye that has not been activated by the previous activation process may be activated by the current activation process. Therefore, by repeating the inactivating process, the activating process, and the imaging process a plurality of times, it is possible to disperse the fluorescence of the first fluorescent dye in each of the first images while causing the first fluorescent dye to emit light evenly. Therefore, bright points based on the first fluorescent dye can be smoothly extracted from each of the first images. Then, by classifying the extracted bright spots into groups corresponding to the first substance, the number of the first substances can be counted. Thereby, the number of the first substance in the test cell can be accurately counted.

  In the first processing step and the second processing step, it is necessary to adjust the intensity of light emitted from the light source 11c for activation so that the first fluorescent dye can be detected at the level of one molecule. In the case of Embodiment 1, the activation efficiency increased in proportion to the product of the intensity of light for activation and the exposure time of light for activation, and the activation efficiency was saturated at a predetermined level. The activation efficiency is the ratio of the first fluorescent dye that is activated by one activation treatment to the quenched first fluorescent dye. The activation efficiency for accurately detecting the first fluorescent dye is 20% or less, preferably 10% or less. By adjusting the intensity of light for activation and the exposure time of light for activation so that the activation efficiency has a desired value, the first fluorescent dye can be detected with high accuracy.

  When the activation efficiency is set low as described above, it is preferable that the number of times of repeating quenching and activation is repeated in order to activate and detect all the first fluorescent dyes. However, if the number of repetitions is large, the measurement time becomes long. Therefore, in the first embodiment, in order to shorten the measurement time as much as possible, the total number of times of quenching and activation in the first processing step and the second processing step was set to 30 times. The number of times of repeating quenching and activation is not limited to 30 times, and may be set to a desired number in consideration of activation efficiency and measurement time. If it is not necessary to detect the first fluorescent dye at the level of one molecule, the activation efficiency may be 50% or more. The activation efficiency is determined by the density in the test cell.

(3) Experiment Next, the experiment conducted by the inventors will be described. In this experiment, a dye that can be switched between a quenching state and an active state was used as the second fluorescent dye for the sake of convenience. However, in the first embodiment, the second fluorescent dye is not used in the first and second processing steps as described above. A fluorescent dye that is not switchable because it is not quenched may be used. As such a second fluorescent dye, for example, a fluorescent dye such as Cy2 can be used.

<Preparation of HER2 FISH stained sample>
A sample for experiment was prepared by the following steps.

  HER2 gene amplification positive calu-3 and negative MCF7 cells on HER2 Dual ISH 3-in-1 control slide (Ventana) using Ventana inform Dual ISH HER2 kit (manufactured by Roche Diagnostics) Was dyed.

[FFPE sample preparation process]
Control slides were dried on a Dry Block Bath THB (As One) for 20 minutes at 65 ° C. Ez Prep was placed on the slide for 5 minutes at 75 ° C. to deparaffinize. After repeating this operation 5 times, it was immersed in Reaction Buffer. Dry Block Bath THB was heated to 90 ° C., CC2 was dropped, and conditioning was performed for 10 minutes. CC2 was added as appropriate to prevent the slides from drying out. After performing this operation 3 times, it was immersed in Reaction Buffer for 4 minutes. This operation was repeated 3 times. 80 μL of ISH Protease II was dropped on the slide, covered with a cover glass, and subjected to enzyme treatment for 16 minutes in a wet box placed in a 37 ° C. incubator.

  It was immersed in 2 × SSC for 4 minutes and washed 3 times. After mixing HybReady and HER2 DNA cocktail probe and dropping 30 μL onto the slide, the slide was covered with a cover glass and sealed with a paper bond. Heat denaturation was performed on a Dry Block Bath THB at 95 ° C for 20 minutes. Then, hybridization was performed overnight at 44 ° C. on a Dry Block Bath THB. It was immersed in 2 × SSC at 62 ° C. for 4 minutes and washed. After repeating this operation 3 times, it was immersed in Reaction Buffer. 500 μL of 1% BSA / Reaction buffer was dropped on the slide, and blocking was performed for 20 minutes in a wet box placed in a 37 ° C. incubator. It was immersed in Reaction buffer and washed.

[Dyeing process]
Rabbit Anti DNP Antibody and Mouse Anti DIG Antibody were mixed, dropped on a slide, covered with a cover glass, and reacted for 20 minutes in a humid box placed in a 37 ° C. incubator. It was immersed in Reaction Buffer for 3 minutes and washed. This was done 3 times. AlexaFluor 647 F (ab ') ₂ fragment of goatanti-rabbit IgG (H + L) (Life Technologies, A-21246), Alexa Fluor 750 Goat Anti Mouse IgG (H + L) (Life Technologies, A-21037), Hoechst 33342 (Life Technologies, H1399) (100 mg diluted in 10 mL of PBS and stored) was diluted 1000 times with 1% BSA / Reaction buffer, 80 μL was dropped on a slide, covered with a cover glass, and placed in a 37 ° C. incubator in a wet box. And reacted for 20 minutes. It was immersed in TBST for 3 minutes and washed. This operation was performed three times. It was immersed in PBS for 3 minutes and washed. This operation was performed three times. It was immersed in purified water and washed. After performing this operation twice, it was dried in an incubator at 37 ° C. for 15 minutes.

  Alexa Fluor 647 corresponds to the above first fluorescent dye. Alexa Fluor 750 corresponds to the above second fluorescent dye. Hoechst 33342 corresponds to the above third fluorescent dye.

[Imaging preparation process]
0.04 μm FluoSphere Dark Red (life technology, F8789) was diluted with PBS, 50 μL was dropped on the slide, and a cover glass was put on the slide, and the mixture was left standing for 10 minutes. After washing off with 500 μL of PBS and dropping 50 μL of mount medium, a cover glass was covered and fixed with nail polish. The composition of Mount Medium is as follows.

5 μL of 1M Tris (pH 7.4)
1M NaCl 1 μL
25% glucose 40 μL
2-mercaptoethanol 1 μL
5000 U / mL Glucose Oxidase 1 μL
1000 μg / mL catalase 1 μL
H ₂ O 51 μL

<Processing result of the first processing step>
The sample was subjected to the first treatment step. In the case of a sample based on HER2 gene amplification positive calu-3, the reference images created in step S112 of FIG. 4 (b) are shown in FIGS. 8 (a) to 8 (d). In FIGS. 8A to 8D, the dotted arrow indicates the first substance, that is, the HER2 gene, and the thick arrow indicates the second substance, that is, CEP17.

  By the treatment of the first treatment step, the ratio calculated in step S116 of FIG. 4 (b) is 6.12 in the case of a sample based on CAL2 of HER2 gene amplification positive, and MCF7 cells of HER2 gene amplification negative. In the case of the sample based on, it was 0.83. According to the Breast Cancer Guidelines, a ratio of more than 2.2 is positive, a ratio of less than 1.8 is negative, and a ratio of 1.8 or more and 2.2 or less is a boundary. Therefore, according to the treatment of the first treatment step, it can be determined that the positive sample can be appropriately determined as positive and the negative sample can be appropriately determined as negative.

<Processing result of the second processing step>
Next, the sample was further processed by the second processing step. In the case of the HER2 gene amplification positive calu-3 based sample, the reference images created in step S132 of FIG. 6B are shown in FIGS. 9A to 9D. In the case of a sample based on HER2 gene amplification negative MCF7 cells, the reference images created in step S132 of FIG. 6 (b) are shown in FIGS. 9 (e) to 9 (g). Also in FIGS. 9A to 9G, as in FIGS. 8A to 8D, the dotted arrow indicates the first substance, that is, the HER2 gene, and the thick arrow indicates the second substance, that is, CEP17. ing.

  By the treatment of the second treatment step, the ratio calculated in step S134 of FIG. 6 (b) is 6.42 in the case of a sample based on calu-3 that is HER2 gene amplification positive, and is HER2 gene amplification negative MCF7 cells. In the case of the sample based on, it was 1.18. Therefore, it can be seen that even in the second treatment step, a positive sample can be properly determined to be positive, and a negative sample can be appropriately determined to be negative.

<Comparative example>
Next, as Comparative Example 1, a sample based on HER2 gene amplification-positive calu-3 was subjected to a process of acquiring a reference image without performing quenching and activation. The reference images created in Comparative Example 1 are shown in FIGS.

  At this time, the calculated ratio was 7.48 in the case of a sample based on calu-3 in which HER2 gene amplification was positive, and 1.16 in the case of a sample based on MCF7 cells in which HER2 gene amplification was negative. Therefore, in Comparative Example 1 as well, it can be seen that the positive sample can be properly determined to be positive, and the negative sample can be appropriately determined to be negative.

  Next, as Comparative Example 2, a sample based on calu-3 having a positive HER2 gene amplification was subjected to a process of obtaining a reference image using a confocal laser microscope without performing quenching and activation. The reference images created in Comparative Example 2 are shown in FIGS. In Comparative Example 2, the image of the second substance, that is, CEP17 is not acquired.

  Comparing the reference images according to the first processing step shown in FIGS. 8A to 8D with the reference images according to Comparative Examples 1 and 2 shown in FIGS. 10A to 10G, the first processing step is better. However, it can be seen that the bright spots corresponding to the HER2 gene are separated. Comparing the reference image by the first processing step shown in FIGS. 8A to 8D with the reference image by the second processing step shown in FIGS. 9A to 9D, the second processing step is better. It can be seen that the bright spots corresponding to the HER2 gene are further separated.

  According to the reference images of FIGS. 8 (a) to 9 (d), the second substance, CEP17, is small in number and spatially well separated even in the sample based on HER2 gene amplification positive calu-3. Has been done. Therefore, the second fluorescent dye that binds to CEP17 has a high resolution and a fluorescent region is specified even without performing the step of reactivating after quenching. Therefore, as described above, for the second substance that can identify the fluorescent region without performing the steps of quenching and reactivating, the second image should be acquired without performing the inactivation treatment for quenching the second fluorescent dye. Thus, the processing can be simplified.

<Other verification>
In the experiment by the treatment of the second treatment step, a portion where the bright spot regions based on the first fluorescent dye are linearly connected was confirmed as shown in the regions surrounded by squares in FIGS. 9B and 9D. . According to the verification by the inventors, even when other HER2 gene amplification-positive test cells are verified by the treatment of the second treatment step, in the super-resolution image, as in FIGS. 9B and 9D, It was confirmed that the bright spot region was linearly connected. It is considered that this is because the HER2 gene, which is the first substance, is significantly amplified as the breast cancer progresses, and the distance between the HER2 genes is shortened, so that the bright spot regions are linearly displayed. Therefore, in breast cancer, the determination based on the above-mentioned ratio and the determination based on the information regarding the arrangement of the first substance, that is, the distance between the first substances can be performed to accurately determine the medical condition and the treatment policy. it is conceivable that. It is considered that this determination can be similarly applied to other diseases other than breast cancer.

  Although the distance between the first substances is focused here, depending on the type of the first substance, along with the progress of the medical condition or amplification, in addition to the distance between the first substances, the position and size of the first substance, etc. It can be assumed that there is a change in the distribution situation. Therefore, it is considered that the determination of the medical condition and the determination of the treatment policy can be accurately performed by further performing the determination based on the distribution state of the first substance.

(4) Display Processing of Embodiment 1 In the processing of the first processing step, the total area of the fluorescent regions is divided by the area of the fluorescent regions corresponding to one fluorescent substance of the first substance to obtain the number of the first substances. It In this method, when the number of the first substances is small and the distance between the first substances is large, the fluorescent regions for each of the first substances are separated in the reactivation diffraction-limited image, so that the first substance You can get the number. Therefore, the negative determination of breast cancer can be appropriately performed even by the treatment of the first treatment step.

  However, as breast cancer progresses and the number of first substances increases, fluorescence regions of the first substances may overlap in the reactivation diffraction-limited image. Therefore, in the processing of the first processing step, the accuracy of the breast cancer determination result based on the ratio may decrease. On the other hand, in the treatment of the second treatment step, even if the number of the first substances is increased in this way, the number of the first substances can be acquired with high accuracy, so that breast cancer can be appropriately determined. Therefore, in the following display processing, first, the processing of the first processing step is performed to determine whether or not the amplification of the disease marker is negative, and the processing of the second processing step is performed only when the amplification is not negative.

  As shown in FIG. 11, in step S141, the processing unit 111 executes the processing of the first processing step shown in FIGS. 4 (a) and 4 (b). Thereby, the processing unit 111 acquires the ratio indicating the presence or absence of amplification of the first substance in step S116 of FIG. 4B. In step S142, the processing unit 111 determines the amplification of the disease marker based on the ratio calculated in step S116 of FIG. Specifically, the processing unit 111 determines that the ratio is greater than 2.2, the result is positive, the ratio is less than 1.8, the result is negative, and the ratio is 1.8 or more and 2.2 or less. To determine.

  Subsequently, in step S143, the processing unit 111 determines whether the determination result of step S142 is negative. When the determination result of step S142 is negative, in step S144, the processing unit 111 treats the ratio indicating the presence or absence of amplification of the first substance acquired in the first processing step and the determination result acquired in step S142. It is displayed on the display unit 120 as index information. In this way, the display process ends. In this case, determination and display based on the second processing step are not performed. On the other hand, when the determination result of step S142 is positive or the boundary, the processing unit 111 advances the process to step S145.

  In step S145, the processing unit 111 executes the processing of the second processing step shown in FIGS. 6 (a) and 6 (b). In step S146, the processing unit 111 determines the amplification of the disease marker based on the ratio calculated in step S134 of FIG. 6B. The determination method is the same as in step S142. As described above, according to the second processing step, the number of the first substance is accurately counted. Therefore, in step S146, the amplification of the first substance that is the target molecule of the molecular target drug is determined based on the number of the first substances that are accurately counted. Therefore, a more accurate determination result of the medical condition can be obtained.

  Subsequently, in step S147, the processing unit 111 determines whether or not the determination result of step S146 is positive. When the determination result of step S146 is positive, the processing unit 111 displays the treatment index information including the determination result acquired in step S146 on the display unit 120 in step S148. In this way, the display process ends. On the other hand, when the determination result of step S146 is negative or the boundary, the processing unit 111 advances the process to step S149.

  In step S149, the processing unit 111 determines whether or not the reference image created in step S132 of FIG. 6B has the linear structure of the first substance. The linear structure of the first substance is the structure shown in the region surrounded by a square in FIGS. 9B and 9D. The processing unit 111 determines that the reference image has a linear structure of the first substance when a predetermined number of consecutive HER2 genes that are the first substance are shorter than the threshold value.

  When the processing unit 111 determines that there is a linear structure of the first substance, the processing unit 111 changes the determination result acquired in step S146 to positive in step S150. On the other hand, if the processing unit 111 determines that there is no linear structure of the first substance, the processing unit 111 advances the process to step S148 without changing the determination result acquired in step S146. In this way, the display process ends.

  In steps S142 and S146, the breast cancer was determined based on the ratio of the number of the first substance and the number of the second substance. However, the number of the first substance for each nucleus is compared with a threshold value to determine the breast cancer. You may be broken. However, when the balance between the number of the first substance and the number of the second substance changes with the progress of the medical condition as described above, the number of the first substance is also referred to and the number of the second substance is also referred to. By making the above determination, the accuracy of determination of the medical condition can be improved.

  As described above, in breast cancer, the number of the HER2 gene, which is the first substance, increases in the nucleus of the test cell due to abnormal cell division, and thus the balance between the number of HER2 genes and the number of CEP17 changes. To do. Therefore, the presence or absence of amplification of the disease marker can be more accurately determined by determining the amplification of the disease marker based on the ratio of the number of the first substance and the number of the second substance as in the above process. The value indicating the balance between the number of the first substances and the number of the second substances is not limited to the ratio, and may be, for example, the difference between the number of the first substances and the number of the second substances.

  In the flowchart of FIG. 11, the process proceeds to step S149 when the determination result of the second processing step is not positive, but the process may proceed to step S149 regardless of the determination result. In addition, when there is a linear structure, the determination result of being negative or boundary is changed to positive, but the determination result is not changed, and even if an indication that there is a linear structure is added to the determination result good.

  As shown in FIGS. 12A and 12B, the screen 200 displayed on the display unit 120 in the display process includes a result area 210 and a comment area 220. The result area 210 displays information acquired by the processing of the first processing step or the second processing step. Specifically, the result area 210 includes the number of the first substance per test cell, the ratio, the treatment index information including the determination result of the medical condition related to the first substance, and the presence or absence of the linear structure. Other treatment index information indicating the reference image and the reference image are displayed. The comment area 220 displays a supplementary explanation about the contents of the result area 210.

  The screens 200 shown in FIGS. 12A and 12B are screens when the processing of the second processing step is executed. In Fig. 12 (a), the test cell does not have a linear structure, and in Fig. 12 (b), the test cell has a linear structure.

  On the screen 200 shown in FIG. 12B, a message indicating that the comment area 220 has a linear structure is displayed. As shown in FIGS. 12 (a) and 12 (b), since the treatment index information including the determination result is displayed on the screen 200, a doctor or the like performs complicated work such as visually counting bright spots. The disease condition can be diagnosed with high accuracy without being required. Since a doctor or the like is not required to have a skilled technique in diagnosing a medical condition, variations in diagnosis among doctors are suppressed. Since the ratio of the number of the first substance and the number of the second substance is displayed in the result area 210, the doctor or the like can determine the treatment policy by referring to this ratio. The displayed treatment index information is not limited to that shown in FIGS. 12 (a) and 12 (b). For example, the display of the number of the first substance may be omitted, or the determination result may not be displayed. It may be omitted.

  When the determination result of the first processing step is not negative, the reference image acquired in the second processing step is displayed in the result area 210. Since this reference image is created based on the super-resolution image of the first fluorescent dye, it is a highly accurate image showing the distribution of bright spots of the first fluorescent dye. Therefore, a doctor or the like can confirm the medical condition in more detail by referring to the distribution of bright spots on this image.

  When the determination result of the first processing step is negative and the processing of the second processing step is skipped, the numerical value based on the processing of the first processing step, the determination result, and the reference image are displayed in the result area 210, and the linear shape is displayed. The structure column is masked.

  As described above, since the number of HER2 genes in the test cells of a patient not suffering from breast cancer is small and it is difficult to approach each other, the number of HER2 genes in the test cells can be accurately determined by the image analysis of the first treatment step. It is possible to count and accurately determine that the amplification of the first substance is negative. Therefore, even if the processing of the second processing step is skipped when the determination of the first processing step is negative, there is no problem in the determination result. Further, in this case, the determination result can be promptly provided to the doctor or the like by skipping the processing of the second processing step. When the determination in the first treatment step is not negative, the treatment in the second treatment step, which can count the number of HER2 genes with higher accuracy, is performed and the determination is performed, so that a highly accurate determination result can be presented to a doctor or the like. .

2. Embodiment 2
In the second embodiment, the second fluorescent dye is also quenched and activated similarly to the first fluorescent dye.

  As shown in FIG. 13, in the second embodiment, steps S201 and S202 are added immediately before step S101 in the processing of the first processing step of FIG. As a result, the second fluorescent dye that binds to the second substance is also quenched in step S201, activated in step S202, and fluorescence is imaged in step S101. The second fluorescent dye is extinguished by the process of step S101. In this case, in step S111 of FIG. 4B, the reactivation diffraction limit image of the second substance is also acquired in the same manner as the first substance, and the acquired reactivation diffraction limit image is acquired in step S112. Image. As a result, in the reference image, the fluorescent region of the second substance is separated as compared with the first embodiment. As a result, the accuracy of the number of the second substances acquired in step S115 of FIG. 4B is increased. The processes of steps S201, S202, and S101 and the processes of steps S103 to S105 may be interchanged so that the order is reversed.

  As shown in FIG. 14A, in the second embodiment, steps S211 to S213 are added before step S121 in the processing of the second processing step of FIG. 6A. The second fluorescent dye that binds to the second substance is also activated in step S211, and then the process of performing imaging and extinction in step S212 is repeated the number of times specified in step S213, and a plurality of second images are obtained. To be acquired.

  In this case, as shown in FIG. 14B, step S221 is added after the step S133 of FIG. 6B. Similar to the first substance, in step S131, the bright points are extracted from the second image to obtain the super-resolution image, and in step S132, the obtained super-resolution image is other. Image. Thereby, in the reference image, the resolution of the bright spot region of the second substance is significantly increased. Further, in step S221, similarly to the acquisition of the number of the first substance in step S133, the bright points of the second substance are classified into groups and the number of the second substance is acquired. This increases the accuracy of the number of second substances. As a result, the accuracy of the ratio calculated in step S134 also increases.

  In the second embodiment, since the second fluorescent dye is also quenched and activated, the distribution of the second substance can be displayed with higher resolution than in the first embodiment.

  When the amplification of the second substance in the test cell becomes significant as the disease progresses, it is possible that the two second substances come close to each other. In this case, if all the second fluorescent dyes that bind to the two adjacent second substances emit fluorescence at the same time, it becomes difficult to separate and capture each fluorescence. In the second embodiment, the second fluorescent dye that binds to the second substance is also subjected to the steps of quenching and reactivating to capture an image of fluorescence. Therefore, when the amplification of the second substance becomes remarkable as the disease progresses. Also, the fluorescence based on the second fluorescent dye can be separated and imaged. Thereby, the accuracy of determining the medical condition can be improved.

  When the super-resolution image is obtained for the second substance as in the second embodiment, the arrangement of the second substance and the distribution state of the second substance may be obtained as in the case of the first substance. The arrangement of the second substances is, for example, the distance between the second substances. It can be assumed that, depending on the type of the second substance, a change in the distance between the second substances and the distribution state of the position, size, etc. of the second substance may be observed as the condition progresses or the amplification progresses. Therefore, further, it is considered that the diagnosis of the medical condition and the determination of the treatment policy can be accurately performed based on the distance between the second substances and the distribution state of the second substances.

<Example of change>
As described above, when quenching and activating the second fluorescent dye as well, the wavelengths of light used for excitation are different from each other, but the wavelengths of light used for activation are common to the first fluorescent dye and the second fluorescent dye. Possible fluorescent dyes can be used. In this modification, such a fluorescent dye is used as the first fluorescent dye and the second fluorescent dye.

  In this case, as shown in FIG. 15A, regarding the processing of the first processing step, step S201 and subsequent steps in FIG. 13 are changed. In steps S301 and S302, the test cells are irradiated with light of different wavelengths to quench the second fluorescent dye and the first fluorescent dye, and in step S303, light of a predetermined wavelength is irradiated to emit the second fluorescent dye and the second fluorescent dye. One fluorescent dye is activated at the same time. Then, in steps S304 and 305, the test cell is irradiated with light that excites the first fluorescent dye and the second fluorescent dye, respectively, and the first image and the second image are acquired. By the processes of steps S304 and S305, the first fluorescent dye and the second fluorescent dye are quenched.

  Further, the processing of the second processing step shown in FIG. 14 (a) is changed as shown in FIG. 15 (b). In step S311, light of a predetermined wavelength is irradiated to simultaneously activate the second fluorescent dye and the first fluorescent dye, and in steps S312 and S313, light for exciting the first fluorescent dye and the second fluorescent dye in the test cell. Are radiated respectively to acquire the first image and the second image. By the processing of steps S312 and S313, the first fluorescent dye and the second fluorescent dye are quenched. The activation, imaging, and extinction processing of steps S311 to S313 is repeated the number of times specified in step S314, and a plurality of first images and second images are acquired.

  As described above, by activating the quenched first fluorescent dye and the quenched second fluorescent dye in the same step, the process can be simplified. In particular, when a large number of quenching and reactivating processes are performed to acquire a super-resolution image, the activation of the first fluorescent dye and the second fluorescent dye can be performed in one step to significantly improve the treatment. Can be simplified.

3. Embodiment 3
In the third embodiment, a part of the optical system is changed as compared with the first embodiment, and the processing unit 111 acquires the position of the bright spot in the optical axis direction of the imaging unit 19.

  As shown in FIG. 16A, in the cell analysis device 10, a beam expander 19c and a phase plate 19d are added to the imaging unit 19. The fluorescence generated from the sample is sequentially transmitted through the beam expander 19c, the phase plate 19d, and the condenser lens 19a, and then imaged by the image sensor 19b.

  The phase plate 19d is arranged on the Fourier plane and has a function of modulating the point spread function so that two focal points appear on the light receiving surface of the image pickup element 19b. Fluorescence generated from one fluorescent dye is imaged at two focal points on the light receiving surface of the image sensor 19b by the action of the phase plate 19d. At this time, the two focal points rotate on the light receiving surface according to the position of the light emitting point of the fluorescence in the optical axis direction of the imaging unit 19, as shown in FIG. That is, the angle formed by the straight line connecting the two focal points and the reference straight line changes on the light receiving surface of the image pickup element 19b according to the position of the fluorescence emission point in the optical axis direction.

  For example, the fluorescence generated from the fluorescent dyes at two different positions in the optical axis direction of the image pickup unit 19 on the slide glass 22 is divided into two by the phase plate 19d, and is irradiated onto the light receiving surface of the image pickup device 19b. . At this time, the straight line connecting the two focal points on the light receiving surface forms an angle of + θ1 with the reference line for one fluorescent dye and the reference line for the other fluorescent dye, as shown in FIG. 16B, for example. Make an angle of + θ2 with the surface. Therefore, if the angle formed by the straight line connecting the two focal points with respect to the reference line is acquired, the position of the fluorescent dye in the optical axis direction can be acquired.

  Specifically, like the second processing step, the processing unit 111 performs Gaussian fitting on the first image obtained by repeating extinction and activation to extract a bright spot, and further, the brightness of the extracted bright spot. To get. Next, the processing unit 111 pairs two bright spots having similar brightness and a distance within a predetermined range. Subsequently, the processing unit 111 fits the two paired bright spots with the template of the two bright spots stored in the storage unit 112 in advance, and the two bright spots that have been fitted with a certain accuracy or more are treated as one bright spot. It is determined that the fluorescence emitted from the fluorescent dye is divided by the phase plate 19d.

  Subsequently, the processing unit 111 sets the midpoint of the two bright points forming a pair on the light receiving surface as the position on the two-dimensional plane at the imaging angle of the fluorescent dye. The processing unit 111 determines the position of the fluorescent dye in the optical axis direction of the imaging unit 19 based on the angle formed by the straight line connecting the two bright points forming a pair and the reference line as described above. In this way, the processing unit 111 specifies the coordinate points of the plurality of fluorescent dyes on the three-dimensional coordinate axis based on the position on the two-dimensional plane and the position in the optical axis direction, and specifies the specified coordinate points on all the first images. A three-dimensional super-resolution image is created by superimposing the above.

  Further, the processing unit 111 classifies the specified coordinate points into groups corresponding to the first substance and acquires the number of the first substances in the test cell. For grouping coordinate points, for example, a reference in which a predetermined reference space is scanned in a three-dimensional coordinate space, the number of coordinate points included in this reference space is greater than a threshold value, and the number of bright points is greater than that of the surroundings. The position of the space is extracted, and the group of bright points included in the reference space at the extracted position is classified into a group corresponding to the first substance.

  Subsequently, the processing unit 111 acquires the range of the nucleus of the test cell in the three-dimensional space. Specifically, the processing unit 111 displaces the objective lens 17 in the optical axis direction of the imaging unit 19 and acquires images at a plurality of focus positions in different optical axis directions. At this time, the region where the fluorescence based on the third fluorescent dye is detected corresponds to the nucleus, and the region where the fluorescence based on the third fluorescent dye is not detected corresponds to other than the nucleus, that is, the cytoplasm. The processing unit 111 acquires the contour of the nucleus from the region where fluorescence is detected for each of the acquired images. Then, the processing unit 111 acquires the range of the nucleus in the three-dimensional space based on each focus position and the contour of the nucleus at that position. Examples of the third fluorescent dye include Hoechst 33342 described above, as well as 7-AAD, DAPI, SYTOX-based fluorescent dye, SYTO-based fluorescent dye, and propidium iodide. Then, the processing unit 111 acquires the number of groups included in the range of the nucleus of the test cell as the number of first substances.

  For example, a three-dimensional super-resolution image is acquired as shown in FIGS. 17 (a) and 17 (b). In each drawing, the XY plane is the two-dimensional plane at the imaging angle, and the Z axis direction is the optical axis direction of the imaging unit 19. FIGS. 17 (a) and 17 (b) are actual three-dimensional super-resolution images acquired for samples with negative and positive breast cancer determination results, respectively. Referring to the three-dimensional super-resolution images of FIGS. 17A and 17B in the Z-axis direction and the Y-axis direction, images as shown in FIGS. 17C and 17D are obtained.

  When a three-dimensional super-resolution image is created, a screen 300 as shown in FIG. 18 is displayed on the display unit 120 in the display process. The screen 300 includes a result area 310 and a comment area 320, similar to the screen 200 shown in FIGS. The result area 310 displays an image when the three-dimensional super-resolution image is referenced in the Z-axis direction and the Y-axis direction. The result area 310 may display a three-dimensional super-resolution image as shown in FIGS. 17 (a) and 17 (b).

  According to the third embodiment, with respect to the first fluorescent dye, a three-dimensional super-resolution image capable of specifying the position in the optical axis direction of the imaging unit 19 together with the position on the two-dimensional plane at the shooting angle is acquired. Bright points that overlap on a dimensional plane can also be separated and extracted. Therefore, the number of the first substances can be counted more correctly, and a more accurate determination result can be presented to a doctor or the like. Further, a doctor or the like can grasp the distribution of the first substance in the optical axis direction by referring to the three-dimensional super-resolution image, and can more accurately determine the medical condition and the treatment policy.

  The distance between the first substances and the distribution state of the first substances may be acquired based on the three-dimensional super-resolution image. According to the three-dimensional super-resolution image, the distance between the first substances and the distribution state of the first substances can be grasped more accurately, so that the medical condition can be determined and the treatment policy can be determined more accurately. Further, a three-dimensional super-resolution image may be acquired for the second substance, and the distance between the second substances and the distribution state of the second substance may be acquired based on the acquired three-dimensional super-resolution image. Also in this case, since the distance between the second substances and the distribution state of the second substances can be grasped more accurately, it is possible to more accurately judge the medical condition and the treatment policy.

  In the first to third embodiments, the disease to be treated is breast cancer, and the first substance serving as a judgment index of the treatment policy is HER2. However, the present invention is not limited to this, and other diseases are treated as the treatment target. The first substance serving as a determination index may be another substance depending on the target disease. Further, the second substance may be another substance depending on the target disease. When the first substance is another substance, it can be assumed that the first substance decreases from the normal time due to a medical condition, unlike the above embodiment. Since the methods of Embodiments 1 to 3 can be appropriately used for detecting the decrease of the first substance, the decrease of the first substance can be detected with high accuracy as in the case of targeting HER2. Moreover, even when the second substance increases or decreases depending on the medical condition, the methods of Embodiments 1 to 3 can be similarly applied.

10 ... Cell analysis device 11 ... Light source part 19 ... Imaging part 111 ... Processing part 120 ... Display part

Claims (10)

A first light source and a second light source for irradiating the test cells containing the first substance bound to the first fluorescent dye with lights of different wavelengths;
An imaging unit for imaging a test cell,
A processing unit,
The processing unit is
In order to activate the first fluorescent dye of the test cell, the test cell is irradiated with the first light source,
In order to inactivate the first fluorescent dye of the test cell, the test cell is irradiated with the second light source,
While irradiating the test cell with the second light source to inactivate the first fluorescent dye, the imaging unit captures a plurality of images,
Counting the first substance contained in the test cell based on the plurality of images captured by the imaging unit,
Cell analyzer.   The cell analysis device according to claim 1, wherein the processing unit counts the first substance contained in the test cell based on a combined image obtained by combining the plurality of images.   The cell analysis device according to claim 2, wherein the processing unit counts the first substance contained in the test cell based on a fluorescent region of the composite image.   The processing unit counts the first substance contained in the test cell based on the area of the fluorescent region of the composite image and the area of the fluorescent region corresponding to one of the first substances, The cell analyzer according to item 3.   The processing unit identifies a nuclear region in the composite image, and counts the first substance contained in the test cell based on the fluorescent region contained in each of the identified regions. The cell analyzer according to.   The processing unit causes the image capturing unit to capture an image of a test cell stained with a nuclear stain fluorescent dye that stains a nucleus, and identifies a nuclear region in the composite image based on the image. Cell analyzer.   The cell analysis device according to claim 2, wherein the processing unit generates the composite image by averaging the plurality of images. The processing unit is
After inactivating the first fluorescent dye by irradiating the test cell with the second light source to the test cell in which the first fluorescent dye is activated, the first fluorescent dye of the test cell is The cell analyzer according to any one of claims 1 to 7, wherein a test cell is irradiated with the first light source for activation.   The processing unit obtains the number of the second substance contained in the test cell, and based on the number of the first substance and the number of the second substance, the treatment index information serving as an index for determining the treatment policy. The cell analyzer according to claim 1, which is obtained. A cell analysis device comprising: a first light source and a second light source for irradiating test cells containing a first substance bound to a first fluorescent dye with lights having wavelengths different from each other; On the computer,
Irradiating the test cell with the first light source to activate the first fluorescent dye of the test cell,
Irradiating the test cell with the second light source to inactivate the first fluorescent dye of the test cell,
Capturing a plurality of images by the image capturing unit while irradiating the test cells with the second light source to inactivate the first fluorescent dye.
A program for executing the step of counting the first substance contained in the test cell based on the plurality of images captured by the imaging unit.