Information processing device, biological sample observation system, and image generation method

Abstract

An information processing device according to one embodiment of this disclosure comprises a simulated image generation unit (131a) that generates a simulated image by superimposing an unstained image including an autofluorescence component and a pigment tile image in which a reference spectrum of a first fluorescent pigment and the image noise of each pixel of the unstained image are associated, a fluorescence separation unit (131b) that generates a separated image from the simulated image by separating a first fluorescent pigment component and the autofluorescence component, and an evaluation unit (131c) that evaluates the degree of the separation of the separated image.

Description

Technical Field

[0001] The present disclosure relates to an information processing apparatus, a biological sample observation system, and an image generation method.

Background Art

[0002] In biological fluorescence imaging, a color separation technique for separating stained fluorescence and unintended autofluorescence derived from biological tissues is required. For example, in multiplex fluorescence imaging technology, color separation techniques using methods such as the least squares method and non-negative matrix factorization have been developed to spectrally separate autofluorescence and extract the target stained fluorescence (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, since the evaluation of color separation accuracy is only a qualitative evaluation by visual inspection, it is difficult to evaluate between algorithms and quantitative evaluation cannot be performed. The reasons for this inability to perform quantitative evaluation include the fact that correct evaluation cannot be performed under staining conditions where the dye luminance levels are not large with respect to the overlap of dyes and autofluorescence, and that comparison between different samples is difficult because the autofluorescence spectrum varies depending on the biological tissue site. Therefore, an evaluation system for quantitatively evaluating color separation accuracy, that is, appropriately evaluating the degree of fluorescence separation, is required.

[0005] Therefore, the present disclosure proposes an information processing apparatus, a biological sample observation system, and an image generation method that can appropriately evaluate the degree of fluorescence separation.

Means for Solving the Problems

[0006] The information processing apparatus according to the embodiment of the present disclosure includes: a simulated image generation unit that generates a simulated image by superimposing an unstained image containing an autofluorescent component with a dye tile image associated with the reference spectrum of a first fluorescent dye and the imaging noise for each pixel of the unstained image; a fluorescence separation unit that separates the component of the first fluorescent dye and the autofluorescent component based on the simulated image and generates a separated image; and an evaluation unit that evaluates the degree of separation of the separated image.

[0007] A biological sample observation system according to the embodiment of the present disclosure comprises an imaging device that acquires an unstained image containing an autofluorescent component, and an information processing device that processes the unstained image, wherein the information processing device includes a simulated image generation unit that superimposes the unstained image with a dye tile image associated with the reference spectrum of a first fluorescent dye and the imaging noise for each pixel of the unstained image to generate a simulated image, a fluorescence separation unit that separates the components of the first fluorescent dye and the autofluorescent component based on the simulated image to generate a separated image, and an evaluation unit that evaluates the degree of separation of the separated image.

[0008] The image generation method according to the embodiment of this disclosure includes superimposing an unstained image containing an autofluorescent component with a dye tile image associated with the reference spectrum of a first fluorescent dye and the pixel-by-pixel imaging noise of the unstained image to generate a simulated image. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows an example of the schematic configuration of an information processing system according to the first embodiment. [Figure 2] This flowchart shows an example of the processing flow by the information processing device according to the first embodiment. [Figure 3] This figure shows an example of a schematic configuration of the analysis unit according to the first embodiment. [Figure 4] This figure illustrates an example of a method for generating a linked fluorescence spectrum according to the first embodiment. [Figure 5] This figure shows an example of a schematic configuration of the analysis unit according to the first embodiment. [Figure 6] It is a diagram for explaining generation of a simulated image according to the first embodiment. [Figure 7] It is a flowchart showing an example of the flow of simulated image generation processing according to the first embodiment. [Figure 8] It is a diagram for explaining shot noise superposition processing according to the first embodiment. [Figure 9] It is a flowchart showing an example of the flow of quantitative evaluation processing according to the first embodiment. [Figure 10] It is a diagram showing an example of a separated image and a histogram according to the first embodiment. [Figure 11] It is a diagram for explaining calculation of a signal separation value based on a histogram according to the first embodiment. [Figure 12] It is a diagram showing an example of a separated image according to the first embodiment. [Figure 13] It is a diagram showing an example of a separated image according to the first embodiment. [Figure 14] It is a diagram showing an example of a separated image according to the first embodiment. [Figure 15] It is a bar graph showing signal separation values for each dye according to the first embodiment. [Figure 16] It is a scatter diagram showing signal separation values for each dye according to the first embodiment. [Figure 17] It is a diagram showing an example of a schematic configuration of a fluorescence observation apparatus according to the first embodiment. [Figure 18] It is a diagram showing an example of a schematic configuration of an observation unit according to the first embodiment. [Figure 19] It is a diagram showing an example of a sample according to the first embodiment. [Figure 20] It is a diagram showing an enlarged area irradiated with line illumination on a sample according to the first embodiment. [Figure 21] It is a diagram showing an example of a schematic configuration of an analysis unit according to the second embodiment. [Figure 22] It is a diagram schematically showing an overall configuration of a microscope system. [Figure 23] It is a diagram showing an example of an imaging method. [Figure 24] This is a diagram showing an example of an imaging method. [Figure 25] This is a diagram showing an example of the schematic configuration of the hardware of an information processing apparatus.

Embodiments for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present disclosure will be described in detail based on the drawings. Note that the apparatus, system, method, etc. according to the present disclosure are not limited by this embodiment. Also, in this specification and the drawings, components having substantially the same functional configuration are basically denoted by the same reference numerals, and redundant description is omitted.

[0011] One or more of the embodiments (including examples and modified examples) described below can be implemented independently. On the other hand, at least a part of the plurality of embodiments described below may be implemented in appropriate combination with at least a part of other embodiments. These plurality of embodiments may include different novel features. Therefore, these plurality of embodiments can contribute to solving different objectives or problems and can exhibit different effects.

[0012] The present disclosure will be described according to the item order shown below. 1. First Embodiment 1-1. Configuration Example of Information Processing System 1-2. Processing Example of Information Processing Apparatus 1-3. Processing Example of Fluorescence Separation 1-4. Configuration Example of Analysis Unit Related to Quantitative Evaluation 1-5. Processing Example of Simulated Image Creation 1-6. Processing Example of Quantitative Evaluation 1-7. Image Example of Separated Image 1-8. Image Example of Evaluation Result Image 1-9. Application Example 1-10. Operations and Effects 2. Second Embodiment 2-1. Configuration Example of Analysis Unit Related to Quantitative Evaluation 2-2. Operations and Effects 3. Other Embodiments 4. Application Examples 5. Example Hardware Configuration 6. Addendum

[0013] <1. First Embodiment> <1-1. Example of an information processing system configuration> An example of the configuration of the information processing system according to this embodiment will be described with reference to Figure 1. Figure 1 is a diagram showing an example of the schematic configuration of the information processing system according to this embodiment. The information processing system is an example of a biological sample observation system.

[0014] As shown in Figure 1, the information processing system according to this embodiment comprises an information processing device 100 and a database 200. The inputs to this information processing system are a fluorescent reagent 10A, a specimen 20A, and a fluorescently stained specimen 30A.

[0015] (Fluorescent reagent 10A) Fluorescent reagent 10A is a chemical used to stain specimen 20A. Fluorescent reagent 10A may include, for example, a fluorescent antibody (including a primary antibody used for direct labeling or a secondary antibody used for indirect labeling), a fluorescent probe, or a nuclear staining reagent, but the type of fluorescent reagent 10A is not particularly limited to these. Furthermore, fluorescent reagent 10A (and the manufacturing lot of fluorescent reagent 10A) is managed with identification information (hereinafter referred to as "reagent identification information 11A") that can identify the fluorescent reagent 10A (and the manufacturing lot of fluorescent reagent 10A). Reagent identification information 11A may be, for example, barcode information (such as one-dimensional barcode information or two-dimensional barcode information), but is not limited to this. Even for identical (same type) products, the properties of fluorescent reagent 10A differ from manufacturing lot to manufacturing lot depending on the manufacturing method and the state of the cells from which the antibody was obtained. For example, in fluorescent reagent 10A, the spectral information, quantum yield, or fluorescence labeling rate (also referred to as "F / P value: Fluorescein / Protein," which refers to the number of fluorescent molecules that label the antibody) may differ from manufacturing lot to manufacturing lot. Therefore, in the information processing system according to this embodiment, the fluorescent reagent 10A is managed for each manufacturing lot by being assigned reagent identification information 11A (in other words, the reagent information for each fluorescent reagent 10A is managed for each manufacturing lot). This allows the information processing device 100 to separate the fluorescent signal and the autofluorescence signal while taking into account even slight differences in properties that may appear for each manufacturing lot. It should be noted that managing the fluorescent reagent 10A on a manufacturing lot basis is merely an example, and the fluorescent reagent 10A may be managed at a finer level than the manufacturing lot.

[0016] (Specimen 20A) Specimen 20A is prepared from a specimen or tissue sample taken from the human body for the purpose of pathological diagnosis or clinical testing. The type of tissue used (e.g., organ or cell), the type of disease being treated, the subject's attributes (e.g., age, sex, blood type, or race), or the subject's lifestyle (e.g., diet, exercise habits, or smoking habits) are not particularly limited for specimen 20A. Furthermore, each specimen 20A is managed with identification information (hereinafter referred to as "specimen identification information 21A") that allows for the identification of each specimen 20A. Similar to reagent identification information 11A, specimen identification information 21A may be, but is not limited to, barcode information (e.g., one-dimensional barcode information or two-dimensional barcode information). The properties of specimen 20A differ depending on the type of tissue used, the type of disease being treated, the subject's attributes, or the subject's lifestyle. For example, in specimen 20A, the measurement channel or spectral information may differ depending on the type of tissue used. Therefore, in the information processing system according to this embodiment, each sample 20A is managed individually by being assigned sample identification information 21A. This allows the information processing device 100 to separate the fluorescence signal and the autofluorescence signal while taking into account even slight differences in properties that appear in each sample 20A.

[0017] (Fluorescent stained specimen 30A) The fluorescently stained specimen 30A is prepared by staining specimen 20A with fluorescent reagent 10A. In this embodiment, it is assumed that specimen 20A is stained with at least one fluorescent reagent 10A, but the number of fluorescent reagents 10A used for staining is not particularly limited. Furthermore, the staining method is determined by the combination of specimen 20A and fluorescent reagent 10A, and is not particularly limited. The fluorescently stained specimen 30A is input to the information processing device 100 and imaged.

[0018] (Information processing device 100) As shown in Figure 1, the information processing device 100 includes an acquisition unit 110, a storage unit 120, a processing unit 130, a display unit 140, a control unit 150, and an operation unit 160.

[0019] (Acquisition part 110) The acquisition unit 110 is configured to acquire information used for various processes of the information processing device 100. As shown in Figure 1, the acquisition unit 110 comprises an information acquisition unit 111 and an image acquisition unit 112.

[0020] (Information acquisition unit 111) The information acquisition unit 111 is configured to acquire reagent information and specimen information. More specifically, the information acquisition unit 111 acquires reagent identification information 11A attached to the fluorescent reagent 10A used to produce the fluorescent stained specimen 30A, and specimen identification information 21A attached to the specimen 20A. For example, the information acquisition unit 111 acquires the reagent identification information 11A and specimen identification information 21A using a barcode reader or the like. Then, the information acquisition unit 111 acquires reagent information based on the reagent identification information 11A and specimen information based on the specimen identification information 21A from the database 200. The information acquisition unit 111 stores this acquired information in the information storage unit 121, which will be described later.

[0021] (Image acquisition unit 112) The image acquisition unit 112 is configured to acquire image information of a fluorescently stained specimen 30A (a specimen 20A stained with at least one fluorescent reagent 10A). More specifically, the image acquisition unit 112 is equipped with an arbitrary image sensor (e.g., a CCD or CMOS), and acquires image information by imaging the fluorescently stained specimen 30A using this image sensor. Here, it should be noted that "image information" is a concept that includes not only the image of the fluorescently stained specimen 30A itself, but also measured values ​​that have not been visualized as an image. For example, the image information may include information about the wavelength spectrum of fluorescence emitted from the fluorescently stained specimen 30A (hereinafter referred to as the fluorescence spectrum). The image acquisition unit 112 stores the image information in the image information storage unit 122, which will be described later.

[0022] (Preservation section 120) The storage unit 120 is configured to store (remember) information used in various processes of the information processing device 100, or information output by various processes. As shown in Figure 1, the storage unit 120 comprises an information storage unit 121, an image information storage unit 122, and an analysis result storage unit 123.

[0023] (Information storage section 121) The information storage unit 121 is configured to store reagent information and sample information acquired by the information acquisition unit 111. After the analysis processing by the analysis unit 131 and the image information generation processing (image information reconstruction processing) by the image generation unit 132, which will be described later, are completed, the information storage unit 121 may increase its free capacity by deleting the reagent information and sample information used in the processing.

[0024] (Image information storage unit 122) The image information storage unit 122 is configured to store image information of the fluorescently stained specimen 30A acquired by the image acquisition unit 112. Similar to the information storage unit 121, after the analysis processing by the analysis unit 131 and the image information generation processing (image information reconstruction processing) by the image generation unit 132 are completed, the image information storage unit 122 may increase its free capacity by deleting the image information used in the processing.

[0025] (Analysis result storage section 123) The analysis result storage unit 123 is configured to store the results of the analysis processing performed by the analysis unit 131, which will be described later. For example, the analysis result storage unit 123 stores the fluorescence signal of the fluorescent reagent 10A or the autofluorescence signal of the sample 20A that has been separated by the analysis unit 131. In addition, the analysis result storage unit 123 provides the results of the analysis processing to the database 200 separately in order to improve the accuracy of the analysis through machine learning or the like. After providing the results of the analysis processing to the database 200, the analysis result storage unit 123 may increase its free capacity by appropriately deleting the analysis processing results it has stored.

[0026] (Processing unit 130) The processing unit 130 has a functional configuration that performs various processing using image information, reagent information, and sample information. As shown in Figure 1, the processing unit 130 comprises an analysis unit 131 and an image generation unit 132.

[0027] (Analysis Department 131) The analysis unit 131 is configured to perform various analysis processes using image information, sample information, and reagent information. For example, the analysis unit 131 performs a process to separate the autofluorescence signal of sample 20A and the fluorescence signal of fluorescent reagent 10A from the image information based on the sample information and reagent information.

[0028] More specifically, the analysis unit 131 recognizes one or more elements constituting the autofluorescence signal based on the measurement channels included in the sample information. For example, the analysis unit 131 recognizes one or more autofluorescence components that constitute the autofluorescence signal. Then, the analysis unit 131 predicts the autofluorescence signal included in the image information using the spectral information of these autofluorescence components included in the sample information. Then, the analysis unit 131 separates the autofluorescence signal and the fluorescence signal from the image information based on the spectral information of the fluorescence component of the fluorescent reagent 10A included in the reagent information and the predicted autofluorescence signal.

[0029] If the sample 20A is stained with two or more fluorescent reagents 10A, the analysis unit 131 separates the fluorescent signals of each of these two or more fluorescent reagents 10A from the image information (or the fluorescent signal after it has been separated from the autofluorescence signal) based on the sample information and reagent information. For example, the analysis unit 131 uses the spectral information of the fluorescent component of each fluorescent reagent 10A contained in the reagent information to separate the fluorescent signals of each fluorescent reagent 10A from the overall fluorescent signal after it has been separated from the autofluorescence signal.

[0030] Furthermore, if the autofluorescence signal is composed of two or more autofluorescence components, the analysis unit 131 separates the autofluorescence signal of each autofluorescence component from the image information (or the autofluorescence signal after it has been separated from the fluorescence signal) based on the sample information and reagent information. For example, the analysis unit 131 uses the spectral information of each autofluorescence component contained in the sample information to separate the autofluorescence signal of each autofluorescence component from the entire autofluorescence signal after it has been separated from the fluorescence signal.

[0031] The analysis unit 131, which separates the fluorescence signal and autofluorescence signal, performs various processing using these signals. For example, the analysis unit 131 may extract the fluorescence signal from the image information of another sample 20A by performing a subtraction process (also called "background subtraction process") on the image information of another sample 20A using the separated autofluorescence signal. If there are multiple identical or similar samples 20A in terms of the tissue used for the sample 20A, the type of disease targeted, the attributes of the subject, and the lifestyle of the subject, the autofluorescence signals of these samples 20A are likely to be similar. Similar samples 20A here include, for example, tissue sections before staining, sections adjacent to the stained section, sections different from the stained section in the same block (sampled from the same location as the stained section), or sections from different blocks in the same tissue (sampled from a different location than the stained section), or sections taken from different patients. Therefore, if the analysis unit 131 is able to extract an autofluorescence signal from a certain sample 20A, it may extract a fluorescence signal from the image information of another sample 20A by removing the autofluorescence signal from that other sample 20A. Furthermore, when the analysis unit 131 calculates the S / N value using the image information of another sample 20A, it can improve the S / N value by using the background after the autofluorescence signal has been removed.

[0032] Furthermore, the analysis unit 131 can perform various processes using the separated fluorescence signal or autofluorescence signal in addition to background subtraction. For example, the analysis unit 131 can use these signals to analyze the fixation state of specimen 20A, or to perform segmentation (or region division) to recognize regions of objects contained in the image information (e.g., cells, intracellular structures (cytoplasm, cell membrane, nucleus, etc.), or tissues (tumor areas, non-tumor areas, connective tissue, blood vessels, blood vessel walls, lymphatic vessels, fibrous structures, necrotic tissue, etc.)). The analysis of the fixation state and segmentation of specimen 20A will be described in detail later.

[0033] (Image generation unit 132) The image generation unit 132 is configured to generate (reconstruct) image information based on the fluorescence signal or autofluorescence signal separated by the analysis unit 131. For example, the image generation unit 132 can generate image information containing only the fluorescence signal or image information containing only the autofluorescence signal. In cases where the fluorescence signal is composed of multiple fluorescence components, or the autofluorescence signal is composed of multiple autofluorescence components, the image generation unit 132 can generate image information for each component. Furthermore, if the analysis unit 131 performs various processing using the separated fluorescence signal or autofluorescence signal (for example, analysis of the immobilization state of the sample 20A, segmentation, or calculation of the S / N value), the image generation unit 132 may generate image information showing the results of those processing. With this configuration, the distribution information of the fluorescent reagent 10A labeled on the target molecule, that is, the two-dimensional spread, intensity, wavelength, and their respective positional relationships of the fluorescence can be visualized, improving visibility for users such as physicians and researchers, especially in the field of tissue image analysis where the information of the target substance is complex.

[0034] Furthermore, the image generation unit 132 may be controlled to distinguish between fluorescent signals and autofluorescence signals based on the fluorescent signals or autofluorescence signals separated by the analysis unit 131, and generate image information. Specifically, it may be controlled to increase the brightness of the fluorescence spectrum of the fluorescent reagent 10A labeled on the target molecule, extract only the fluorescence spectrum of the labeled fluorescent reagent 10A and change its color, extract the fluorescence spectra of two or more fluorescent reagents 10A from a sample 20A labeled with two or more fluorescent reagents 10A and change each to a different color, extract only the autofluorescence spectrum of the sample 20A and divide or subtract it, improve the dynamic range, etc., in order to generate image information. This makes it possible for the user to clearly distinguish the color information derived from the fluorescent reagent bound to the target substance, thereby improving the user's visibility.

[0035] (Display section 140) The display unit 140 is configured to present image information generated by the image generation unit 132 to the user by displaying it on a screen. The type of screen used for the display unit 140 is not particularly limited. Furthermore, although not described in detail in this embodiment, the image information generated by the image generation unit 132 may also be presented to the user by projecting it using a projector or printing it using a printer (in other words, the method of outputting the image information is not particularly limited).

[0036] (Control unit 150) The control unit 150 has a functional configuration that comprehensively controls all processes performed by the information processing device 100. For example, the control unit 150 controls the start and end of various processes described above (for example, imaging of the fluorescently stained specimen 30A, analysis, image information generation (image information reconstruction), and image information display) based on user input via the operation unit 160. The control contents of the control unit 150 are not particularly limited. For example, the control unit 150 may control processes that are commonly performed in general-purpose computers, PCs, tablet PCs, etc. (for example, processes related to the OS (Operating System)).

[0037] (Operation unit 160) The operation unit 160 is configured to receive user input. More specifically, the operation unit 160 is equipped with various input means such as a keyboard, mouse, buttons, touch panel, or microphone, and the user can perform various inputs to the information processing device 100 by operating these input means. Information regarding the operation input performed via the operation unit 160 is provided to the control unit 150.

[0038] (Database 200) Database 200 is a device that manages sample information, reagent information, and the results of the analysis process. More specifically, database 200 manages sample identification information 21A in association with sample information, and reagent identification information 11A in association with reagent information. As a result, the information acquisition unit 111 can acquire sample information from database 200 based on the sample identification information 21A of the sample 20A to be measured, and reagent information based on the reagent identification information 11A of the fluorescent reagent 10A.

[0039] As described above, the sample information managed by database 200 includes information on the measurement channels and spectral information specific to the autofluorescence components contained in sample 20A. However, in addition to this, the sample information may also include target information for each sample 20A, specifically the type of tissue used (e.g., organs, cells, blood, body fluids, ascites, pleural fluid, etc.), the type of disease targeted, the attributes of the subject (e.g., age, sex, blood type, or race), or the subject's lifestyle (e.g., diet, exercise habits, or smoking habits). The information including the measurement channels and spectral information specific to the autofluorescence components contained in sample 20A and the target information may be linked for each sample 20A. This makes it easy to trace the information including the measurement channels and spectral information specific to the autofluorescence components contained in sample 20A from the target information, and for example, it becomes possible to shorten the measurement time by having the analysis unit 131 perform similar separation processing that has been performed in the past based on the similarity of the target information in multiple samples 20A. Furthermore, the term "tissue used" is not specifically limited to tissue collected from the subject, and may also include in vivo tissues and cell lines from humans or animals, as well as solutions, solvents, solutes, and materials contained in the object being measured.

[0040] Furthermore, the reagent information managed by database 200 includes spectral information of fluorescent reagent 10A, as described above. However, the reagent information may also include other information about fluorescent reagent 10A, such as manufacturing lot, fluorescent component, antibody, clone, fluorescence labeling rate, quantum yield, fade coefficient (information indicating how easily the fluorescence intensity of fluorescent reagent 10A decreases), and absorption cross-section (or molar extinction coefficient). Moreover, the sample information and reagent information managed by database 200 may be managed in different configurations, and in particular, the reagent information may be a reagent database that presents the optimal reagent combination to the user.

[0041] Here, it is assumed that sample information and reagent information are provided by the manufacturer or measured independently within the information processing system related to this disclosure. For example, the manufacturer of fluorescent reagent 10A often does not measure and provide spectral information or fluorescence labeling rates for each manufacturing lot. Therefore, by independently measuring and managing this information within the information processing system related to this disclosure, the separation accuracy of fluorescent signals and autofluorescence signals can be improved. Furthermore, for the sake of simplifying management, the database 200 may use catalog values ​​published by the manufacturer or literature values ​​listed in various documents as sample information and reagent information (especially reagent information). However, since actual sample information and reagent information generally differ from catalog values ​​and literature values, it is more preferable that the sample information and reagent information be independently measured and managed within the information processing system related to this disclosure as described above.

[0042] Furthermore, the accuracy of the analysis process (for example, the separation of fluorescent signals and autofluorescence signals) can be improved by machine learning techniques that utilize the sample information, reagent information, and results of the analysis process managed in the database 200. The entity that performs learning using machine learning techniques is not particularly limited, but in this embodiment, the case in which the analysis unit 131 of the information processing device 100 performs learning will be described as an example. For example, the analysis unit 131 uses a neural network to generate a classifier or estimator that has been trained using learning data in which the separated fluorescent signals and autofluorescence signals are linked to the image information, sample information, and reagent information used for separation. When new image information, sample information, and reagent information are acquired, the analysis unit 131 can input that information into the classifier or estimator to predict and output the fluorescent signals and autofluorescence signals contained in the image information.

[0043] Furthermore, the system may calculate similar separation processes performed in the past (separation processes using similar image information, sample information, or reagent information) that are more accurate than the predicted fluorescence and autofluorescence signals, statistically or regressively analyze the content of those processes (information and parameters used in the process, etc.), and output a method to improve the separation process of fluorescence signals and autofluorescence signals based on the analysis results. The machine learning method is not limited to the above, and known machine learning techniques may be used. In addition, the separation process of fluorescence signals and autofluorescence signals may be performed by artificial intelligence. Moreover, not only the separation process of fluorescence signals and autofluorescence signals, but also various processes using the separated fluorescence signal or autofluorescence signal (e.g., analysis of the immobilization state of sample 20A, or segmentation) may be improved by machine learning techniques.

[0044] The above describes an example configuration of the information processing system according to this embodiment. Note that the above configuration described with reference to Figure 1 is merely an example, and the configuration of the information processing system according to this embodiment is not limited to this example. For example, the information processing device 100 does not necessarily have to have all of the functional configurations shown in Figure 1. Also, the information processing device 100 may have a database 200 internally. The functional configuration of the information processing device 100 can be flexibly modified according to specifications and operation.

[0045] Furthermore, the information processing device 100 may perform processing other than that described above. For example, by including information such as the quantum yield, fluorescence labeling efficiency, and absorption cross-section (or molar extinction coefficient) of the fluorescent reagent 10A in the reagent information, the information processing device 100 may use the image information from which the autofluorescence signal has been removed and the reagent information to calculate the number of fluorescent molecules in the image information, the number of antibodies bound to the fluorescent molecules, and so on.

[0046] <1-2. Examples of processing by information processing devices> An example of the processing of the information processing device 100 according to this embodiment will be described with reference to Figure 2. Figure 2 is a flowchart showing an example of the processing flow of the information processing device 100 according to this embodiment.

[0047] As shown in Figure 2, in step S1000, the user determines the fluorescent reagent 10A and sample 20A to be used for analysis. In step S1004, the user prepares a fluorescently stained sample 30A by staining sample 20A with the fluorescent reagent 10A.

[0048] In step S1008, the image acquisition unit 112 of the information processing device 100 acquires image information by imaging the fluorescently stained specimen 30A. In step S1012, the information acquisition unit 111 acquires reagent information and specimen information from the database 200 based on the reagent identification information 11A attached to the fluorescent reagent 10A used to produce the fluorescently stained specimen 30A, and the specimen identification information 21A attached to the specimen 20A.

[0049] In step S1016, the analysis unit 131 separates the autofluorescence signal of sample 20A and the fluorescence signal of fluorescent reagent 10A from the image information based on the sample information and reagent information. If the fluorescence signal contains signals from multiple fluorescent dyes (step S1020 / Yes), in step S1024, the analysis unit 131 separates the fluorescence signals of each fluorescent dye. If the fluorescence signal does not contain signals from multiple fluorescent dyes (step S1020 / No), the separation process of the fluorescence signals of each fluorescent dye is not performed in step S1024.

[0050] In step S1028, the image generation unit 132 generates image information using the fluorescence signals separated by the analysis unit 131. For example, the image generation unit 132 generates image information from which the autofluorescence signal has been removed, or generates image information in which the fluorescence signals are displayed for each fluorescent dye. In step S1032, the display unit 140 displays the image information generated by the image generation unit 132, thus completing the series of processes.

[0051] It should be noted that each step in the flowchart in Figure 2 does not necessarily have to be processed chronologically in the order indicated. In other words, each step in the flowchart may be processed in a different order than indicated, or may be processed in parallel.

[0052] For example, instead of separating the autofluorescence signal of sample 20A and the fluorescence signal of fluorescent reagent 10A from the image information in step S1016 and then separating the fluorescence signals of each fluorescent dye in step S1024, the analysis unit 131 may directly separate the fluorescence signals of each fluorescent dye from the image information. Alternatively, the analysis unit 131 may separate the fluorescence signals of each fluorescent dye from the image information and then separate the autofluorescence signal of sample 20A from the image information.

[0053] Furthermore, the information processing device 100 may also perform processes not shown in Figure 2. For example, the analysis unit 131 may not only separate signals but also perform segmentation based on the separated fluorescence signals or autofluorescence signals, or analyze the immobilization state of the sample 20A.

[0054] <1-3. Examples of fluorescence separation processes> An example of fluorescence separation processing according to this embodiment will be described with reference to Figures 3 and 4. Figure 3 is a diagram showing an example of the schematic configuration of the analysis unit 131 according to this embodiment. Figure 4 is a diagram illustrating an example of a method for generating a linked fluorescence spectrum according to this embodiment.

[0055] As shown in Figure 3, the analysis unit 131 comprises a linking unit 1311, a color separation unit 1321, and a spectrum extraction unit 1322. This analysis unit 131 is configured to perform various processes, including fluorescence separation. For example, the analysis unit 131 is configured to link fluorescence spectra as a pretreatment for fluorescence separation and to separate the linked fluorescence spectra for each molecule.

[0056] (Connection part 1311) The concatenation unit 1311 is configured to generate a concatenated fluorescence spectrum by concatenating at least a portion of the multiple fluorescence spectra acquired by the image acquisition unit 112 in the wavelength direction. For example, the concatenation unit 1311 extracts data of a predetermined width from each fluorescence spectrum so as to include the maximum fluorescence intensity in each of the four fluorescence spectra (A to D in Figure 4) acquired by the image acquisition unit 112. The width of the wavelength band from which the concatenation unit 1311 extracts data can be determined based on reagent information, excitation wavelength, or fluorescence wavelength, and may differ for each fluorescent substance (in other words, the width of the wavelength band from which the concatenation unit 1311 extracts data may differ for each fluorescence spectrum shown in A to D in Figure 4). Then, as shown in Figure 4E, the concatenation unit 1311 generates a single concatenated fluorescence spectrum by concatenating the extracted data in the wavelength direction. Note that since the concatenated fluorescence spectrum is composed of data extracted from multiple fluorescence spectra, the wavelengths are not continuous at the boundaries of each concatenated data point.

[0057] At this time, the coupling unit 1311 performs the above coupling after aligning the excitation light intensity corresponding to each of the multiple fluorescence spectra based on the excitation light intensity (in other words, after correcting the multiple fluorescence spectra). More specifically, the coupling unit 1311 performs the above coupling after aligning the excitation light intensity corresponding to each of the multiple fluorescence spectra by dividing each fluorescence spectrum by the excitation power density, which is the excitation light intensity. This allows for the determination of fluorescence spectra when excitation light of the same intensity is irradiated. Furthermore, if the intensity of the irradiated excitation light is different, the intensity of the spectrum absorbed by the fluorescently stained specimen 30A (hereinafter referred to as the "absorption spectrum") will also differ according to that intensity. Therefore, by aligning the excitation light intensity corresponding to each of the multiple fluorescence spectra as described above, the absorption spectrum can be appropriately evaluated.

[0058] Here, Figures 4A to 4D show specific examples of fluorescence spectra acquired by the image acquisition unit 112. Figures 4A to 4D show specific examples of fluorescence spectra acquired when a fluorescently stained specimen 30A contains four types of fluorescent substances, such as DAPI, CK / AF488, PgR / AF594, and ER / AF647, and is irradiated with excitation light having excitation wavelengths of 392 nm (Figure 4A), 470 nm (Figure 4B), 549 nm (Figure 4C), and 628 nm (Figure 4D), respectively. Note that the fluorescence wavelength is shifted to a longer wavelength than the excitation wavelength due to the release of energy for fluorescence emission (Stokes shift). Furthermore, the fluorescent substances contained in the fluorescently stained specimen 30A and the excitation wavelength of the irradiated excitation light are not limited to those described above.

[0059] In detail, the coupling unit 1311 extracts fluorescence spectrum SP1 in the excitation wavelength band of 392 nm to 591 nm from the fluorescence spectrum shown in Figure 4A, extracts fluorescence spectrum SP2 in the excitation wavelength band of 470 nm to 669 nm from the fluorescence spectrum shown in Figure 4B, extracts fluorescence spectrum SP3 in the excitation wavelength band of 549 nm to 748 nm from the fluorescence spectrum shown in Figure 4C, and extracts fluorescence spectrum SP4 in the excitation wavelength band of 628 nm to 827 nm from the fluorescence spectrum shown in Figure 4D. Next, the coupling unit 1311 corrects the wavelength resolution of the extracted fluorescence spectrum SP1 to 16 nm (no intensity correction), corrects the intensity of fluorescence spectrum SP2 by 1.2 times and corrects the wavelength resolution to 8 nm, corrects the intensity of fluorescence spectrum SP3 by 1.5 times (no wavelength resolution correction), corrects the intensity of fluorescence spectrum SP4 by 4.0 times and corrects the wavelength resolution to 4 nm. The coupling unit 1311 then sequentially couples the corrected fluorescence spectra SP1 to SP4 to generate a coupled fluorescence spectrum as shown in Figure 4E.

[0060] Figure 4 shows the case where the merging unit 1311 extracts fluorescence spectra SP1 to SP4 with a predetermined bandwidth (200 nm width in Figure 4) from the excitation wavelengths when each fluorescence spectrum is acquired and then merges them. However, the bandwidths of the fluorescence spectra extracted by the merging unit 1311 do not need to match for each fluorescence spectrum; they may be different. In other words, the region extracted by the merging unit 1311 from each fluorescence spectrum only needs to include the peak wavelength of each fluorescence spectrum, and the wavelength band and bandwidth can be changed as appropriate. In this case, the shift in spectral wavelength due to Stokes shift may also be taken into consideration. By narrowing the wavelength band to be extracted in this way, the amount of data can be reduced, making it possible to perform fluorescence separation processing at a faster speed.

[0061] Furthermore, the intensity of the excitation light in this explanation may be the excitation power or excitation power density, as described above. The excitation power or excitation power density may be the power or power density obtained by actually measuring the excitation light emitted from the light source, or it may be the power or power density obtained from the driving voltage applied to the light source. In addition, the intensity of the excitation light in this explanation may be a value obtained by correcting the above excitation power density with the absorption rate of each excitation light of the intercept that is the object of observation, or the amplification rate of the detection signal in the detection system (image acquisition unit 112, etc.) that detects the fluorescence emitted from the intercept. That is, the intensity of the excitation light in this explanation may be the power density of the excitation light that actually contributed to the excitation of the fluorescent substance, or a value obtained by correcting that power density with the amplification rate of the detection system, etc. By considering the absorption rate, amplification rate, etc., it becomes possible to appropriately correct the intensity of the excitation light that changes in accordance with changes in machine state, environment, etc., and thus it becomes possible to generate a linked fluorescence spectrum that enables higher accuracy color separation.

[0062] Furthermore, the correction value (also called the intensity correction value) based on the intensity of the excitation light for each fluorescence spectrum is not limited to a value that equalizes the intensity of the excitation light corresponding to each of the multiple fluorescence spectra, but can be modified in various ways. For example, the signal intensity of a fluorescence spectrum with an intensity peak on the longer wavelength side tends to be lower than the signal intensity of a fluorescence spectrum with an intensity peak on the shorter wavelength side. Therefore, if a linked fluorescence spectrum contains both fluorescence spectra with an intensity peak on the longer wavelength side and fluorescence spectra with an intensity peak on the shorter wavelength side, the fluorescence spectra with an intensity peak on the longer wavelength side may be hardly considered, and only the fluorescence spectra with an intensity peak on the shorter wavelength side may be extracted. In such cases, for example, by setting a larger intensity correction value for fluorescence spectra with an intensity peak on the longer wavelength side, it is possible to improve the separation accuracy of fluorescence spectra with an intensity peak on the shorter wavelength side.

[0063] (Color separation section 1321) The color separation unit 1321 comprises, for example, a first color separation unit 1321a and a second color separation unit 1321b, and separates the coupled fluorescence spectrum of the stained section (also called a stained sample) input from the coupling unit 1311 into molecules by color.

[0064] More specifically, the first color separation unit 1321a separates the linked fluorescence spectrum of the stained sample input from the linking unit 1311 into molecular spectra by performing a color separation process using the linked fluorescence reference spectrum contained in the reagent information and the linked autofluorescence reference spectrum contained in the sample information, both input from the information storage unit 121. The color separation process can be performed using methods such as least squares (LSM), weighted least squares (WLSM), non-negative matrix factorization (NMF), or Gram matrix analysis. t Non-negative matrix factorization using AA may also be used.

[0065] The second color separation unit 1321b separates the linked fluorescence spectrum into molecular spectra by performing a color separation process on the linked fluorescence spectrum of the stained sample input from the linking unit 1311 using the adjusted linked autofluorescence reference spectrum input from the spectrum extraction unit 1322. Similar to the first color separation unit 1321a, the color separation process can utilize methods such as least squares (LSM), weighted least squares (WLSM), non-negative matrix factorization (NMF), and Gram matrix analysis. t Non-negative matrix factorization using AA may also be used.

[0066] Here, the least squares method calculates the color mixing ratio by fitting the concatenated fluorescence spectrum generated by the concatenation unit 1311 to a reference spectrum. Furthermore, in the weighted least squares method, weighting is applied to emphasize errors at low signal levels by utilizing the fact that the noise of the measured concatenated fluorescence spectrum (Signal) follows a Poisson distribution. However, the upper limit for which weighting is not applied in the weighted least squares method is defined as the Offset value. The Offset value is determined by the characteristics of the sensor used for measurement, and separate optimization is required when an image sensor is used as the sensor.

[0067] (Spectral extraction unit 1322) The spectral extraction unit 1322 is configured to improve the linked autofluorescence reference spectrum so that more accurate color separation results can be obtained. It adjusts the linked autofluorescence reference spectrum included in the sample information input from the information storage unit 121 to obtain more accurate color separation results based on the color separation results from the color separation unit 1321.

[0068] The spectral extraction unit 1322 performs spectral extraction processing on the concatenated autofluorescence reference spectrum input from the information storage unit 121 using the color separation results input from the first color separation unit 1321a, and improves the concatenated autofluorescence reference spectrum by adjusting it based on the results, thereby obtaining a more accurate color separation result. For example, non-negative matrix factorization (NMF) or singular value decomposition (SVD) may be used for spectral extraction processing.

[0069] In Figure 3, an example is shown where the linked autofluorescence reference spectrum is adjusted only once. However, the system is not limited to this. Alternatively, the color separation result from the second color separation unit 1321b may be input to the spectrum extraction unit 1322, and the process of adjusting the linked autofluorescence reference spectrum in the spectrum extraction unit 1322 may be repeated one or more times before obtaining the final color separation result.

[0070] As described above, the first color separation unit 1321a and the second color separation unit 1321b can output a unique spectrum as a separation result by performing fluorescence separation processing using reference spectra linked in the wavelength direction (linked autofluorescence reference spectrum and linked fluorescence reference spectrum) (the separation result is not separated for each excitation wavelength). Therefore, the operator can obtain the correct spectrum more easily. In addition, since the reference spectrum for autofluorescence used for separation (linked autofluorescence reference spectrum) is automatically acquired and fluorescence separation processing is performed, the operator does not have to extract the spectrum corresponding to autofluorescence from an appropriate space in the unstained section.

[0071] Conventionally, there has been no method to quantitatively evaluate the above-mentioned color separation algorithms (e.g., color separation accuracy) using images of actually stained tissues. The reasons for this are: 1. When images are taken of biological samples that have been actually stained, it is impossible to determine where the dye has stained, and therefore it is impossible to judge whether the dye and autofluorescence have been successfully separated (the correct answer is unknown); 2. Systems used in FCM (flow cytometry) that create panels with good dye separation using the dye spectrum and the wavelength resolution characteristics of the detection system cannot be used when there is significant overlap of dyes or the influence of autofluorescence; 3. Systems that determine the panel from antigen expression rate, antibody dye labeling rate, dye brightness, and excitation efficiency cannot be used for spatial composite evaluation because the characteristics of autofluorescence differ depending on the tissue site; 4. In the above two systems, the spectral shape of the measured autofluorescence, the level to be added, and the noise level of the measurement system are unknown and cannot be considered during panel design.

[0072] Therefore, using simulated images is effective for quantitative evaluation of color separation algorithms and the like. For example, in this embodiment, a dye tile image (fluorescence image) is generated by superimposing a dye spectrum with noise characteristics corresponding to the shooting parameters onto an unstained image acquired by shooting in a tile pattern. The dye tile image and the unstained image are then combined to create an image that simulates the actual measurement (simulated image). This makes it possible to reproduce staining conditions in which the dye brightness level is not large relative to the autofluorescence, and to distinguish between pixels with dye and pixels with autofluorescence. As a result, the accuracy of color separation can be quantitatively determined as a signal separation value from the mean and variance of the pixels. This quantitative evaluation will be explained in detail below.

[0073] <1-4. Example of the configuration of the analysis unit related to quantitative evaluation> An example of the configuration of the analysis unit 131 related to quantitative evaluation according to this embodiment will be described with reference to Figures 5 and 6. Figure 5 is a diagram showing an example of the schematic configuration of the analysis unit 131 according to this embodiment. Figure 6 is a diagram for explaining the generation of a simulated image according to this embodiment.

[0074] As shown in Figure 5, the analysis unit 131 comprises a simulated image generation unit 131a, a fluorescence separation unit 131b, and an evaluation unit 131c. The fluorescence separation unit 131b corresponds to the color separation unit 1321.

[0075] As shown in Figure 6, the simulated image generation unit 131a generates a simulated image by superimposing an unstained image (background image) containing autofluorescence components with a dye tile image (fluorescence image). The dye tile image is a group of dye tiles having multiple dye tiles. This dye tile image is, for example, an image in which the standard spectrum (reference spectrum) of a fluorescent dye (first fluorescent dye) and the imaging noise of each pixel in the unstained image are associated.

[0076] For example, the intensity of the dye added to the autofluorescence intensity of an unstained image is determined by factors such as antigen expression rate, antibody labeling rate, dye excitation efficiency, and dye emission efficiency. The autofluorescence component is intrinsic noise inherent in the tissue sample. In addition to the autofluorescence component of the unstained image, other intrinsic noises include, for example, the standard spectra of other fluorescent dyes (secondary fluorescent dyes) in the unstained image. Furthermore, imaging noise is noise that changes depending on, for example, the imaging conditions of the unstained image. The degree of this imaging noise is quantified and visualized for each pixel. The imaging conditions of the unstained image include, for example, laser power, gain, and exposure time.

[0077] Examples of imaging noise (measurement system noise) include: 1. Unwanted signal noise due to autofluorescence, 2. Random noise originating from sensor circuits such as COMS (e.g., readout noise, dark current noise, etc.), and 3. Shot noise (random) that increases according to the square root of the detected charge amount. To simulate imaging noise, the noise associated with, i.e., added to, the standard spectrum (tile image) is mainly the shot noise described in 3 above. This is because 1 and 2 are included in the unstained background image (autofluorescence image). By superimposing the tile and the background, it is possible to represent all of the imaging noise (measurement system noise) described in 1 to 3 above. The amount of shot noise to be added in 3 above can be determined from the number of photons (or charge amount) of the dye signal added to the tile. For example, in this embodiment, the charge amount of the unstained background image is calculated, the charge amount of the dye is determined from that value, and then the amount of shot noise is determined. Shot noise is also called photon noise and is caused by the physical fluctuation of the amount of photons reaching the sensor, which does not take a constant value. This shot noise cannot be eliminated no matter how much the measurement system's circuitry is improved.

[0078] In the example shown in Figure 6, the dye tile consists of 10 × 10 pixels (display pixels) (approximately 0.3 μm / pixel). This is the case when an unstained image is captured at a magnification of 20x; when the magnification is changed, the size of the dye tile needs to be changed to match the cell size. The size of one dye tile corresponds to the size of the cell, and the number of pixels in the dye tile image corresponds to the number of pixels in the cell size. The smallest unit of a pixel is equal to the size of the cell. The dye tile image contains standard spectra for multiple types of dye tiles with different dyes, i.e., multiple fluorescent dyes. It is also possible to evaluate the color separation performance under double staining or triple staining conditions by mixing multiple dyes in one dye tile, rather than using one dye per dye tile.

[0079] In the example in Figure 6, nine colors of pigment (pigment tiles) are used. The color scheme of the nine pigment tiles is a pattern in which pigment tiles of the same color are arranged in diagonal stripes, but it is not limited to this. For example, the color scheme of each pigment tile may be a pattern in which pigment tiles of the same color are arranged in vertical stripes, horizontal stripes, checkerboard patterns, etc., as long as it is a predetermined color scheme that defines which pigment tile is in which location.

[0080] Specifically, the simulated image generation unit 131a acquires an unstained image (unstained tissue image) and imaging parameters as input parameters. The imaging parameters are examples of imaging conditions and include, for example, laser power, gain, and exposure time. The simulated image generation unit 131a generates dye tiles by adding noise characteristics corresponding to the imaging parameters to the dye spectrum, and repeatedly places dye tiles for the number of dyes that the user wants to stain, thereby generating a dye tile image as a dataset.

[0081] The fluorescence separation unit 131b separates the first fluorescent dye component and the autofluorescence component based on the simulated image generated by the simulated image generation unit 131a, and generates a separated image. This fluorescence separation unit 131b performs color separation calculations on the simulated image (dataset) and generates a separated image. The fluorescence separation unit 131b is the color separation unit 1321 and performs the same processing as the color separation unit 1321. Color separation methods include, for example, LSM and NMF.

[0082] The evaluation unit 131c evaluates the degree of separation of the separated images generated by the fluorescence separation unit 131b. This evaluation unit 131c determines the degree of separation of the separated images (the quality of the panel) from the mean and variance of the color separation calculation results. For example, the evaluation unit 131c generates a histogram from the separated images, calculates the signal separation value between the dye and non-dye from the histogram, and evaluates the degree of separation based on the signal separation value. As an example, the evaluation unit 131c represents the color-separated positive and negative pixels in a histogram and generates a graph showing the signal separation value, which is a numerical value of the color separation accuracy calculation result.

[0083] The display unit 140 displays information or images showing the evaluation results of the evaluation unit 131c (for example, signal separation values ​​for each dye). For example, the display unit 140 displays graphs or diagrams showing the signal separation values ​​for each dye generated by the evaluation unit 131c. This allows the user to understand the evaluation results of the evaluation unit 131c.

[0084] <1-5. Example of process for creating simulated images> An example of the process for creating a simulated image according to this embodiment will be described with reference to Figures 7 and 8. Figure 7 is a flowchart showing an example of the flow of the simulated image generation process according to this embodiment. Figure 8 is a diagram illustrating the shot noise superposition process according to this embodiment.

[0085] As shown in Figure 7, in step S11, the user selects a combination of antibody and dye to be stained. In step S12, the simulated image generation unit 131a determines the spectral intensity of the dye to be applied from the autofluorescence intensity of the superimposed unstained image. In step S13, the simulated image generation unit 131a repeatedly arranges dye tiles, adding noise (imaging noise) that takes into account the noise level at the time of imaging measurement for each pixel, to create a fluorescence image (dye tile image). The simulated image generation unit 131a superimposes the created fluorescence image onto the unstained image. This completes the simulated image.

[0086] In detail, in step S12 above, the spectral intensity of the dye to be added to the autofluorescence intensity of the unstained image (background image) is determined. For example, the brightness of the dye spectrum to be added to the autofluorescence intensity of the unstained image is determined in the following sequence from (a) to (c).

[0087] (a) Calculation of the peak position intensity of the pigment The simulated image generation unit 131a acquires the intensity of each dye spectrum at a peak position of 16 nm (2 channels from the maximum value) and integrates the values.

[0088] (b) Peak position intensity of autofluorescence The simulated image generation unit 131a acquires the autofluorescence intensity of the background image. For example, the simulated image generation unit 131a integrates the spectral intensities of the background image corresponding to two channels of the peak position of each dye. In this case, the spectral intensity of the wavelength channel of the background image is the average value of all pixels.

[0089] (c) Determination of the dye intensity to be imparted to the autofluorescence intensity The simulated image generation unit 131a determines the dye intensity to be applied to the autofluorescence intensity of the background image based on factors such as antigen expression rate, antibody labeling rate, dye excitation efficiency, and dye emission efficiency. The simulated image generation unit 131a adjusts the dye spectrum by calculating the magnification ratio from the spectral intensities obtained in (a) and (b) above so that it matches the set dye intensity. The magnification ratio can be calculated from the following equation (1). Equation (1) is an equation relating to the method for determining the dye intensity relative to autofluorescence.

[0090]

number

[0091] Furthermore, in step S13 described above, noise superposition corresponding to the imaging parameters is performed. For example, the noise characteristics of a CMOS recording device consist of dark current and readout noise, which increase in proportion to the exposure time, and shot noise, which is proportional to the square root of the signal intensity. In this evaluation system, since the dark current noise and readout noise components are already included in the measured unstained image, only the shot noise component needs to be added to the superimposed dye spectrum. Shot noise superposition is performed in the following order from (a) to (d).

[0092] (a) The simulated image generation unit 131a divides the dye spectrum by the wavelength calibration data (conversion coefficient from camera output value to spectral radiance) to return it to an AD value.

[0093] (b) The simulated image generation unit 131a calculates the AD value from the gain and pixel saturation charge amount during background image capture and the charge amount e - Convert to [the appropriate value].

[0094]

number

[0095] Equation (2) is a charge quantity conversion equation. F(λ): standard spectrum of the dye, Cor(λ): wavelength calibration data, H: conversion coefficient, E(λ): charge quantity.

[0096] (c) The simulated image generation unit 131a has σ=S 1 / 2 (S: charge amount per pixel e) - ) is superimposed as shot noise.

[0097]

number

[0098] Equation (3) is the shot noise superposition formula. newE(λ): standard spectrum of the dye superimposed with shot noise, Nrand: normally distributed random number with σ=1, S: charge per pixel e - That is the case.

[0099] (d) After superimposing shot noise as described in (c) above, the simulated image generation unit 131a converts the dye spectrum back to spectral radiance in the reverse order of (a) to (b).

[0100] Figure 8 shows the process described in (a) to (d) above. Since the dye spectrum created in the process described in (a) to (d) above corresponds to one pixel of the image, it is repeatedly arranged as a 10x10 pixel dye tile to create a fluorescence image (dye tile image).

[0101] <1-6. Examples of quantitative evaluation processes> An example of the quantitative evaluation process according to this embodiment will be described with reference to Figures 9 to 11. Figure 9 is a flowchart showing an example of the quantitative evaluation process according to this embodiment. Figure 10 is a diagram showing an example of separated images and histograms according to this embodiment. Figure 11 is a diagram illustrating the calculation of signal separation values ​​based on the histogram according to this embodiment.

[0102] As shown in Figure 9, in step S21, the fluorescence separation unit 131b receives a simulated image. In step S22, the fluorescence separation unit 131b performs a color separation calculation on the simulated image. In step S23, the evaluation unit 131c creates a histogram from the separated images. In step S24, the evaluation unit 131c calculates the signal separation value.

[0103] In detail, in step S22 described above, the fluorescence separation unit 131b performs color separation using a color separation algorithm (e.g., LSM or NMF) to be evaluated, with the set of dye spectra used and the set of autofluorescence spectra as input values.

[0104] Furthermore, in step S23 described above, the evaluation unit 131c generates a histogram from the separated images for each pigment after the color separation calculation, as shown in Figure 10.

[0105] Furthermore, in step S24 described above, the evaluation unit 131c treats the average brightness of one tile, which corresponds to one cell (10 × 10 pixels), as a single signal, and calculates the signal separation value from the average value μ and standard deviation σ of the brightness of all tiles, as shown in Figure 11. For example, if the signal separation value exceeds the detection limit value of 3.29σ = 1.645, the color separation performance (e.g., color separation accuracy) is considered sufficient.

[0106]

number

[0107] Equation (4) is the formula for calculating the signal separation value. μ_0: the mean value of tiles other than the dye being evaluated, μ_1: the mean value of tiles with the dye being evaluated, σ_1: the standard deviation of tiles with the dye being evaluated, and σ_2: the standard deviation of tiles other than the dye being evaluated (see Figure 11).

[0108] <1-7. Examples of separated images> Examples of separated images according to this embodiment will be described with reference to Figures 12 to 14. Figures 12 to 14 are diagrams showing examples of separated images according to this embodiment.

[0109] Figure 12 shows a good example of a separated image, Figure 13 shows a bad example 1 of a separated image (autofluorescence leakage), and Figure 14 shows a bad example 2 of a separated image (autofluorescence leakage). These images are displayed by the display unit 140 as needed. Whether or not this display is shown may be selectable by user input to the operation unit 160.

[0110] As shown in Figure 12, there is no autofluorescence leakage in the separated image. In the example in Figure 12, a magnified section is shown, and even in this magnified section, there is no autofluorescence leakage. On the other hand, as shown in Figure 13, there is autofluorescence leakage in the separated image. In the example in Figure 13, a magnified section of the area with autofluorescence leakage is shown, and there is strong autofluorescence leakage. Also, similar to Figure 13, as shown in Figure 14, there is autofluorescence leakage in the separated image. In the example in Figure 14, similar to Figure 13, a magnified section of the area with autofluorescence leakage is shown, and there is strong autofluorescence leakage.

[0111] <1-8. Example of evaluation result image> Examples of evaluation result images according to this embodiment will be described with reference to Figures 15 and 16. Figure 15 is a bar graph showing the signal separation values ​​for each dye according to this embodiment. Figure 16 is a scatter plot showing the signal separation values ​​for each dye according to this embodiment.

[0112] As shown in Figure 15, a bar graph showing the signal separation value for each dye is displayed by the display unit 140. Also, as shown in Figure 16, a scatter plot showing the signal separation value for each dye is displayed by the display unit 140. This scatter plot shows the leakage between dyes with similar excitation levels. These bar flags and dispersion plots are generated by the evaluation unit 131c and output to the display unit 140. The bar graphs and dispersion plots are images showing the evaluation results of the evaluation unit 131c and are merely examples. The presence or absence of this display and the display mode (for example, the display mode of bar graphs and dispersion plots) may be selectable by user input to the operation unit 160.

[0113] As described above, according to the information processing system of this embodiment, a stained image (simulated image) that simulates actual measurements is created by repeatedly arranging a number of dye tiles corresponding to the number of dyes to be stained and the number of pixels corresponding to the size of the cells, while superimposing noise characteristics corresponding to shooting parameters such as gain and exposure time onto the dye spectrum of each pixel, and superimposing them onto an unstained image. This makes it possible to reflect the spectral shape and noise level characteristics of the measured autofluorescence, so that a simulated image can be created under any shooting conditions.

[0114] Furthermore, by creating a simulated image with repeatedly arranged dye tiles, it becomes possible to distinguish between pixels with superimposed dye and other pixels containing autofluorescence. This allows for the quantitative calculation of the color separation accuracy as a signal separation value from the mean and standard deviation of each pixel. In addition, the dye intensity to be applied to the autofluorescence spectrum of the unstained image can be set based on antigen expression rate, antibody labeling rate, dye excitation efficiency, and dye emission efficiency, making it possible to evaluate color separation accuracy under any staining conditions.

[0115] In other words, the simulated image generation unit 131a generates a dye tile image by superimposing a dye spectrum with noise characteristics corresponding to the shooting parameters onto the unstained image acquired during shooting in a tile pattern, and then synthesizes the dye tile image and the unstained image to create an image that simulates the actual measurement (simulated image). This makes it possible to reproduce staining conditions in which the dye brightness level is not large relative to the autofluorescence, and to distinguish between pixels with dye and pixels with autofluorescence. As a result, the accuracy of color separation can be quantitatively determined as a signal separation value from the mean and variance of the pixels.

[0116] For example, the accuracy of a color separation algorithm can be quantitatively determined as a signal separation value calculated from variance and mean. Furthermore, the evaluation of combinations of dyes and combinations of dyes and reagents can also be quantitatively determined numerically. Quantitative evaluation is also possible for tissue parts with different autofluorescence spectra (different tissues), enabling complex evaluations.

[0117] Normally, the accuracy of a color separation algorithm is evaluated qualitatively by visual inspection, but according to this embodiment, quantitative evaluation can be performed to select the optimal color separation algorithm. Furthermore, although there are the challenges described in 1 to 4 above, the accuracy of color separation can be quantitatively evaluated under all staining conditions. In addition, since complex evaluation is possible, a more optimal panel design can be achieved. Moreover, evaluation can be performed even when the effects of dye overlap and autofluorescence are significant. Furthermore, although the characteristics of autofluorescence differ depending on the tissue site, spatial complex evaluation can also be performed. Panel design simulations can be performed while taking into account the noise level of the measurement system.

[0118] For example, by staining the superimposed unstained image only with DAPI (4',6-Diamidino-2-phenylindole,dihydrochloride), it becomes possible to perform simulations with the user's selected dye plus DAPI. Furthermore, it is possible to evaluate color separation algorithms and design panels while taking into account factors such as DAPI leakage.

[0119] <1-9. Application Examples> The technology described herein can be applied, for example, to a fluorescence observation device 500 (an example of a microscope system). Hereinafter, examples of the configuration of the fluorescence observation device 500 to which the technology may be applied will be described with reference to Figures 17 and 18. Figure 17 is a diagram showing an example of the schematic configuration of the fluorescence observation device 500 according to this embodiment. Figure 18 is a diagram showing an example of the schematic configuration of the observation unit 1 according to this embodiment.

[0120] As shown in Figure 17, the fluorescence observation device 500 includes an observation unit 1, a processing unit 2, and a display unit 3.

[0121] Observation unit 1 includes an excitation unit (irradiation unit) 10, a stage 20, a spectroscopic imaging unit 30, an observation optical system 40, a scanning mechanism 50, a focusing mechanism 60, and a non-fluorescence observation unit 70.

[0122] The excitation unit 10 irradiates the object to be observed with multiple illumination lights of different wavelengths. For example, the excitation unit 10 irradiates the pathological specimen (pathological sample), which is the object to be observed, with multiple line illuminations of different wavelengths arranged parallel to each other on opposite axes. The stage 20 is a stand that supports the pathological specimen and is configured to be movable perpendicular to the direction of the line light from the line illumination by the scanning mechanism 50. The spectroscopic imaging unit 30 includes a spectrometer and acquires the fluorescence spectrum (spectroscopic data) of the pathological specimen excited in a line shape by the line illumination.

[0123] In other words, observation unit 1 functions as a line spectrometer that acquires spectral data corresponding to line illumination. Observation unit 1 also functions as an imaging device that captures multiple fluorescence images generated by the imaging target (pathological specimen) for each of multiple fluorescence wavelengths for each line, and acquires the data of the multiple fluorescence images in the order of the lines.

[0124] Here, "parallel by different axes" means that multiple line lights are parallel and on different axes. "Paragonal by different axes" means that they are not coaxial, and the distance between the axes is not particularly limited. "Parallel" is not limited to strictly parallel, but also includes a state that is nearly parallel. For example, there may be distortions from optical systems such as lenses or deviations from parallel due to manufacturing tolerances, and in this case, they are also considered parallel.

[0125] The excitation unit 10 and the spectroscopic imaging unit 30 are connected to the stage 20 via an observation optical system 40. The observation optical system 40 has a function to track the optimal focus by a focusing mechanism 60. A non-fluorescence observation unit 70 for performing dark-field observation, bright-field observation, etc., may be connected to the observation optical system 40. In addition, a control unit 80 that controls the excitation unit 10, spectroscopic imaging unit 30, scanning mechanism 50, focusing mechanism 60, non-fluorescence observation unit 70, etc., may be connected to the observation unit 1.

[0126] The processing unit 2 includes a storage unit 21, a data calibration unit 22, and an image forming unit 23. Based on the fluorescence spectrum of the pathological specimen (hereinafter also referred to as sample S) acquired by the observation unit 1, the processing unit 2 typically forms an image of the pathological specimen or outputs the distribution of the fluorescence spectrum. The image referred to here includes the composition ratio of the pigments and autofluorescence derived from the sample that constitute the spectrum, the waveform converted to RGB (red, green, blue) color, and the brightness distribution of a specific wavelength band.

[0127] The storage unit 21 includes a non-volatile storage medium, such as a hard disk drive or flash memory, and a storage control unit that controls the writing and reading of data to and from the storage medium. The storage unit 21 stores spectral data showing the correlation between each wavelength of light emitted by each of the multiple line illuminations included in the excitation unit 10 and the fluorescence received by the camera of the spectral imaging unit 30. The storage unit 21 also pre-stores information showing the standard spectrum of autofluorescence related to the sample to be observed (pathological specimen) and information showing the standard spectrum of individual dyes used to stain the sample.

[0128] The data calibration unit 22 configures the spectral data stored in the storage unit 21 based on the captured image taken by the camera of the spectral imaging unit 30. The image forming unit 23 forms a fluorescence image of the sample based on the spectral data and the interval Δy of multiple line illuminations irradiated by the excitation unit 10. For example, the processing unit 2, including the data calibration unit 22 and the image forming unit 23, is realized by hardware elements used in computers, such as a CPU (Central Processing Unit), RAM (Random Access Memory), and ROM (Read Only Memory), and the necessary programs (software). Instead of or in addition to the CPU, a PLD (Programmable Logic Device) such as an FPGA (Field Programmable Gate Array), or a DSP (Digital Signal Processor), or other ASIC (Application Specific Integrated Circuit) may be used.

[0129] The display unit 3 displays various information, such as images based on the fluorescent image formed by the image forming unit 23. This display unit 3 may be, for example, a monitor integrally attached to the processing unit 2, or a display device connected to the processing unit 2. The display unit 3 comprises, for example, a display element such as a liquid crystal device or an organic EL device, and a touch sensor, and is configured as a UI (User Interface) for displaying input settings for shooting conditions and captured images.

[0130] Next, the details of the observation unit 1 will be described with reference to Figure 18. Here, the excitation unit 10 will be described as including two line illuminations Ex1 and Ex2, each emitting light at two wavelengths. For example, line illumination Ex1 emits light at wavelengths of 405 nm and 561 nm, and line illumination Ex2 emits light at wavelengths of 488 nm and 645 nm.

[0131] As shown in Figure 18, the excitation unit 10 has multiple (four in this example) excitation light sources L1, L2, L3, and L4. Each excitation light source L1 to L4 is composed of a laser light source that outputs laser light with wavelengths of 405 nm, 488 nm, 561 nm, and 645 nm, respectively. For example, each excitation light source L1 to L4 is composed of a light-emitting diode (LED) or a laser diode (LD).

[0132] Furthermore, the excitation unit 10 includes a plurality of collimator lenses 11, a plurality of laser line filters 12, a plurality of dichroic mirrors 13a, 13b, 13c, a homogenizer 14, a condenser lens 15, and an entrance slit 16, corresponding to each excitation light source L1 to L4.

[0133] The laser beams emitted from excitation light source L1 and excitation light source L3 are made parallel by collimator lens 11, then pass through laser line filter 12 to cut off the tails of their respective wavelength bands, and are made coaxial by dichroic mirror 13a. The two coaxial laser beams are then beam-shaped by homogenizer 14 such as a fly-eye lens and condenser lens 15 to form line illumination Ex1.

[0134] The laser light emitted from the excitation light source L2 and the laser light emitted from the excitation light source L4 are similarly coaxialized by the respective dichroic mirrors 13b and 13c, and are illuminated as a line illumination Ex2 which is on a different axis from the line illumination Ex1. The line illuminations Ex1 and Ex2 each form a line illumination (primary image) on a different axis, separated by a distance Δy, in the incident slit 16 (slit conjugate), which has multiple slit sections that can be passed through.

[0135] In this embodiment, we describe an example where four lasers are arranged in two coaxial and two anomalous configurations. However, other configurations are also possible, such as two lasers arranged in two anomalous configurations, or four lasers arranged in four anomalous configurations.

[0136] The primary image is projected onto the sample S on the stage 20 via the observation optical system 40. The observation optical system 40 includes a condenser lens 41, dichroic mirrors 42 and 43, an objective lens 44, a bandpass filter 45, and a condenser lens (an example of an imaging lens) 46. Line illumination Ex1 and Ex2 are made into parallel light by the condenser lens 41 paired with the objective lens 44, reflected by the dichroic mirrors 42 and 43, transmitted through the objective lens 44, and projected onto the sample S on the stage 20.

[0137] Here, Figure 19 shows an example of sample S according to this embodiment. In Figure 19, sample S is shown as viewed from the irradiation direction of line illumination Ex1 and Ex2, which are excitation light. Sample S typically consists of a slide containing an object to be observed, such as a tissue section, as shown in Figure 19, but of course, other types of objects may be used. The object to be observed, Sa, is a biological sample such as nucleic acid, cells, proteins, bacteria, or viruses. Sample S (object to be observed, Sa) is stained with multiple fluorescent dyes. Observation unit 1 magnifies and observes sample S to a desired magnification.

[0138] Figure 20 is an enlarged view of region A in which line illuminations Ex1 and Ex2 are irradiated onto sample S according to this embodiment. In the example of Figure 20, two line illuminations Ex1 and Ex2 are arranged in region A, and the imaging areas R1 and R2 of the spectral imaging unit 30 are arranged so as to overlap with each of the line illuminations Ex1 and Ex2. The two line illuminations Ex1 and Ex2 are parallel to each other in the Z-axis direction and are arranged at a predetermined distance Δy in the Y-axis direction.

[0139] Line illuminations Ex1 and Ex2 are formed on the surface of sample S as shown in Figure 20. The fluorescence excited in sample S by these line illuminations Ex1 and Ex2 is focused by the objective lens 44, reflected by the dichroic mirror 43, passes through the dichroic mirror 42 and the bandpass filter 45 which cuts out the excitation light, is focused again by the condenser lens 46, and enters the spectral imaging unit 30, as shown in Figure 18.

[0140] As shown in Figure 18, the spectroscopic imaging unit 30 includes an observation slit (aperture) 31, an image sensor 32, a first prism 33, a mirror 34, a diffraction grating 35 (wavelength dispersion element), and a second prism 36.

[0141] In the example shown in Figure 18, the image sensor 32 is composed of two image sensors 32a and 32b. This image sensor 32 captures (receives) multiple light (fluorescence, etc.) whose wavelengths are dispersed by the diffraction grating 35. For example, a two-dimensional imager such as a CCD (Charge Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor) is used for the image sensor 32.

[0142] The observation slit 31 is positioned at the focal point of the condenser lens 46 and has the same number of slits as the number of excitation lines (two in this example). The fluorescence spectra from the two excitation lines that have passed through the observation slit 31 are separated by the first prism 33 and further separated into fluorescence spectra for each excitation wavelength by reflection from the lattice plane of the diffraction grating 35 via the mirror 34. The four separated fluorescence spectra are incident on the image sensors 32a and 32b via the mirror 34 and the second prism 36, and are expanded as spectral data, expressed as spectral data (x,λ) in terms of position x in the line direction and wavelength λ. Spectral data (x,λ) is the pixel value of the pixel in the image sensor 32 at position x in the row direction and wavelength λ in the column direction. Spectral data (x,λ) is sometimes simply described as spectral data.

[0143] The pixel size [nm / Pixel] of the image sensors 32a and 32b is not particularly limited and can be set to, for example, 2 [nm / Pixel] or more and 20 [nm / Pixel] or less. This dispersion value may be achieved by the pitch of the diffraction grating 35 or optically, or by using hardware binning of the image sensors 32a and 32b. In addition, a dichroic mirror 42 or a bandpass filter 45 is inserted in the optical path to prevent the excitation light (line illumination Ex1 and Ex2) from reaching the image sensor 32.

[0144] Each line illumination Ex1 and Ex2 is not limited to being composed of a single wavelength, but may also be composed of multiple wavelengths. When line illumination Ex1 and Ex2 are composed of multiple wavelengths, the fluorescence excited by them will also include multiple spectra. In this case, the spectroscopic imaging unit 30 has a wavelength-dispersing element for separating the fluorescence into spectra derived from the excitation wavelength. The wavelength-dispersing element is composed of a diffraction grating, a prism, or the like, and is typically placed in the optical path between the observation slit 31 and the image sensor 32.

[0145] The stage 20 and scanning mechanism 50 constitute an XY stage, and move the sample S in the X-axis and Y-axis directions to acquire a fluorescence image of the sample S. In WSI (Whole slide imaging), the sample S is scanned in the Y-axis direction, then moved in the X-axis direction, and then scanned again in the Y-axis direction, and this process is repeated. By using the scanning mechanism 50, dye spectra (fluorescence spectra) excited at different excitation wavelengths, which are spatially separated by a distance Δy on the sample S (object Sa), can be continuously acquired in the Y-axis direction.

[0146] The scanning mechanism 50 changes the position to which the illumination light hits the sample S over time. For example, the scanning mechanism 50 scans the stage 20 in the Y-axis direction. This scanning mechanism 50 allows multiple line illuminations Ex1 and Ex2 to be scanned relative to the stage 20 in the Y-axis direction, that is, in the direction of the arrangement of each line illumination Ex1 and Ex2. This is not limited to this example, and multiple line illuminations Ex1 and Ex2 may be scanned in the Y-axis direction by a galvanometer mirror placed in the middle of the optical system. Since the data from each line illumination Ex1 and Ex2 (for example, 2D data or 3D data) is shifted by a distance Δy in the Y-axis direction, it is corrected and output based on a pre-stored distance Δy or a value of distance Δy calculated from the output of the image sensor 32.

[0147] As shown in Figure 18, the non-fluorescent observation unit 70 consists of a light source 71, a dichroic mirror 43, an objective lens 44, a condenser lens 72, an image sensor 73, and the like. In the example in Figure 18, the non-fluorescent observation unit 70 shows an observation system using dark-field illumination.

[0148] The light source 71 is positioned on the side of the stage 20 opposite the objective lens 44, and illuminates the sample S on the stage 20 with illumination light from the opposite side of the line illumination Ex1 and Ex2. In the case of dark-field illumination, the light source 71 illuminates from outside the NA (numerical aperture) of the objective lens 44, and the light diffracted by the sample S (dark-field image) is captured by the image sensor 73 via the objective lens 44, dichroic mirror 43, and condenser lens 72. By using dark-field illumination, even seemingly transparent samples such as fluorescently stained samples can be observed with contrast.

[0149] Furthermore, this dark-field image may be observed simultaneously with fluorescence and used for real-time focusing. In this case, the illumination wavelength should be selected to avoid affecting fluorescence observation. The non-fluorescence observation unit 70 is not limited to an observation system that acquires dark-field images, but may also consist of an observation system capable of acquiring non-fluorescence images such as bright-field images, phase-contrast images, phase images, and in-line hologram images. For example, various observation methods such as the Schlieren method, phase-contrast method, polarization observation method, and reflected illumination method can be used to acquire non-fluorescence images. The position of the illumination light source is not limited to below the stage 20, but may be above the stage 20 or around the objective lens 44. In addition, other methods such as a pre-focus map method, in which the focus coordinates (Z coordinate) are recorded in advance, may be used, rather than just a method that performs focus control in real time.

[0150] In the above description, the line illumination as excitation light consisted of two lines, line illumination Ex1 and Ex2, but it is not limited to this, and may consist of three, four, or five or more lines. Furthermore, each line illumination may include multiple excitation wavelengths selected so as not to degrade the color separation performance as much as possible. In addition, even with only one line illumination, if the excitation light source consists of multiple excitation wavelengths, and each excitation wavelength is linked to the data acquired by the image sensor 32 and recorded, a multicolor spectrum can be obtained, although the separation performance will not be as high as that of parallel-axis illumination.

[0151] The above describes an example of applying the technology described herein to a fluorescence observation device 500. Note that the above configuration described with reference to Figures 17 and 18 is merely an example, and the configuration of the fluorescence observation device 500 according to this embodiment is not limited to this example. For example, the fluorescence observation device 500 does not necessarily have to include all of the configurations shown in Figures 17 and 18, and may include configurations not shown in Figures 17 and 18.

[0152] <1-10. Actions and Effects> As described above, according to the first embodiment, a simulated image generation unit 131a is provided that generates a simulated image by superimposing an unstained image containing an autofluorescence component with a dye tile image associated with the standard spectrum (reference spectrum) of the first fluorescent dye and the imaging noise for each pixel of the unstained image; a fluorescence separation unit 131b is provided that separates the component of the first fluorescent dye and the autofluorescence component based on the simulated image and generates a separated image; and an evaluation unit 131c is provided that evaluates the degree of separation of the separated image. Thus, a simulated image is generated, a color separation process is performed on the simulated image to generate a separated image, and the degree of separation of the separated image is evaluated. By using a simulated image in this way, it becomes possible to quantitatively evaluate the color separation accuracy, and thus the degree of fluorescence separation can be appropriately evaluated.

[0153] Furthermore, the dye tile image may include the standard spectrum of the second fluorescent dye in addition to the first fluorescent dye, and may be an image in which the individual standard spectra of the first and second fluorescent dyes are associated with the pixel-by-pixel imaging noise of the unstained image. This makes it possible to generate simulated images corresponding to multiple fluorescent dyes.

[0154] Furthermore, the imaging noise may vary depending on the imaging conditions of the unstained image. This makes it possible to generate a simulated image that corresponds to the imaging conditions of the unstained image.

[0155] Furthermore, the imaging conditions for the unstained image may include at least one or all of the following: laser power, gain, and exposure time. This allows for the generation of a simulated image corresponding to that information.

[0156] Furthermore, the pigment tile image may be a group of pigment tiles containing multiple pigment tiles. This allows for the generation of a simulated image corresponding to each pigment tile.

[0157] Furthermore, the individual sizes of multiple pigment tiles may be the same as the size of the cells. This allows for the generation of simulated images corresponding to each pigment tile that is the same size as the cells.

[0158] Furthermore, multiple pigment tiles may be arranged in a predetermined color scheme pattern. This makes it possible to perform color separation processing on the simulated image corresponding to each pigment tile based on the predetermined color scheme pattern, thus enabling efficient color separation processing.

[0159] Furthermore, the degree of imaging noise may be quantified or visualized for each dye tile. This allows for the generation of simulated images corresponding to the quantified degree of imaging noise when the degree is quantified. Additionally, visualization of the degree of imaging noise allows the user to understand the actual level of noise.

[0160] Furthermore, the simulated image generation unit 131a may repeatedly arrange a number of pigment tiles specified by the user to generate a pigment tile image. This makes it possible to generate a simulated image corresponding to a number of pigment tiles specified by the user.

[0161] Furthermore, the simulated image generation unit 131a may create a dye tile by mixing multiple dyes. This makes it possible to evaluate the color separation performance (e.g., color separation accuracy) under double staining conditions, triple staining conditions, etc.

[0162] Furthermore, the simulated image generation unit 131a may determine the spectral intensity of the dye to be applied relative to the autofluorescence intensity of the unstained image. This makes it possible to reproduce staining conditions in which the dye brightness level is not large relative to the autofluorescence intensity, and to distinguish between pixels with dye and pixels with autofluorescence.

[0163] Furthermore, the simulated image generation unit 131a may superimpose imaging noise onto the standard spectrum of the first fluorescent dye. This allows the dye tile image to be generated by relating the standard spectrum and the imaging noise.

[0164] Furthermore, the superimposed imaging noise may also be shot noise. This makes it possible to generate a dye tile image that corresponds to the shot noise.

[0165] Furthermore, the fluorescence separation unit 131b may separate the components of the first fluorescent dye from the autofluorescent components by a color separation calculation that includes at least one of the following: least squares method, weighted least squares method, or non-negative matrix factorization. This enables accurate color separation processing.

[0166] Furthermore, the evaluation unit 131c may generate a histogram from the separated images, calculate the signal separation value between the dye and non-dye elements from the histogram, and evaluate the degree of separation based on the signal separation value. This allows for accurate evaluation of the degree of separation. For example, if the signal separation value exceeds a predetermined value (e.g., 1.645), the degree of separation is evaluated as good.

[0167] <2. Second Embodiment> <2-1. Example of the configuration of the analysis unit related to quantitative evaluation> An example of the configuration of the analysis unit 131 related to quantitative evaluation according to this embodiment will be described with reference to Figure 21. Figure 21 is a diagram showing an example of the schematic configuration of the analysis unit 131 according to this embodiment.

[0168] As shown in Figure 21, the analysis unit 131 includes a simulated image generation unit 131a, a fluorescence separation unit 131b, and an evaluation unit 131c according to the first embodiment, as well as a recommendation unit 131d.

[0169] The recommendation unit 131d recommends the optimal reagent (fluorescent reagent 10A) from among the dyes specified by the user, based on the degree of separation evaluated by the evaluation unit 131c. For example, the recommendation unit 131d generates an image (e.g., a table or figure) to present the user with spatial information evaluation of tissues with different autofluorescence spectra and the optimal dye combination for a given tissue, and the display unit 140 displays the image generated by the recommendation unit 131d. This allows the user to visually inspect the displayed image and understand the optimal dye combination.

[0170] For example, the evaluation unit 131c calculates signal separation values ​​for combinations of dyes used for staining and combinations of dyes and reagents. The recommendation unit 131d generates an image to show the user which combination is optimal based on the calculation results (e.g., signal separation values ​​for each combination). For example, the recommendation unit 131d excludes dyes whose signal separation value does not exceed 1.645 and generates an image showing the optimal combination. In addition to generating the optimal combination, the system may also generate an image (e.g., a table or figure) showing multiple recommended combinations along with their color separation performance (e.g., signal separation values). Furthermore, an image (e.g., a table) showing matrix information of antibody and dye combinations may be displayed for reference.

[0171] <2-2. Action and Effects> As described above, the second embodiment provides the same effects as the first embodiment. Furthermore, a recommendation section 131d is provided that recommends the optimal reagent (fluorescent reagent 10A) corresponding to the dye specified by the user based on the degree of separation. This allows the user to identify the optimal reagent, thereby improving user convenience.

[0172] Furthermore, the recommendation section 131d may generate an image (e.g., a table or diagram) showing the combination of dyes or the combination of dyes and reagents. This allows the user to understand the combination of dyes or the combination of dyes and reagents, thereby improving user convenience.

[0173] Furthermore, the recommended section 131d may generate an image (e.g., a diagram) showing the combination of antibody and dye. This allows the user to understand the combination of antibody and dye, thereby improving user convenience.

[0174] <3. Other Embodiments> The processes described in the above-described embodiments (or variations) may be carried out in various other forms (variations) besides those described in the above embodiments. For example, all or part of the processes described as being performed automatically in the above embodiments may be performed manually, or all or part of the processes described as being performed manually may be performed automatically by known methods. In addition, the processing procedures, specific names, and information including various data and parameters shown in the above document and drawings may be changed at will unless otherwise specified. For example, the various information shown in each figure is not limited to the information shown.

[0175] Furthermore, the components of each illustrated device are functionally conceptual and do not necessarily need to be physically configured as shown. In other words, the specific forms of distribution and integration of each device are not limited to those shown, and all or part of them can be functionally or physically distributed and integrated in any unit according to various loads and usage conditions.

[0176] Furthermore, the embodiments (or modifications) described above can be combined as appropriate, provided that the processing content is not contradictory. Also, the effects described herein are merely examples and not limiting, and other effects may exist.

[0177] <4. Application Examples> The technology disclosed herein can be applied, for example, to a microscope system. An example configuration of a microscope system 5000 to which this technology can be applied will be described below with reference to Figures 22 to 24. A microscope device 5100, which is part of the microscope system 5000, functions as an imaging device.

[0178] An example configuration of the microscope system of this disclosure is shown in Figure 22. The microscope system 5000 shown in Figure 22 includes a microscope device 5100, a control unit 5110, and an information processing unit 5120. The microscope device 5100 includes a light irradiation unit 5101, an optical unit 5102, and a signal acquisition unit 5103. The microscope device 5100 may further include a sample placement unit 5104 on which a biological sample S is placed. The configuration of the microscope device is not limited to that shown in Figure 22. For example, the light irradiation unit 5101 may be located outside the microscope device 5100, and a light source not included in the microscope device 5100 may be used as the light irradiation unit 5101. Also, the light irradiation unit 5101 may be arranged so that the sample placement unit 5104 is sandwiched between the light irradiation unit 5101 and the optical unit 5102, for example, it may be located on the side where the optical unit 5102 is located. The microscope apparatus 5100 may be configured to perform one or more of the following: bright-field observation, phase-contrast observation, differential interference contrast observation, polarization observation, fluorescence observation, and dark-field observation.

[0179] The microscope system 5000 may be configured as a so-called WSI (Whole Slide Imaging) system or a digital pathology imaging system and can be used for pathological diagnosis. Alternatively, the microscope system 5000 may be configured as a fluorescence imaging system, particularly a multi-fluorescence imaging system.

[0180] For example, the microscope system 5000 may be used for intraoperative pathological diagnosis or remote pathological diagnosis. In intraoperative pathological diagnosis, the microscope device 5100 may acquire data from a biological sample S obtained from the patient undergoing surgery and transmit this data to the information processing unit 5120. In remote pathological diagnosis, the microscope device 5100 may transmit the acquired data from the biological sample S to the information processing unit 5120 located in a different location (such as another room or building). In these diagnoses, the information processing unit 5120 receives and outputs the data. Based on the output data, the user of the information processing unit 5120 can perform a pathological diagnosis.

[0181] (Biologically derived samples) The biological sample S may be a sample containing biological components. The biological components may be tissues, cells, liquid components of a living organism (such as blood or urine), cultures, or living cells (such as cardiomyocytes, nerve cells, and fertilized eggs). The biological sample may be a solid, and may be a specimen fixed with a fixative such as paraffin or a solid formed by freezing. The biological sample may be a section of the solid. A specific example of the biological sample is a section of a biopsy specimen.

[0182] The biological sample may be treated with staining or labeling. This treatment may be staining to show the morphology of the biological component or to show the substances (such as surface antigens) present in the biological component, and examples include HE (Hematoxylin-Eosin) staining and immunohistochemistry staining. The biological sample may be treated with one or more reagents, and these reagents may be fluorescent dyes, chromogenic reagents, fluorescent proteins, or fluorescently labeled antibodies.

[0183] The aforementioned specimen may be prepared from a tissue sample for the purpose of pathological diagnosis or clinical testing. Furthermore, the aforementioned specimen may be derived not only from the human body, but also from animals, plants, or other materials. The characteristics of the aforementioned specimen will differ depending on the type of tissue used (e.g., organs or cells), the type of disease being treated, the attributes of the subject (e.g., age, sex, blood type, or race), or the subject's lifestyle (e.g., diet, exercise habits, or smoking habits). Each of the aforementioned specimens may be managed with identifiable identification information (e.g., a barcode or QR code®).

[0184] (Light irradiation area) The light irradiation unit 5101 is a light source for illuminating a biological sample S, and an optical unit for guiding the light emitted from the light source to the sample. The light source can irradiate the biological sample with visible light, ultraviolet light, or infrared light, or a combination thereof. The light source may be one or more of the following: halogen light source, laser light source, LED light source, mercury light source, and xenon light source. There may be multiple types of light sources and / or wavelengths for fluorescence observation, and these may be appropriately selected by those skilled in the art. The light irradiation unit may have a transmission type, reflection type, or reflected type (coaxial reflected type or side-emitting type).

[0185] (Optical Department) The optical unit 5102 is configured to guide light from a biological sample S to a signal acquisition unit 5103. The optical unit may be configured to enable the microscope device 5100 to observe or image the biological sample S. The optical unit 5102 may include an objective lens. The type of objective lens may be appropriately selected by a person skilled in the art depending on the observation method. The optical unit may also include a relay lens for relaying the image magnified by the objective lens to the signal acquisition unit. The optical unit may further include optical components other than the objective lens and the relay lens, such as an eyepiece, a phase plate, and a condenser lens. The optical unit 5102 may further include a wavelength separation unit configured to separate light having a predetermined wavelength from the light from the biological sample S. The wavelength separation unit may be configured to selectively allow light of a predetermined wavelength or wavelength range to reach the signal acquisition unit. The wavelength separation unit may include, for example, one or more of a filter that selectively transmits light, a polarizer, a prism (Wollaston prism), and a diffraction grating. The optical components included in the wavelength separation unit may be arranged, for example, in the optical path from the objective lens to the signal acquisition unit. The wavelength separation unit is provided in the microscope apparatus when fluorescence observation is performed, especially when an excitation light irradiation unit is included. The wavelength separation unit may be configured to separate fluorescence from each other or to separate white light from fluorescence.

[0186] (Signal acquisition unit) The signal acquisition unit 5103 may be configured to receive light from a biological sample S and convert the light into an electrical signal, particularly a digital electrical signal. The signal acquisition unit may be configured to acquire data relating to the biological sample S based on the electrical signal. The signal acquisition unit may be configured to acquire image data (images, particularly still images, time-lapse images, or moving images) of the biological sample S, and may be configured to acquire image data magnified by the optical unit. The signal acquisition unit includes one or more image sensors, such as a CMOS or CCD, having a plurality of pixels arranged in one or two dimensions. The signal acquisition unit may include an image sensor for low-resolution image acquisition and an image sensor for high-resolution image acquisition, or it may include an image sensor for sensing, such as for AF, and an image output image sensor for observation, etc. The image sensor may include, in addition to the plurality of pixels, a signal processing unit (including one or more of a CPU, DSP, and memory) that performs signal processing using the pixel signals from each pixel, and an output control unit that controls the output of image data generated from the pixel signals and processed data generated by the signal processing unit. The image sensor including the plurality of pixels, the signal processing unit, and the output control unit may preferably be configured as a single-chip semiconductor device. The microscope system 5000 may further include an event detection sensor. The event detection sensor may include a pixel that converts incident light into photoelectric light and may be configured to detect an event when the brightness change of the pixel exceeds a predetermined threshold. The event detection sensor may be particularly asynchronous.

[0187] (Control Unit) The control unit 5110 controls imaging by the microscope device 5100. For imaging control, the control unit may drive the movement of the optical unit 5102 and / or the sample mounting unit 5104 to adjust the positional relationship between the optical unit and the sample mounting unit. The control unit 5110 may move the optical unit and / or the sample mounting unit toward or toward each other (for example, in the direction of the optical axis of the objective lens). The control unit may also move the optical unit and / or the sample mounting unit in any direction in a plane perpendicular to the optical axis. For imaging control, the control unit may control the light irradiation unit 5101 and / or the signal acquisition unit 5103.

[0188] (Sample placement area) The sample placement section 5104 may be configured to fix the position of the biological sample on the sample placement section, and may be a so-called stage. The sample placement section 5104 may be configured to move the position of the biological sample in the direction of the optical axis of the objective lens and / or in a direction perpendicular to the optical axis.

[0189] (Information Processing Department) The information processing unit 5120 may acquire data (such as imaging data) acquired by the microscope device 5100 from the microscope device 5100. The information processing unit may perform image processing on the imaging data. This image processing may include unmixing, particularly spectral unmixing. This unmixing may include processes that extract data of light components of a predetermined wavelength or wavelength range from the imaging data to generate image data, or processes that remove data of light components of a predetermined wavelength or wavelength range from the imaging data. This image processing may also include autofluorescence separation processing to separate autofluorescence components and pigment components of tissue sections, and fluorescence separation processing to separate wavelengths between pigments with different fluorescence wavelengths. In the autofluorescence separation processing, the autofluorescence component may be removed from the image information of another specimen using an autofluorescence signal extracted from one of the multiple specimens that are identical or have similar properties. The information processing unit 5120 may transmit data for imaging control to the control unit 5110, and the control unit 5110, upon receiving the data, may control imaging by the microscope device 5100 according to the data.

[0190] The information processing unit 5120 may be configured as an information processing device such as a general-purpose computer, and may be equipped with a CPU, RAM, and ROM. The information processing unit may be contained within the housing of the microscope device 5100, or it may be located outside the housing. Furthermore, various processing or functions performed by the information processing unit may be implemented by a server computer or cloud connected via a network.

[0191] The imaging method for biological samples S using the microscope device 5100 may be appropriately selected by those skilled in the art, depending on the type of biological sample and the purpose of imaging. An example of such imaging method is described below.

[0192] One example of an imaging method is as follows: First, the microscope device can identify the imaging target area. This imaging target area may be defined to cover the entire area where the biological sample is located, or it may be defined to cover a specific part of the biological sample (the part where the target tissue section, target cells, or target lesion is located). Next, the microscope device divides the imaging target area into multiple divided areas of a predetermined size, and the microscope device sequentially images each divided area. In this way, an image of each divided area is acquired.

[0193] As shown in Figure 23, the microscope device identifies an imaging target area R that covers the entire biological sample S. The microscope device then divides the imaging target area R into 16 divided regions. The microscope device then images divided region R1, and then may image any region within the imaging target area R, such as a region adjacent to divided region R1. This imaging of divided regions continues until there are no unimaged divided regions left. Regions other than the imaging target area R may also be imaged based on the image information of the divided regions. After imaging a divided region, the positional relationship between the microscope device and the sample mounting unit is adjusted in order to image the next divided region. This adjustment may be performed by moving the microscope device, moving the sample mounting unit, or moving both. In this example, the imaging device that images each divided region may be a two-dimensional image sensor (area sensor) or a one-dimensional image sensor (line sensor). The signal acquisition unit may image each divided region via the optical unit. Furthermore, imaging of each divided region may be performed continuously while moving the microscope device and / or the sample mounting unit, or the movement of the microscope device and / or the sample mounting unit may be stopped when imaging each divided region. The imaging target area may be divided such that parts of each divided region overlap, or it may be divided so that the imaging target area does not overlap. Each divided region may be imaged multiple times with different imaging conditions such as focal length and / or exposure time. In addition, the information processing device can stitch together multiple adjacent divided regions to generate image data of a wider area. By performing this stitching process over the entire imaging target area, an image of a wider area can be obtained for the imaging target area. Furthermore, lower-resolution image data can be generated from the images of the divided regions or from the stitched images.

[0194] Other examples of imaging methods are as follows: First, the microscope device may identify the imaging target area. This imaging target area may be identified to cover the entire area where the biological sample is located, or it may be identified to cover a specific part of the biological sample (the part where the target tissue section or target cells are located). Next, the microscope device scans a portion of the imaging target area (also called a "segmented scan area") in one direction (also called the "scanning direction") in a plane perpendicular to the optical axis and images it. Once scanning of the segmented scan area is complete, the microscope device then scans the adjacent segmented scan area. These scanning operations are repeated until the entire imaging target area is imaged. As shown in Figure 24, the microscope device identifies the area where the tissue section is located (gray area) of the biological sample S as the imaging target area Sa. Then, the microscope device scans the segmented scan area Rs within the imaging target area Sa in the Y-axis direction. Once scanning of the segmented scan area Rs is complete, the microscope device then scans the adjacent segmented scan area in the X-axis direction. This operation is repeated until scanning of the entire imaging target area Sa is completed. The positional relationship between the microscope device and the sample holder is adjusted for scanning each segmented scan area, and for imaging the next segmented scan area after imaging a certain segmented scan area. This adjustment may be performed by moving the microscope device, moving the sample holder, or moving both. In this example, the imaging device that images each segmented scan area may be a one-dimensional image sensor (line sensor) or a two-dimensional image sensor (area sensor). The signal acquisition unit may image each segmented area via a magnifying optical system. Furthermore, imaging of each segmented scan area may be performed continuously while moving the microscope device and / or the sample holder. The imaging target area may be divided such that parts of each segmented scan area overlap, or it may be divided so that parts of the imaging target area do not overlap. Each segmented scan area may be imaged multiple times with different imaging conditions such as focal length and / or exposure time. Furthermore, the information processing device can stitch together multiple adjacent segmented scan areas to generate image data of a wider area.By performing this stitching process across the entire target area, it is possible to acquire images of a wider area of ​​the target region. Furthermore, lower-resolution image data can be generated from the images of the segmented scan areas or from the stitched images.

[0195] <5. Hardware Configuration Examples> Examples of the hardware configuration of the information processing device 100 according to each embodiment (or each modified example) will be described with reference to Figure 25. Figure 25 is a block diagram showing an example of the schematic configuration of the hardware of the information processing device 100. Various processes performed by the information processing device 100 are realized, for example, through the cooperation of software and the hardware described below.

[0196] As shown in Figure 25, the information processing device 100 includes a CPU (Central Processing Unit) 901, a ROM (Read Only Memory) 902, a RAM (Random Access Memory) 903, and a host bus 904a. The information processing device 100 also includes a bridge 904, an external bus 904b, an interface 905, an input device 906, an output device 907, a storage device 908, a drive 909, a connection port 911, a communication device 913, and a sensor 915. The information processing device 100 may have a processing circuit such as a DSP or ASIC in place of, or together with, the CPU 901.

[0197] The CPU 901 functions as both an arithmetic processing unit and a control unit, controlling the overall operation of the information processing unit 100 according to various programs. The CPU 901 may also be a microprocessor. The ROM 902 stores programs and arithmetic parameters used by the CPU 901. The RAM 903 temporarily stores programs used in the execution of the CPU 901 and parameters that change as needed during its execution. For example, the CPU 901 can embody at least the processing unit 130 and the control unit 150 of the information processing unit 100.

[0198] The CPU 901, ROM 902, and RAM 903 are interconnected by a host bus 904a, which includes the CPU bus. The host bus 904a is connected to an external bus 904b, such as a PCI (Peripheral Component Interconnect / Interface) bus, via a bridge 904. It is not necessary to configure the host bus 904a, bridge 904, and external bus 904b separately; these functions may be implemented on a single bus.

[0199] The input device 906 can be implemented by a device into which information is input by the user, such as a mouse, keyboard, touch panel, button, microphone, switch, and lever. The input device 906 may also be a remote control device utilizing infrared or other radio waves, or an external device such as a mobile phone or PDA that is compatible with the operation of the information processing device 100. Furthermore, the input device 906 may include, for example, an input control circuit that generates an input signal based on the information input by the user using the above-mentioned input means and outputs it to the CPU 901. By operating this input device 906, the user can input various data to the information processing device 100 or instruct it to perform processing operations. The input device 906 can, for example, embody at least the operation unit 160 of the information processing device 100.

[0200] The output device 907 is formed of a device capable of visually or audibly notifying the implementer of the acquired information. Examples of such devices include display devices such as CRT displays, liquid crystal displays, plasma displays, EL displays, and lamps, as well as audio output devices such as speakers and headphones, and printers. The output device 907 may, for example, embody at least the display unit 140 of the information processing device 100.

[0201] The storage device 908 is a device for storing data. The storage device 908 can be implemented by, for example, a magnetic storage device such as an HDD, a semiconductor storage device, an optical storage device, or a magneto-optical storage device. The storage device 908 may also include a storage medium, a recording device for recording data on the storage medium, a reading device for reading data from the storage medium, and a deletion device for deleting data recorded on the storage medium. This storage device 908 stores programs executed by the CPU 901, various data, and various data acquired from external sources. The storage device 908 can, for example, embody at least the storage unit 120 of the information processing device 100.

[0202] Drive 909 is a reader / writer for storage media and is either built into or external to the information processing unit 100. Drive 909 reads information recorded on removable storage media such as magnetic disks, optical disks, magneto-optical disks, or semiconductor memory and outputs it to RAM 903. Drive 909 can also write information to removable storage media.

[0203] Connection port 911 is an interface for connecting to external devices, and is a connection port for external devices that can transmit data via, for example, USB (Universal Serial Bus).

[0204] The communication device 913 is, for example, a communication interface formed by a communication device for connecting to the network 920. The communication device 913 is, for example, a communication card for wired or wireless LAN (Local Area Network), LTE (Long Term Evolution), Bluetooth (registered trademark), or WUSB (Wireless USB). Alternatively, the communication device 913 may be a router for optical communication, a router for ADSL (Asymmetric Digital Subscriber Line), or a modem for various types of communication. This communication device 913 can, for example, send and receive signals to and from the Internet or other communication devices in accordance with a predetermined protocol such as TCP / IP.

[0205] In this embodiment, the sensor 915 includes a sensor capable of acquiring a spectrum (e.g., an image sensor), but it may also include other sensors (e.g., an acceleration sensor, a gyroscope, a geomagnetic sensor, a pressure sensor, a sound sensor, or a distance measuring sensor). The sensor 915 can, for example, embody at least the image acquisition unit 112 of the information processing device 100.

[0206] Network 920 is a wired or wireless transmission path for information transmitted from devices connected to Network 920. For example, Network 920 may include public networks such as the Internet, telephone networks, and satellite communication networks, as well as various LANs (Local Area Networks) and WANs (Wide Area Networks), including Ethernet®. Network 920 may also include dedicated network lines such as IP-VPN (Internet Protocol-Virtual Private Network).

[0207] The above describes an example of a hardware configuration that can realize the functions of the information processing device 100. Each of the above components may be realized using general-purpose materials, or it may be realized using hardware specialized for the function of each component. Therefore, it is possible to change the hardware configuration used as appropriate, depending on the level of technology at the time of implementing this disclosure.

[0208] Furthermore, it is possible to create computer programs to realize each of the functions of the information processing device 100 as described above and implement them on a PC or the like. A computer-readable recording medium containing such computer programs can also be provided. The recording medium includes, for example, magnetic disks, optical disks, magneto-optical disks, and flash memory. Alternatively, the computer programs may be distributed without using a recording medium, for example, via a network.

[0209] <6. Addendum> Furthermore, this technology can also be configured as follows. (1) A simulated image generation unit superimposes an unstained image containing autofluorescence components with a dye tile image in which the reference spectrum of the first fluorescent dye and the imaging noise for each pixel of the unstained image are associated to generate a simulated image. A fluorescence separation unit that separates the components of the first fluorescent dye and the autofluorescent components based on the simulated image and generates a separated image, An evaluation unit for evaluating the degree of separation of the separated images, An information processing device equipped with the following features. (2) The aforementioned dye tile image includes the reference spectrum of the second fluorescent dye in addition to the first fluorescent dye, and is an image in which the individual reference spectra of the first and second fluorescent dyes are associated with the pixel-by-pixel imaging noise of the unstained image. The information processing device described in (1) above. (3) The aforementioned imaging noise is noise that changes depending on the imaging conditions of the unstained image. The information processing device described in (1) or (2) above. (4) The imaging conditions for the unstained image include at least one or all of the following: laser power, gain, and exposure time. The information processing device described in (3) above. (5) The aforementioned color tile image is a group of color tiles having multiple color tiles. An information processing device as described in any one of the above (1) to (4). (6) The individual size of the aforementioned plurality of pigment tiles is the same as the size of a cell. The information processing device described in (5) above. (7) The plurality of colored tiles are arranged in a predetermined color scheme pattern. The information processing device described in (5) or (6) above. (8) The degree of the aforementioned imaging noise is quantified or visualized for each of the aforementioned dye tiles. An information processing device as described in any one of (5) to (7) above. (9) The simulated image generation unit repeatedly arranges the number of pigment tiles specified by the user and generates the pigment tile image. An information processing device as described in any one of the above (5) to (8). (10) The simulated image generation unit mixes multiple dyes to create the dye tile. An information processing device as described in any one of the above (5) to (9). (11) The simulated image generation unit determines the spectral intensity of the dye to be imparted to the autofluorescence intensity of the unstained image. An information processing device as described in any one of the above (1) through (10). (12) The simulated image generation unit superimposes the imaging noise onto the reference spectrum of the first fluorescent dye. An information processing device as described in any one of the above (1) through (11). (13) The aforementioned imaging noise is shot noise. The information processing device described in (12) above. (14) The fluorescence separation unit separates the components of the first fluorescent dye from the autofluorescent components by a color separation calculation that includes at least one of the least squares method, weighted least squares method, or non-negative matrix factorization. An information processing device as described in any one of the above (1) through (13). (15) The evaluation unit described above, A histogram is generated from the aforementioned separated images, From the aforementioned histogram, the signal separation values ​​for the dye and non-dye components are calculated. The degree of separation is evaluated based on the signal separation value. An information processing device as described in any one of the above (1) through (14). (16) The system further includes a recommendation section that recommends the optimal reagent corresponding to the dye specified by the user, based on the degree of separation. An information processing device as described in any one of the above (1) through (15). (17) The recommendation unit generates an image showing a combination of dyes or a combination of dyes and the reagent. The information processing device described in (16) above. (18) The recommended section generates an image showing the combination of antibody and dye. The information processing device described in (16) above. (19) An imaging device that acquires unstained images containing autofluorescence components, An information processing device for processing the aforementioned unstained image, Equipped with, The aforementioned information processing device is A simulated image generation unit superimposes the aforementioned unstained image with a dye tile image in which the reference spectrum of the first fluorescent dye and the imaging noise for each pixel of the unstained image are associated, to generate a simulated image. A fluorescence separation unit that separates the components of the first fluorescent dye and the autofluorescent components based on the simulated image and generates a separated image, An evaluation unit for evaluating the degree of separation of the separated images, A biological sample observation system having the following features. (20) An image generation method comprising superimposing an unstained image containing autofluorescence components with a dye tile image in which the reference spectrum of a first fluorescent dye and the imaging noise for each pixel of the unstained image are associated to generate a simulated image. (twenty one) A biological sample observation system equipped with an information processing device described in any one of the above (1) to (18). (twenty two) An image generation method that generates an image using an information processing device described in any one of the above (1) to (18). [Explanation of Symbols]

[0210] 1 Observation Unit 2 Processing Units 3 Display section 10 Excitation section 10A Fluorescent reagent 11A Reagent identification information 20 Stage 20A Specimen 21 Memory unit 21A Specimen identification information 22 Data calibration unit 23 Image formation unit 30 Spectral imaging unit 30A Fluorescently stained specimen 40 Observation optical system 50 Scanning mechanism 60 Focus mechanism 70 Non-fluorescent observation unit 80 Control unit 100 Information processing device 110 Acquisition unit 111 Information acquisition unit 112 Image acquisition unit 120 Storage unit 121 Information storage unit 122 Image information storage unit 123 Analysis result storage unit 130 Processing unit 131 Analysis unit 131a Simulated image generation unit 131b Fluorescence separation unit 131c Evaluation unit 131d Recommendation unit 132 Image generation unit 140 Display unit 150 Control unit 160 Operation unit 200 Database 500 Fluorescence observation device 1311 Connection unit 1321 Color separation unit 1321a First color separation unit 1321b Second color separation unit 1322 Spectrum extraction unit 5000 Microscope system 5100 Microscope device 5101 Light irradiation unit 5102 Optical unit 5103 Signal acquisition unit 5104 Sample placement section 5110 Control Unit 5120 Information Processing Unit

Claims

1. A simulated image generation unit superimposes an unstained image containing autofluorescence components with a dye tile image in which the reference spectrum of the first fluorescent dye and the imaging noise for each pixel of the unstained image are associated to generate a simulated image. A fluorescence separation unit that separates the components of the first fluorescent dye and the autofluorescent components based on the simulated image and generates a separated image, An evaluation unit for evaluating the degree of separation of the separated images, An information processing device equipped with the following features.

2. The aforementioned dye tile image includes the reference spectrum of the second fluorescent dye in addition to the first fluorescent dye, and is an image in which the individual reference spectra of the first and second fluorescent dyes are associated with the pixel-by-pixel imaging noise of the unstained image. The information processing apparatus according to claim 1.

3. The aforementioned imaging noise is noise that changes depending on the imaging conditions of the unstained image. The information processing apparatus according to claim 1.

4. The imaging conditions for the unstained image include at least one or all of the following: laser power, gain, and exposure time. The information processing apparatus according to claim 3.

5. The aforementioned color tile image is a group of color tiles having multiple color tiles. The information processing apparatus according to claim 1.

6. The individual size of the aforementioned plurality of pigment tiles is the same as the size of a cell. The information processing apparatus according to claim 5.

7. The plurality of colored tiles are arranged in a predetermined color scheme pattern. The information processing apparatus according to claim 5.

8. The degree of the aforementioned imaging noise is quantified or visualized for each of the aforementioned dye tiles. The information processing apparatus according to claim 5.

9. The simulated image generation unit repeatedly arranges the number of pigment tiles specified by the user and generates the pigment tile image. The information processing apparatus according to claim 5.

10. The simulated image generation unit mixes multiple dyes to create the dye tile. The information processing apparatus according to claim 5.

11. The simulated image generation unit determines the spectral intensity of the dye to be imparted to the autofluorescence intensity of the unstained image. The information processing apparatus according to claim 1.

12. The simulated image generation unit superimposes the imaging noise onto the reference spectrum of the first fluorescent dye. The information processing apparatus according to claim 1.

13. The aforementioned imaging noise is shot noise. The information processing apparatus according to claim 12.

14. The fluorescence separation unit separates the components of the first fluorescent dye from the autofluorescent components by a color separation calculation that includes at least one of the least squares method, weighted least squares method, or non-negative matrix factorization. The information processing apparatus according to claim 1.

15. The evaluation unit, A histogram is generated from the aforementioned separated images, From the aforementioned histogram, the signal separation values ​​for the dye and non-dye components are calculated. The degree of separation is evaluated based on the signal separation value. The information processing apparatus according to claim 1.

16. The system further includes a recommendation section that recommends the optimal reagent corresponding to the dye specified by the user, based on the degree of separation. The information processing apparatus according to claim 1.

17. The recommendation unit generates an image showing a combination of dyes or a combination of dyes and the reagent. The information processing apparatus according to claim 16.

18. The recommended section generates an image showing the combination of antibody and dye. The information processing apparatus according to claim 16.

19. An imaging device that acquires unstained images containing autofluorescence components, An information processing device for processing the aforementioned unstained image, Equipped with, The aforementioned information processing device is A simulated image generation unit superimposes the unstained image with a dye tile image in which the reference spectrum of the first fluorescent dye and the imaging noise for each pixel of the unstained image are associated, to generate a simulated image. A fluorescence separation unit that separates the components of the first fluorescent dye and the autofluorescent components based on the simulated image and generates a separated image, An evaluation unit for evaluating the degree of separation of the separated images, A biological sample observation system having the following features.

20. An image generation method comprising superimposing an unstained image containing autofluorescence components with a dye tile image in which the reference spectrum of a first fluorescent dye and the imaging noise for each pixel of the unstained image are associated to generate a simulated image.

Images