WO2020209217A1 - 画像処理システム、画像処理方法及びプログラム - Google Patents

画像処理システム、画像処理方法及びプログラム Download PDF

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Publication number
WO2020209217A1
WO2020209217A1 PCT/JP2020/015483 JP2020015483W WO2020209217A1 WO 2020209217 A1 WO2020209217 A1 WO 2020209217A1 JP 2020015483 W JP2020015483 W JP 2020015483W WO 2020209217 A1 WO2020209217 A1 WO 2020209217A1
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Prior art keywords
image
region
tissue
processing system
image processing
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English (en)
French (fr)
Japanese (ja)
Inventor
武寿 磯田
北斗 田中
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Konica Minolta Inc
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Konica Minolta Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T1/00—General purpose image data processing

Definitions

  • the present invention relates to an image processing system, an image processing method and a program.
  • the virtual microscope is a system that digitizes an image observed by an optical microscope and allows the tissue sample to be observed on a display as if the optical microscope was actually used (see, for example, Patent Document 1).
  • the entire tissue sample on the slide glass is photographed, the obtained image is converted into digital data and saved in a database, and observation is performed using viewer software installed on a personal computer or the like.
  • the reason why it is called a virtual microscope is that it can be observed while performing operations such as moving up and down, left and right, and scaling, as in the case of observation using an optical microscope.
  • Digital image data of the entire tissue specimen called a whole slide image (WSI)
  • WSSI whole slide image
  • the stored digital image data can be accessed via the Internet or the like, for example, a rapid pathological diagnosis by a pathologist at a remote location and a rare tissue specimen can be made available to anyone.
  • the present invention has been made in view of the above problems, and an object of the present invention is to provide an image processing system, an image processing method, and a program capable of efficiently performing high-precision analysis.
  • the image processing system of the present invention An acquisition method for acquiring a tissue image of a tissue sample, An extraction means for extracting an image of a specific region to be rephotographed by the acquisition means from the tissue image acquired by the acquisition means, and an extraction means. Have.
  • the image processing method of the present invention The acquisition process to acquire the tissue image of the tissue sample, An extraction step of extracting an image of a specific region for rephotographing from the tissue image acquired by the acquisition step, and an extraction step. Have.
  • the program of the present invention Computer, An acquisition method for acquiring a tissue image of a tissue sample, An extraction means for extracting an image of a specific region to be rephotographed by the acquisition means from the tissue image acquired by the acquisition means, and an extraction means. It is a program to function as.
  • FIG. 1 shows a schematic configuration of the image processing system 1 (WSI creation system) in the present invention.
  • the image processing system 1 includes a microscope device 10, a control device 60, a display device 70, and a database 80.
  • the microscope device 10 includes a first image acquisition unit 20, a second image acquisition unit 30, and a stage 40.
  • the tissue specimen 50 after immunostaining is placed in the stage 40.
  • the tissue sample 50 is an example of a biological sample.
  • FIG. 2 shows a schematic configuration of the first image acquisition unit 20.
  • the first image acquisition unit 20 acquires a bright-field image of the tissue sample 50.
  • the first image acquisition unit 20 includes a bright field light source 21, a first image sensor 22, and a light guide lens 23.
  • the bright-field light source 21 is a light source that irradiates the tissue sample 50 with light for generating a light image for acquiring a bright-field image, and is installed so as to irradiate the light from below the stage 40.
  • the tissue sample 50 is irradiated by the bright-field light source 21 and an optical image is generated, the optical image is guided to the first image sensor 22 via the light guide lens 23, and the light image of the tissue sample 50 is brightened by the first image sensor 22.
  • a field image is taken.
  • the first image sensor 22 is an image sensor such as a two-dimensional CCD sensor capable of acquiring a two-dimensional image of the optical image of the tissue specimen 50.
  • FIG. 3 shows a schematic configuration of the second image acquisition unit 30.
  • the second image acquisition unit 30 acquires a fluorescence image of the tissue sample 50.
  • the second image acquisition unit 30 includes a transmission light source 31, an excitation light source 32, a second image sensor 33, an objective lens 34, a fluorescence cube 35, and an imaging lens 36.
  • the fluorescent cube 35 includes an excitation filter 351, a dichroic mirror 352, and an absorption filter 353.
  • the transmission light source 31 is a light source used when acquiring a transmission observation image of the tissue specimen 50, and is installed so as to irradiate light from below the stage 40.
  • the excitation light source 32 is a lamp that emits excitation light by a light source such as a discharge tube.
  • the excitation filter 351 is a filter that transmits only excitation light.
  • the dichroic mirror 352 is a mirror that reflects or transmits light having a predetermined wavelength as a boundary, and here, it reflects excitation light and transmits fluorescence.
  • the absorption filter 353 is a filter that blocks excitation light and transmits only fluorescence.
  • the excitation light passes through the excitation filter 351 and is reflected by the dichroic mirror 352, passes through the objective lens 34, and irradiates the tissue sample 50.
  • fluorescence is emitted from the tissue specimen 50, and the fluorescence is focused by the objective lens 34 and transmitted through the dichroic mirror 352 and the absorption filter 353.
  • the fluorescence is guided to the second image sensor 33 as a fluorescence image via the image pickup lens 36, and is imaged by the second image sensor 33.
  • the objective lens 34 includes a low-magnification objective lens (for example, 20 times) and a high-magnification objective lens (for example, 40 times).
  • the second image sensor 33 is an image sensor such as a one-dimensional CCD camera capable of acquiring a one-dimensional image or a two-dimensional image having a predetermined direction as the longitudinal direction, and acquires a high-resolution fluorescent image of the tissue sample 50. be able to.
  • a control device 60 for controlling these is connected to the microscope device 10.
  • the control device 60 includes a control unit (acquisition means, extraction means, detection means) 61, a storage unit 62, an image processing unit 63, and a communication unit 64.
  • the control unit 61 is configured to include a CPU (Central Processing Unit), a RAM (Random Access Memory), and the like, and executes various processes in cooperation with various programs stored in the storage unit 62 to execute various processes of the microscope device 10. Control the operation comprehensively.
  • the control unit 61 is connected to the stage 40 and can control the ascent and descent of the stage 40 to control the focusing position (Z coordinate) of the tissue sample 50 installed on the stage 40. Further, the control unit 61 is connected to the first image acquisition unit 20 and controls the bright field light source 21 and the first image sensor 22 to take a bright field image. Further, it is connected to the control unit 61 second image acquisition unit 30 and controls the transmission light source 31, the excitation light source 32, and the second image sensor 33 to take a fluorescence image.
  • a CPU Central Processing Unit
  • RAM Random Access Memory
  • the storage unit 62 is composed of, for example, an HDD (Hard Disk Drive), a semiconductor non-volatile memory, or the like.
  • the storage unit 62 stores a program for taking a bright field image and a fluorescent image.
  • the image processing unit 63 performs image processing on the fluorescent image taken by the microscope device 10 to create a hall slide image (WSI). As will be described later, according to the instruction of the control unit 61, the captured partial images are combined to create an entire image of the tissue sample 50, and the image data is A / D converted into a digital image to create a WSI. .. In addition, based on the created WSI, a fluorescence brightness map used for quantitative analysis of the target substance is created.
  • WSI hall slide image
  • the communication unit 64 is an interface for transmitting and receiving data to and from an external device such as a personal computer.
  • a user who wants to refer to the WSI can read the WSI stored in the database 80 into a personal computer or the like via the communication unit 64 and observe it on the display.
  • a display device 70 is connected to the control device 60.
  • the display device 70 is configured to include, for example, a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display), and displays various screens according to an instruction of a display signal input from the control unit 61. ..
  • the display device 70 functions as an output means for outputting a captured fluorescent image or the like.
  • a database 80 is further connected to the control device 60.
  • the database 80 includes, for example, an HDD (Hard Disk Drive) and stores the WSI synthesized by the image processing unit 63.
  • the WSI is stored in the database 80 as described above.
  • the storage area is not limited to the database 80, and the database 80 is not provided. There may be.
  • the WSI may be stored in the storage unit 62, or may be stored in an external server (not shown) to form a database.
  • tissue sample 50 is a tissue section containing the target substance and is stained with an immunostaining agent, and the stained tissue sample 50 is placed on the stage 40.
  • Target substance is a substance that is present in a tissue section and is a target of immunostaining using a fluorescent label mainly for detection or quantification from the viewpoint of pathological diagnosis.
  • it is a protein (antigen).
  • Typical target substances include biological substances that are expressed in the cell membranes of various cancer tissues and can be used as biomarkers for proteins, RNA, and the like.
  • the target substance may be a substance introduced from outside the body, such as a drug.
  • units smaller than proteins such as peptides can be immunostained.
  • Immunostaining agent antibody-fluorescent nanoparticle conjugate
  • the primary antibody and fluorescent nanoparticles indirectly use, that is, an antigen-antibody reaction, etc., other than covalent bonds. It is preferable to use a complex linked by the binding of.
  • a complex in which fluorescent nanoparticles are directly linked to the primary antibody or the secondary antibody can also be used as the immunostaining agent.
  • immunostaining agent examples include [primary antibody against the target substance] ... [antibody against the primary antibody (secondary antibody)] to [fluorescent nanoparticles].
  • “...” Indicates that the bond is bound by an antigen-antibody reaction, and the mode of binding indicated by “ ⁇ ” is not particularly limited.
  • covalent bond, ionic bond, hydrogen bond, coordination bond, antigen-antibody bond examples thereof include biotin avidin reaction, physical adsorption, and chemisorption, and may be mediated by a linker molecule if necessary.
  • an antibody (IgG) that specifically recognizes and binds to the target substance as an antigen can be used.
  • an anti-HER2 antibody can be used
  • HER3 is the target substance
  • an anti-HER3 antibody can be used.
  • an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
  • Both the primary antibody and the secondary antibody may be polyclonal antibodies, but monoclonal antibodies are preferable from the viewpoint of quantitative stability.
  • the type of animal (immunized animal) that produces an antibody is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like as in the conventional case.
  • Fluorescent nanoparticles are nano-sized particles that fluoresce when irradiated with excitation light, and emit fluorescence of sufficient intensity to represent the target substance as a bright spot one by one. Fluorescent particles.
  • fluorescent nanoparticles in the present invention, fluorescent substance integrated nanoparticles (PID: Phosphor Integrated Dot nanoparticles) are used.
  • Fluorescent substance-accumulated nanoparticles is based on particles made of an organic substance or an inorganic substance, and a plurality of fluorescent substances (for example, the quantum dots, organic fluorescent dyes, etc.) are contained therein and /. Alternatively, it is a nano-sized particle having a structure adsorbed on its surface.
  • fluorescent substance used for PID shows emission of visible to near infrared light having a wavelength in the range of 400 to 900 nm when excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm. It is preferable that the mother body and the fluorescent substance have substituents or sites having opposite charges, and an electrostatic interaction acts.
  • the average particle size of the PID used in the present invention is not particularly limited, but one having a diameter of about 30 to 800 nm can be used.
  • the average particle size is more preferably in the range of 40 to 500 nm.
  • the reason why the average particle size is set to 40 to 500 nm is that if it is less than 40 nm, an expensive detection system is required, and if it exceeds 500 nm, the quantification range is narrowed due to the physical size. ..
  • the average particle size is determined by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number of particles, and using each measured value as the area of a circle. Can be obtained as.
  • the organic substances are generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin.
  • Resins generally classified as thermoplastic resins such as styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); poly Other resins such as lactic acid; polysaccharides can be exemplified.
  • the inorganic substance in the mother body include silica and glass.
  • Quantum dot integrated nanoparticles have a structure in which the quantum dots are contained in the mother body and / or adsorbed on the surface thereof. When the quantum dots are contained in the mother body, the quantum dots need only be dispersed inside the mother body, and may or may not be chemically bonded to the mother body itself.
  • quantum dots semiconductor nanoparticles containing a II-VI group compound, a III-V group compound, or an IV group element are used.
  • CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge and the like can be mentioned.
  • a quantum dot having the above quantum dot as a core and a shell provided on the core.
  • the core is CdSe and the shell is ZnS
  • CdSe / ZnS when the core is CdSe and the shell is ZnS, it is described as CdSe / ZnS.
  • CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO2, Si / ZnS, Ge / GeO2, Ge / ZnS and the like can be used, but are not limited thereto.
  • the quantum dots may be surface-treated with an organic polymer or the like.
  • an organic polymer or the like for example, CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like can be mentioned.
  • Quantum dot integrated nanoparticles can be produced by a known method.
  • the silica nanoparticles containing quantum dots can be synthesized with reference to the synthesis of CdTe-encapsulating silica nanoparticles described in New Journal of Chemistry, Vol. 33, p. 561 (2009).
  • the silica nanoparticles encapsulating quantum dots refer to the synthesis of silica nanoparticles in which particles of CdSe / ZnS capped with 5-amino-1-pentanol and APS described on page 2670 (2009) of Chemical Communication are integrated on the surface. Can be synthesized into.
  • Polymer nanoparticles containing quantum dots can be produced by using the method of impregnating polystyrene nanoparticles with quantum dots described in Nature Biotechnology, Vol. 19, p. 631 (2001).
  • the fluorescent dye-accumulated nanoparticles have a structure in which a fluorescent dye is contained in the mother body and / or is adsorbed on the surface thereof.
  • the fluorescent dye include organic fluorescent dyes such as rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyromesene-based dye molecules. it can.
  • Alexa Fluor registered trademark, manufactured by Invigen
  • BODIPY registered trademark, manufactured by Invigen
  • Cy registered trademark, manufactured by GE Healthcare
  • HiLite registered trademark, Trademarks, Anaspec
  • DyLight registered trademark, Thermoscientific
  • ATTO registered trademark, ATTO-TEC
  • MFP registered trademark, Mobitec
  • Dye molecule CF (registered trademark, manufactured by Biotium) dye molecule
  • DY registered trademark, manufactured by DYOMICS
  • CAL registered trademark, manufactured by BioSearch Technologies
  • the fluorescent dye When the fluorescent dye is encapsulated in the mother body, the fluorescent dye may or may not be chemically bonded to the mother body itself as long as it is dispersed inside the mother body.
  • Fluorescent dye-accumulated nanoparticles can be produced by a known method.
  • silica nanoparticles containing a fluorescent dye can be synthesized with reference to the synthesis of FITC-encapsulating silica particles described in Langmuir Vol. 8, p. 2921 (1992).
  • a desired fluorescent dye instead of FITC
  • various fluorescent dye-accumulated nanoparticles can be synthesized.
  • Polystyrene nanoparticles containing a fluorescent dye can be obtained by a copolymerization method using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982) or polystyrene nanoparticles described in US Pat. No. 5,326,692 (1992). It can be prepared by using a method of impregnating particles with a fluorescent dye.
  • the method for preparing a tissue section to which this staining method can be applied (also referred to simply as a “section” and including a section such as a pathological section) is not particularly limited, and a tissue section prepared by a known procedure can be used.
  • Specimen preparation step (5.1) Specimen preparation step (5.1.1) Deparaffinization treatment
  • the section is immersed in a container containing xylene to remove paraffin.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, xylene may be replaced during immersion.
  • the section is immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, ethanol may be replaced during immersion.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, the water may be replaced during immersion.
  • the activation treatment of the target substance is performed according to a known method.
  • the activation conditions are not particularly specified, but the activation solution is 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, and 0.1 M Tris-hydrochloric acid buffer.
  • a liquid or the like can be used.
  • the pH condition is such that a signal is output from the range of pH 2.0 to 13.0 depending on the tissue section to be used and the tissue roughness is such that the signal can be evaluated. Normally, the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
  • an autoclave As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.
  • the section after activation treatment is immersed in a container containing PBS and washed.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, PBS may be replaced during immersion.
  • (5.2) Immunostaining Step in order to stain the target substance, a solution of an immunostaining agent containing fluorescent nanoparticles having a site capable of directly or indirectly binding to the target substance is applied to a section. Place and react with the target substance.
  • the solution of the immunostaining agent used in the immunostaining step may be prepared in advance before this step.
  • immunostaining is performed with a plurality of immunostaining agents corresponding to the target substances.
  • the plurality of immunostaining agents used in this case may be those containing at least one immunostaining agent using PID (PID staining agent), and if the antibody and the fluorescent substance (fluorescence wavelength) are different from each other, PID staining may be performed. It is also possible to detect multiple target substances by multiple staining using multiple agents or by combining PID staining agents with immunostaining agents using fluorescent labels such as organic fluorescent substances and quantum dots. is there. In this case, a solution of each immunostaining agent is prepared, placed on a section, and reacted with the target substance.
  • each immunostaining agent may be mixed in advance when the solution is placed on the section, or separately. It may be placed in order.
  • the conditions for performing the immunostaining step should be appropriately adjusted so as to obtain an appropriate signal according to the conventional immunostaining method. Can be done.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the reaction time is preferably 30 minutes or more and 24 hours or less. It is preferable to drop a known blocking agent such as PBS containing BSA or a surfactant such as Tween 20 before performing the treatment as described above.
  • tissue specimen after the immunostaining step is subjected to treatments such as immobilization / dehydration, permeation, and encapsulation so as to be suitable for observation.
  • the tissue section may be immersed in a fixing treatment solution (crosslinking agent such as formalin, paraformaldehyde, glutaaldehyde, acetone, ethanol, methanol).
  • the permeation treatment may be performed by immersing the tissue section that has undergone the immobilization / dehydration treatment in a permeation solution (xylene or the like).
  • the tissue section that has undergone the permeation treatment may be immersed in the encapsulation liquid.
  • the conditions for performing these treatments for example, the temperature and the immersion time when the tissue section is immersed in a predetermined treatment solution, may be appropriately adjusted so as to obtain an appropriate signal according to the conventional immunostaining method. it can.
  • Bright-field morphological observation staining step Separate from the immunostaining step, morphological observation staining is performed so that the morphology of cells, tissues, organs, etc. can be observed in the bright-field.
  • the morphological observation dyeing step can be performed according to a conventional method.
  • staining with eosin in which the cytoplasm, interstitium, various fibers, erythrocytes, and keratinocytes are stained in red to deep red, is standardly used.
  • hematoxylin which stains the cell nucleus, lime, cartilage tissue, bacteria, and mucus in blue-blue to pale blue
  • the method of performing these two stainings at the same time is hematoxylin / eosin staining. Known as (HE staining)).
  • HE staining HE staining
  • FIG. 4 shows the overall flow of the operation of the image processing system 1 in the present invention.
  • the operations of the image processing system 1 include a focusing step (step S101), a low-magnification imaging step (step S102), a specific region extraction step (step S103), a high-magnification imaging step (step S104), and , Has an analysis step (step S105).
  • step S101 a focusing step
  • step S102 low-magnification imaging step
  • step S103 a specific region extraction step
  • step S104 a high-magnification imaging step
  • Has an analysis step step S105
  • the first image acquisition unit 20 acquires a bright-field image of the tissue sample 50, sets an imaging region to be created for WSI based on the bright-field image, and performs focusing based on the bright-field image. Further, the tissue sample 50 is fluorescently labeled with excitation light by the second image acquisition unit 30, and the PID is irradiated with excitation light, and more rigorous focusing is performed with reference to the fluorescent bright spot of the detected PID.
  • the control unit 61 controls the first image acquisition unit 20 to acquire a bright field image for focusing of the entire slide glass (step S1).
  • This bright-field image is used for setting high-resolution imaging conditions such as a fluorescence image described later, and is a low-magnification image using a low-magnification objective lens.
  • the control unit 61 sets an imaging region R including the tissue sample 50 as shown in FIG. 6 (step S2). Specifically, the control unit 61 binarizes the entire image of the tissue sample 50 depending on the presence or absence of the tissue sample 50, and detects the region where the tissue sample 50 exists in each of the X-axis direction and the Y-axis direction.
  • the imaging region R is determined.
  • the imaging region R may be manually set on the display device 70 while the user is observing the bright field image of the entire tissue specimen 50, but it is preferably set automatically.
  • the tissue sample 50 is focused based on the bright field image. Focusing based on the bright-field image may be performed manually by the user, but it is preferably performed automatically under the control of the control unit 61.
  • a method of automatically creating a focus map under the control of the control unit 61 and performing focusing will be described.
  • the first focus measurement position P1 is set on the imaging region R (step S3).
  • the control unit 61 divides the imaging region R into the X-axis direction and the Y-axis direction to set a small region, and obtains the XY coordinates of each small region.
  • the XY coordinates are the center coordinates of each subregion, but the coordinates are not limited to this, and for example, the coordinates of the upper left end of each subregion can be set as the XY coordinates.
  • the control unit 61 assigns numbers such as 1, 2, 3, ... In each of the X-axis direction and the Y-axis direction to each small area, and sets the array number.
  • the control unit 61 sets the first focus measurement position P1 for each small area.
  • P1 is the central coordinate position of each small area, but the present invention is not limited to this, and for example, the upper left end of each small area can be set as the first focus measurement position P1.
  • the tissue sample 50 may not exist on the center coordinates, for example, as in the region of the sequence number (1, 1) in FIG. In this case, the first focus measurement position P1 can be moved to an arbitrary coordinate on the tissue sample 50.
  • step S4 focusing is performed on the first focus measurement position P1 in each small area.
  • the control unit 61 adjusts the optical axis position to the first focus measurement position P1 while moving the stage 40 in the XY direction, and focuses the bright field by actual measurement with respect to each first focus measurement position P1. Find the position (Z coordinate).
  • the control unit 61 Based on the bright field focus position obtained in this way, the control unit 61 creates a focus map as shown in FIG. 8 (step S5).
  • the focus map stores the array number of each small area and the corresponding stage coordinates.
  • the stage coordinates correspond to the center coordinates of each small area for the X-axis and the Y-axis, and the bright field focusing position for the Z-axis. This completes the focusing of the tissue sample 50 based on the bright-field image.
  • step S6 onward focusing is performed on the PID bright spot based on the focusing information obtained based on the bright field image. That is, as described above, the bright-field focusing position of the tissue sample 50 based on the bright-field image is specified by the processes of steps S1 to S3, and the PID bright spot is further focused based on this. By performing the above, a more precisely focused fluorescence image can be obtained.
  • a fluorescence image of the PID for focusing is acquired (step S6). That is, the control unit 61 controls the excitation light source 32 to irradiate the tissue sample 50 with the excitation light of the PID, and acquires the fluorescence image of the PID by the second image sensor 33.
  • the fluorescence bright spot of an arbitrary PID on the obtained fluorescence image is selected, and the second focus measurement position P2 is set (step S7).
  • the user manually sets the second focus measurement position P2, and as shown in FIG. 9, one or a plurality of second focus measurement positions P2 on the tissue sample 50 are set.
  • the second focus measurement position P2 may be automatically set.
  • step S8 focusing is performed on the set second focus measurement position P2 (step S8).
  • the control unit 61 adjusts the optical axis position to the second focus measurement position P2 while moving the stage 40 in the XY direction, and refers to the focus position of the focus map created in step 3. , Finely adjust in the Z coordinate direction to obtain the fluorescence focusing position (Z coordinate) with respect to the second focus measurement position P2.
  • control unit 61 modifies the focus map created in step S3 by using the obtained new focus position (step S9). As described above, the focusing on the tissue sample 50 is completed.
  • the focusing method described above is merely an example, and the method applicable to the present invention is not limited to this.
  • the fluorescently labeled body labeled on the tissue specimen 50 is excited (step S10).
  • the control unit 61 controls the excitation light source 32 to irradiate the tissue specimen 50 with excitation light that excites the labeled PID.
  • a partial image of the tissue sample 50 is acquired (step S11).
  • the control unit 61 moves and controls the stage 40, and controls the second image acquisition unit 30 to acquire a partial fluorescence image. That is, the optical axis position and the focusing position are moved to the XYZ coordinates indicated by the stage coordinates stored in the focus map, and the second image sensor 33 is controlled to capture an image for each small area.
  • a high-magnification objective lens as the objective lens 34, a high-resolution image can be acquired.
  • a strip-shaped scan image as shown in FIG. 11 is acquired as a partial image.
  • imaging of the tissue specimen 50 is started from the upper left end.
  • the control unit 61 irradiates the excitation light and scans the image pickup position by the second image pickup element 33 while moving in the positive direction of the Y axis of the tissue section 51 to acquire a partial image A.
  • the control unit 61 moves the image pickup position by the second image pickup element 33 in the positive direction of the X axis to acquire the partial image B.
  • the partial images are acquired in the order of the partial image C, ..., The partial image N, the imaging is completed.
  • control unit 61 controls the image processing unit 63 as the creating means to synthesize the captured partial images to create the entire fluorescent image of the imaging region R (step S12). That is, by arranging and pasting the partial images A to N in the X-axis direction, a high-resolution fluorescence image of the entire tissue sample 50 can be obtained. Further, the image processing unit 63 A / D-converts the entire fluorescent image of the obtained imaging region R into a digital image (step S13). With the above, the creation of WSI is completed.
  • the created WSI is stored in the database 80 as a storage means.
  • a user who wants to refer to the WSI can read the image data into a personal computer or the like via the communication unit 64 and observe it on the display.
  • the WSI of the bright field image can also be created in the same manner.
  • whether the WSI is a bright field image or a fluorescent image is not particularly limited, and (1) an image in which the cell morphology is visualized and (2) a target substance are visualized as necessary. It is sufficient that there is at least one of the images shown.
  • Examples of the image in which the cell morphology is visualized include an image in which the cell nucleus is visualized. Even if it is a fluorescent image, for example, if the cell nucleus is visualized, it can be used as an image showing the morphology of the cell.
  • Examples of the image in which the target substance is visualized include the above-mentioned image of the fluorescence bright spot of PID.
  • the specific region extraction step uses the created WSI (that is, at least one of (1) an image in which the cell morphology is visualized and (2) an image in which the target substance is visualized). This is a process of extracting a specific region of interest.
  • the "specific region” is not limited as long as it is a region useful for pathological diagnosis, and may include a normal tissue region as well as a lesion portion such as a cancer region.
  • a region in which the effect of the drug appears or a region in which the drug is present can also be included in a specific region because it is considered to be useful for pathological diagnosis.
  • a region specified by the color of the bright-field image or a region specified by the shade of the fluorescence image may also be useful, and the size of each region can be appropriately specified, preferably about 100 um square. Is.
  • specifying a region using the number of fluorescent bright spots as an index may be useful for pathological diagnosis, and identifying and extracting a region having a large number, a region having a medium number, and a region having a small number, respectively. You can also. Areas with a large number are sometimes called hot spots, and areas with a small number are sometimes called cold spots.
  • a plurality of methods can be considered for the extraction process of a specific area.
  • the first to third three processes will be described by way of example, but the present invention is not limited to this, and measures can be taken to achieve the purpose of extraction.
  • a process of generating a frequency spectrum by DFT processing (high-pass filter), performing reduction processing on the generated ROI information, and extracting a region in which the average luminance value is equal to or higher than a preset first threshold value is explained using as a means of achieving the purpose, but the present invention is not limited to this.
  • the control unit 61 reads a predetermined fluorescence image (WSI) from the database 80 with image processing software (step S21), and performs an autofluorescence suppression process on the read fluorescence image (step S22). Specifically, the control unit 61 generates a frequency spectrum by DFT processing (high-pass filter), and multiplies the frequency spectrum by a high-pass filter image. Next, a low-frequency component-suppressed image is generated by IDFT. Since the size of the fluorescent image is huge, it is preferable to divide and process the fluorescent image according to the memory load used when the processing by DFT is executed. That is, the above-mentioned processing may be executed for each of the divided images of the fluorescence image, and finally they may be combined.
  • DFT processing high-pass filter
  • the fluorescence signal by PID and the fluorescence signal by autofluorescence have different spatial frequency profiles.
  • autofluorescence contains a large amount of low-frequency components
  • PID fluorescence has a steep peak shape
  • low-frequency components are less than autofluorescence.
  • the frequency spectrum can be reversibly converted into a spatial signal by IDFT / IFFT, and as described above, the frequency spectrum in a state where the brightness component due to autofluorescence is selectively suppressed is inversely transformed into spatial information by IDFT / IFFT, thereby autofluorescent. A fluorescent image with suppressed is obtained.
  • control unit 61 sets the analysis target region (excludes non-specific fluorescence) (step S23). This is a process for analyzing a region one size smaller than the peripheral edge in the large section because the fluorescence at the peripheral edge of the tissue may become strong due to uneven staining of the automatic staining machine.
  • the control unit 61 specifies the search start point at the end of the tissue section image by a conventionally known method, and obtains the ROI information of the edge of the section by the contour tracking algorithm that allows the pixel value change within a specific range. Generate. As a conventionally known method, for example, Imagej's WandTool (National Institutes of Health, MD, USA) can be mentioned. Next, the control unit 61 performs a reduction process of several hundred pixels (about one field of view of the microscope) on the generated ROI information (that is, the designated pixel area is reduced inward). Next, the control unit 61 excludes the non-analysis target region by superimposing the generated ROI information on the HPF image and deleting the fluorescence pixel value outside the region (luminance is zero).
  • the analysis target area is narrowed down.
  • narrowing down the area is not essential in cases where non-specific uneven staining around the tissue does not occur.
  • the autofluorescent region of cells without nuclei such as erythrocytes may be excluded as the region not to be analyzed.
  • various known methods can be applied in addition to the above-mentioned method of deleting the fluorescence pixel value outside the region (luminance is zero).
  • control unit 61 cuts out the analysis target region (the region surrounded by the broken line in FIG. 13 (A)) from the autofluorescence suppressed image to generate the analysis target image ( Step S24).
  • the control unit 61 scans the image to be analyzed in a rectangular region (spot) 200 having the same size as the field of view of the microscope, and calculates the average luminance value for each region ( Step S25).
  • the scanning process is started from a predetermined point set in advance, and scans the entire analysis target image evenly.
  • the control unit 61 extracts a region whose average luminance value is equal to or higher than a preset first threshold value as a hot spot (specific region) 300 (step S26).
  • the top N elements having a high average luminance value may be extracted as hot spots. In this case, it is preferable to extract the top N spots from the areas that do not overlap each other.
  • the former can compare the expression level of the target substance and the distribution of spots among the samples by unifying the threshold values. The latter makes it possible to extract a target spot in a sample even in a sample having a significantly different order of expression level.
  • a magnified image magnified at a higher magnification than the photographing magnification of the tissue image 50 is taken with respect to the presented hot spot 300, and a pathological diagnosis is performed.
  • hot spots whose average luminance value is equal to or greater than the first threshold value as a specific area
  • cold spots are areas where the average luminance value is smaller than the first threshold value and less than the second threshold value. It may be extracted as (specific area).
  • a standard value region (standard spot) of the entire analysis target can be extracted.
  • the standard value region can be defined as, for example, a region between a first threshold and a second threshold smaller than the first threshold.
  • the hot spot, the cold spot, and the standard spot may be extracted from the histogram of the average luminance value without using the first threshold value and the second threshold value.
  • the first threshold value and the second threshold value may be adaptively set based on the extracted standard spot.
  • the control unit 61 reads a predetermined fluorescence image (WSI) from the database 80 with image processing software, and displays the intensity of the fluorescence brightness as a fluorescence brightness map by expressing it by a shade of color or the like. Specifically, the control unit 61 calculates the average value of the fluorescence brightness for each predetermined pixel, reads out the set value of the color density corresponding to the fluorescence brightness stored in the storage unit 62, and associates it with the WSI. Is subjected to image processing.
  • the pixel unit can be arbitrarily specified by the user.
  • the control unit 61 measures the detection sensitivity of the target substance by the PID and the fluorescent dye as the number of bright spots, and detects the number of bright spots per cell. Then, cells having a number of bright spots equal to or greater than the first threshold value (for example, 25) are extracted, and among the extracted cells, cells having a center of gravity within 100 ⁇ m are connected by a line. Next, the cells connected by the line are assigned to one cluster, and the clusters having a predetermined number (for example, 20) or more of cells are extracted as a specific region (hot spot). As a result, as shown in FIG. 14, the hot spot 300 is extracted. Generally, about 20 to 100 cell clusters are hot spots.
  • the hot spot it was decided to extract cells having a number of bright spots equal to or greater than the first threshold value, and to extract clusters having a predetermined number or more of extracted cells as a specific region (hot spot).
  • Cells smaller than the threshold of 1 and less than the second threshold may be extracted, and clusters in which the number of extracted cells is a predetermined number or more may be extracted as a specific region (cold spot).
  • a standard value region (standard spot) of the entire analysis target can be extracted.
  • the standard value region can be defined as, for example, a region between a first threshold and a second threshold smaller than the first threshold.
  • the hot spot, the cold spot, and the standard spot may be extracted from the histogram of the number of bright spots without using the first threshold value and the second threshold value.
  • the method for creating cell clusters as described above is applied as the creation of clusters based on another index after extracting a specific region based on one index with a low-magnification (low-sensitivity) whole slide scanner. You can also do it. That is, it is the extraction of a specific region of another viewpoint existing in the specific region.
  • the control unit 61 reads a predetermined bright-field image (WSI) from the database 80 with image processing software (step S31: acquisition step), and extracts the read bright-field image from the nuclear peripheral region.
  • the bright field image is an image in which the cell nucleus is stained by HE staining or the like as shown in FIG. 16 (A). With respect to this image, the cell nucleus region can be extracted by color separation of hematoxylin (H) staining to generate a nuclear region mask image as shown in FIG. 16 (B).
  • the control unit 61 generates an image obtained by extracting the hematoxylin (H) dye from the bright field image, and performs binarization and region expansion processing to generate a nuclear region mask image. Then, the peripheral region of the nucleus is extracted from the mask image of the nuclear region.
  • control unit 61 performs a calculation process of the density feature amount of the extracted peripheral region, and calculates the kernel density feature amount data for each of a plurality of divided regions of the image (step S33). Specifically, for example, a kernel density heat map showing the high and low densities of the binarized kernel region mask image by color shading, a kernel density histogram, etc. are created, and the kernel density feature amount for each region of the image is calculated. calculate.
  • control unit 61 reads a predetermined fluorescence image (WSI) from the database 80 with image processing software (step S34: acquisition step), and from the read fluorescence image, in the same manner as in steps S21 to S24 described above. , As shown in FIG. 16C, an image to be analyzed is created (step S35).
  • WSI predetermined fluorescence image
  • control unit 61 performs a process of calculating the expression feature amount of the target substance in the target region from the analysis target image, and calculates the expression feature amount data of the target substance for each of a plurality of divided regions of the image (step S36). ). Specifically, the control unit 61 creates an expression level heat map or the like in which the average luminance value of the image to be analyzed is represented by a shade of color or the like, and calculates the expression feature amount of the target substance for each region of the image. Instead of the average brightness value of the image to be analyzed, the number of fluorescence bright spots of the image to be analyzed may be used to calculate the expression feature amount of the target substance.
  • the control unit 61 performs arithmetic processing using the nuclear density feature amount and the expression feature amount of the target substance, and executes the calculation process of the composite feature amount (step S37). Specifically, the control unit 61 calculates a value obtained by multiplying the nuclear density feature amount for each region by the expression feature amount of the target substance as a composite feature amount. Alternatively, the control unit 61 calculates a value obtained by dividing the expression feature amount of the target substance for each region by the kernel density feature amount as the composite feature amount.
  • the kernel density feature and the expression feature of the target substance are constant, such as addition of a certain offset value, normalization, or conversion to another dimension such as n-step stratification, in addition to using the values as they are.
  • the composite feature amount may be calculated after the processing of. Further, after calculating a plurality of the above-mentioned composite feature quantities at the same time, the secondary composite feature quantity may be calculated by the four arithmetic operations of the composite feature quantities for each region.
  • the control unit 61 extracts a hot spot (specific region) based on the calculated composite feature amount, as shown in FIG. 14 (step S38: extraction step). For example, when a value obtained by multiplying the nuclear density feature amount for each region and the expression feature amount of the target substance is calculated as a composite feature amount, the region whose value is equal to or higher than the first threshold value is extracted as a hot spot (specific region). can do. At this time, the region equal to or higher than the first threshold value can be estimated to be, for example, a region that may be a population of cancer cells.
  • the region whose value is equal to or higher than the first threshold value is extracted as a hot spot (specific region). be able to.
  • the region above the first threshold value can be estimated to be, for example, a region that may be a population of cancer cells having a high expression level of the target substance.
  • the specific region to be extracted is not limited to the above example, and for example, when a value obtained by multiplying the nuclear density feature amount for each region and the expression feature amount of the target substance is calculated as a composite feature amount, the value is calculated from the first threshold value.
  • a region that is less than a small second threshold value may be extracted as a cold spot (specific region).
  • the region below the second threshold value can be estimated to be, for example, a normal / noise cell / immune region.
  • the region smaller than the first threshold value and less than the second threshold value is a cold spot (cold spot (). It may be extracted as a specific area).
  • the region below the second threshold is estimated to be, for example, a region of a normal cell population, a noise cell population, or an immune cell population because the expression level of the target substance is relatively small and the cell density is high. it can.
  • step S36 when calculating the expression feature amount data of the target substance from the fluorescence image (analysis target image), as shown in FIG. 17, the fluorescence brightness value outside the nucleus peripheral region, which is not the analysis target, is invalidated (0). ) May be used. That is, for example, the brightness value of an autofluorescent region such as a blood cell without a nucleus may be set to 0.
  • the high-magnification imaging step is a step of photographing the specific area extracted as described above at a magnification higher than the imaging magnification of the tissue image. Specifically, the position of the extracted specific region is observed with the high-magnification objective lens of the second image acquisition unit 30, and an image is acquired again.
  • a high-magnification microscope such as BX63 + DP80 (Olympus) can also be used. By taking an enlarged image of the extracted specific area in this way, more accurate analysis becomes possible. It should be noted that such an embodiment can be taken in the case of a hall slide scanner having a low magnification (low sensitivity).
  • the analysis step is a step in which the control unit 61 (detection means) analyzes using the image (re-photographed image) taken at high magnification as described above.
  • a step of evaluating the fluorescence bright spot derived from PID which will be described later, and a step of quantitatively evaluating the target substance can be mentioned.
  • the step of evaluating the fluorescent bright spots derived from PID include a step of measuring the number of fluorescent bright spots and a step of measuring the number of PID particles corresponding to the number of fluorescent bright spots.
  • the step of quantitatively evaluating the target substance include a step of calculating a PID score.
  • the control unit 61 acquires a tissue image obtained by photographing the tissue sample 50, and from the acquired tissue image, an image of a specific region for re-imaging. Is extracted.
  • the tissue image includes at least one of an image in which the target substance in the tissue sample 50 is visualized and an image in which the cell morphology in the tissue sample 50 is visualized. Therefore, it is possible to efficiently perform high-precision analysis by suppressing unnecessarily high-magnification imaging by the low-magnification imaging step, the specific region extraction process, and the high-magnification imaging process, that is, the analysis. It is possible to achieve high accuracy and high efficiency at the same time.
  • the tissue image is a whole slide image obtained by photographing the entire tissue sample 50. Therefore, in pathological diagnosis using a hole slide image, it is possible to efficiently perform highly accurate analysis.
  • control unit 61 extracts an image of a region where the average luminance value is equal to or higher than the first threshold value as an image of a specific region. Therefore, the image can be extracted with the hot spot whose average luminance value is equal to or higher than the first threshold value as a specific region.
  • control unit 61 extracts an image of a region where the average luminance value is less than the second threshold value as an image of the specific region. Therefore, the image can be extracted with the cold spot whose average luminance value is less than the second threshold value as a specific region.
  • the image in which the target substance in the tissue sample 50 is visualized is a fluorescent image obtained by photographing the fluorescently labeled target substance, and the control unit 61 measures the number of bright spots in the fluorescent image. , An image of a specific area is extracted based on the measured number of bright spots. Therefore, an image of a specific region can be extracted based on the number of bright spots.
  • control unit 61 extracts an image of a region in which the number of bright spots is equal to or greater than the first threshold value as an image of a specific region. Therefore, the image can be extracted with the hot spot whose number of bright spots is equal to or greater than the first threshold value as a specific region.
  • the control unit 61 extracts an image of a specific region from the image excluding the region to be analyzed from the tissue image.
  • the image excluding the region to be analyzed is an image excluding the region having non-specific uneven staining in the tissue image (image excluding the peripheral edge of the tissue specimen 50), and a cell without a nucleus. It is an image excluding the autofluorescent region (an image excluding the autofluorescent region of erythrocytes) or an image excluding a fluorescence signal derived from autofluorescence in a tissue image. Therefore, more accurate analysis becomes possible.
  • the acquisition means are a first photographing means for acquiring a tissue image (a low-magnification objective lens of the first image acquisition unit 20 or the second image acquisition unit 30) and a first image.
  • a second photographing means (such as a high-magnification objective lens of the second image acquisition unit 30) having a higher photographing magnification than the photographing means is provided, and the re-photographing is performed by the second photographing means in the extracted specific area. Therefore, it is possible to take an enlarged image of the extracted specific area, and more accurate analysis becomes possible.
  • a low-magnification microscope may be used in addition to the whole slide scanner as long as the WSI can be obtained at a low magnification.
  • the first photographing means and the second photographing means may be configured as different photographing means in the same photographing device, or may be configured as two different photographing devices.
  • control unit 61 re-photographs the extracted specific region and measures the number of bright spots in the re-photographed image acquired by the re-photographing.
  • control unit 61 quantifies and evaluates the target substance based on the number of bright spots in the re-photographed image. Therefore, it is possible to measure the number of bright spots and quantify and evaluate the target substance more accurately and efficiently.
  • a tissue section is targeted as a biological sample, and the tissue sample 50 is stained with an immunostaining agent containing fluorescent substance-accumulated nanoparticles as a fluorescent label.
  • the target of the biological sample may be a cultured cell or a gene (DNA).
  • 0.1 mg of the above Texas red accumulated melamine resin particles are dispersed in 1.5 mL of ethanol, 2 ⁇ L of aminopropyltrimethoxysilane (“LS-3150”, manufactured by Shinetsu Chemical Industry Co., Ltd.) is added, and the mixture is reacted for 8 hours.
  • a surface amination treatment was performed to convert the hydroxyl groups present on the surface of the resin particles into amino groups.
  • the concentration of the Texas red accumulated melamine resin particles was adjusted to 3 nM using a phosphate buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA).
  • PBS phosphate buffered saline
  • EDTA ethylenediaminetetraacetic acid
  • SM (PEG) 12 succinimidyl-[(N-maleimidepropionamide) -dodecaethylene glycol] ester, Thermo Fisher Scientific so that the final concentration is 10 mM with respect to the dispersion of Texas red accumulated melamine resin particles whose concentration has been adjusted.
  • Manufactured by Tiffic was mixed and reacted at 20 ° C. for 1 hour to obtain a mixed solution containing Texas red accumulated melamine resin particles whose particle surface was modified with a maleimide group.
  • streptavidin manufactured by Wako Pure Chemical Industries, Ltd.
  • SATA N-succinimidyl-S-acetylthioacetic acid
  • a thiol group was introduced into streptavidin.
  • gel filtration was performed to separately prepare streptavidin capable of binding to fluorescent dye-accumulating particles.
  • the stained slide was irradiated with excitation light corresponding to Texas red used for the fluorescence labeling of the target protein HER2 to emit fluorescence, and an immunostained image in that state was photographed (“fluorescent image” in the inspection support method of the present invention. Corresponds to "acquisition process”).
  • the wavelength of the excitation light was set to 575 to 600 nm using an optical filter for excitation light provided in the fluorescence microscope, and the wavelength of fluorescence to be observed was set to 612 to 692 nm using an optical filter for fluorescence.
  • the intensity of the excitation light during observation with a fluorescence microscope and image capture was set so that the irradiation energy near the center of the visual field was 900 W / cm 2 .
  • the exposure time at the time of image capture was adjusted within a range in which the brightness of the image was not saturated, and was set to, for example, 4000 ⁇ sec.
  • the immunostained image and the stained image for morphology were photographed in the same visual field, and then the same operation was repeated by changing the visual field, and 5 visual fields were taken for each staining slide.
  • Image processing / measurement process Image processing software "ImageJ” (open source) was used for image processing in this process.
  • Streptavidin-modified Texas red accumulation labeled with HER2 protein expressed on the cell membrane by identifying the cell shape (position of the cell membrane) by image processing using the stained image for morphology and superimposing it on the immunostained image.
  • the bright spots representing the melamine resin particles were extracted and measured as the number of PID particles.
  • those having a brightness equal to or higher than a predetermined value were converted into the number of particles by dividing the brightness by the brightness per one Texas Red integrated particle (PID) particle. Since HER2 is not expressed in the stromal cell region, the bright spots located in the stromal cells were treated as non-specific signals, that is, noise.
  • the number of bright spots derived from HER2 is measured in 5 fields of view per dyed slide, converted into the number of fluorescent nanoparticles per unit area (100 ⁇ m 2 ) as described above, and the average value is calculated. Then, it was set as the "PID score" of the sample slide.
  • Tables I to III show the results of analysis of the extracted specific regions on the prepared HER2 stained slides and Table IV on the prepared HER3 stained slides, respectively, at three facilities. In the present invention without arbitrariness, the disparity between facilities was small, and good results were obtained.
  • the observer selects a region of interest and does not automatically inherit the position coordinates of a specific region to the high-magnification microscope, there is some variation in the number of bright spots, and it is preferable to perform it automatically. Met. It was considered that the selection of a specific area was biased depending on the facility, and that it lacked accuracy when the number of bright spots was large due to the lack of automatic inheritance.
  • the present invention can be used in an image processing system, an image processing method, and a program that enable highly accurate analysis to be performed efficiently.
  • Image processing system 10
  • Microscope device 20
  • First image acquisition unit (first imaging means) 21
  • Bright field light source 22
  • First image sensor 30
  • Second image acquisition unit (first imaging means, second imaging means) 31
  • Transmission light source 32
  • Excitation light source 33
  • Second image sensor 40
  • Stage 50
  • Tissue specimen 60
  • Control device 61
  • Storage unit 63
  • Image processing unit 64
  • Communication unit 70
  • Display device 80 Database 80

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