WO2015002082A1 - 画像処理装置、病理診断支援システム、画像処理プログラム及び病理診断支援方法 - Google Patents
画像処理装置、病理診断支援システム、画像処理プログラム及び病理診断支援方法 Download PDFInfo
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Definitions
- the present invention relates to an image processing apparatus, a pathological diagnosis support system, an image processing program, and a pathological diagnosis support method.
- the collected tissue is first dehydrated to fix it, and then treated with paraffin, and then cut into thin slices with a thickness of 2 to 8 ⁇ m, the paraffin is removed, stained, and observed under a microscope.
- a pathologist makes a diagnosis based on morphological information such as a change in the size and shape of a cell nucleus and a change in a pattern as a tissue, and staining information.
- morphological feature values such as cell nuclear area and nucleus concentration and cell malignancy are calculated in a microscopic image, and these morphological feature values and cell malignancy are observed on a monitor as a cell image. I can do it.
- HE staining hematoxylin-eosin staining
- DAB diaminobenzidine
- identifying proteins that are over-expressed in tissue sections and their expression levels can be very important information in determining prognosis and subsequent treatment plans.
- the HER2 protein encoded by the HER2 gene is a receptor-type glycoprotein that penetrates the cell membrane, and is composed of three domains, extracellular, transmembrane, and intracellular. It is activated and is involved in cell proliferation and malignant transformation through signal transduction pathways. Overexpression of HER2 protein is observed in breast cancer, lung cancer, colon cancer, stomach cancer, bladder cancer and the like.
- Patent Document 2 a cell nucleus is extracted from an image of a biological tissue stained by the DAB method, a cell membrane is specified from the image of the biological tissue based on the cell nucleus, a staining state of the cell membrane is determined, and based on this determination result A system for evaluating the expression of HER2 protein is described.
- JP-A-57-153367 JP 2009-115599 A Japanese Patent Application No. 2012-078722
- Patent Document 3 a cancer region in which a specific protein is overexpressed can be efficiently grasped from the entire tissue section image, which is considered to be extremely useful.
- the present inventor has repeatedly studied the technique of Patent Document 3, as described above, when the cell morphology image and the entire fluorescent image are usually overlaid, basically, misalignment is a major problem.
- alignment is particularly important when only the cell nucleus is to be observed.
- the cells to be observed (cell nuclei to be observed) extracted from the cell morphology image are often very small, and in order to accurately quantify the expression of a specific protein there, it is necessary to align the images very accurately Therefore, further improvement that can accurately quantify the expression of a specific protein in a cell to be observed is desired.
- a main object of the present invention is to provide an image processing apparatus capable of accurately quantifying the expression of a specific protein in an observation target cell, and also pathological diagnosis using the image processing apparatus.
- An object is to provide a support system, an image processing program, and a pathological diagnosis support method.
- An input means for inputting a cell morphology image representing the morphology of the cells in the tissue section and a fluorescence image representing the expression of a specific protein in the same range of the tissue section as a fluorescent bright spot;
- An alignment means for aligning the cell morphology image and the fluorescence image based on an information source commonly detected in the cell morphology image and the fluorescence image;
- An image processing apparatus is provided.
- the image processing device An image acquisition device used in the image processing device for acquiring the cell morphology image and the fluorescence image; A pathological diagnosis support system is provided.
- Computer An input means for inputting a cell morphology image representing the morphology of a cell in a tissue section and a fluorescence image representing the expression of a specific protein in the same range of the tissue section as a fluorescent bright spot; An alignment means for aligning the cell morphology image and the fluorescence image based on an information source commonly detected in the cell morphology image and the fluorescence image; An image processing program for functioning as
- a pathological diagnosis support method characterized by comprising:
- the cell morphology image and the fluorescence image can be accurately aligned, and the expression of a specific protein in the observation target cell can be accurately quantified.
- FIG. 1 It is a figure which shows the system configuration
- FIG. 1 shows an example of the overall configuration of a pathological diagnosis support system 100 in the present embodiment.
- the pathological diagnosis support system 100 acquires a microscopic image of a tissue section of a human body stained with a predetermined staining reagent, and analyzes the acquired microscopic image, thereby expressing the expression of a specific biological material in the tissue section to be observed. This is a system that outputs feature quantities quantitatively.
- the pathological diagnosis support system 100 is configured by connecting a microscope image acquisition apparatus 1A and an image processing apparatus 2A so as to be able to transmit and receive data via an interface such as a cable 3A.
- the connection method between the microscope image acquisition device 1A and the image processing device 2A is not particularly limited.
- the microscope image acquisition apparatus 1A and the image processing apparatus 2A may be connected via a LAN (Local Area Network) or may be configured to be connected wirelessly.
- LAN Local Area Network
- the microscope image acquisition apparatus 1A is a known optical microscope with a camera, and acquires a microscope image of a tissue section on a slide placed on a slide fixing stage and transmits it to the image processing apparatus 2A.
- the microscope image acquisition apparatus 1A includes an irradiation unit, an imaging unit, an imaging unit, a communication I / F, and the like.
- the irradiating means includes a light source, a filter, and the like, and irradiates light to a tissue section on the slide placed on the slide fixing stage.
- the imaging means is composed of an eyepiece lens, an objective lens, and the like, and forms an image of transmitted light, reflected light, or fluorescence emitted from the tissue section on the slide by the irradiated light.
- the imaging means is a microscope-installed camera that includes a CCD (Charge Coupled Device) sensor and the like, captures an image formed on the imaging surface by the imaging means, and generates digital image data of the microscope image.
- the communication I / F transmits image data of the generated microscope image to the image processing apparatus 2A.
- the microscope image acquisition apparatus 1A includes a bright field unit that combines an irradiation unit and an imaging unit suitable for bright field observation, and a fluorescence unit that combines an irradiation unit and an imaging unit suitable for fluorescence observation. It is possible to switch between bright field / fluorescence by switching units.
- the microscope image acquisition apparatus 1A is not limited to a microscope with a camera.
- a virtual microscope slide creation apparatus for example, a special microscope scan apparatus that acquires a microscope image of an entire tissue section by scanning a slide on a microscope slide fixing stage). Table 2002-514319
- the virtual microscope slide creation device it is possible to acquire image data that allows a display unit to view a whole tissue section on a slide at a time.
- the image processing device 2A calculates a feature amount quantitatively indicating the expression level of a specific biological material in the tissue section to be observed by analyzing the microscope image transmitted from the microscope image acquisition device 1A. Output feature values.
- FIG. 2 shows a functional configuration example of the image processing apparatus 2A. As shown in FIG. 2, the image processing apparatus 2 ⁇ / b> A includes a control unit 21, an operation unit 22, a display unit 23, a communication I / F 24, a storage unit 25, and the like, and each unit is connected via a bus 26. Yes.
- the control unit 21 includes a CPU (Central Processing Unit), a RAM (Random Access Memory), and the like.
- the control unit 21 executes various processes in cooperation with various programs stored in the storage unit 25, and performs image processing 2A. Overall control of the operation.
- the control unit 21 executes image analysis processing (see FIG. 10) in cooperation with a program stored in the storage unit 25, and realizes functions as an alignment unit, a calculation unit, a determination unit, and a determination unit. To do.
- the operation unit 22 includes a keyboard having character input keys, numeric input keys, various function keys, and the like, and a pointing device such as a mouse, and a key pressing signal pressed by the keyboard and an operation signal by the mouse. Are output to the control unit 21 as an input signal.
- the display unit 23 includes, for example, a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display), and displays various screens in accordance with display signal instructions input from the control unit 21.
- the display unit 23 functions as an output unit for outputting an image analysis result.
- the communication I / F 24 is an interface for transmitting and receiving data to and from external devices such as the microscope image acquisition device 1A.
- the communication I / F 24 functions as a bright field image and fluorescent image input unit.
- the storage unit 25 includes, for example, an HDD (Hard Disk Drive), a semiconductor nonvolatile memory, or the like. As described above, the storage unit 25 stores various programs, various data, and the like.
- the image processing apparatus 2A may include a LAN adapter, a router, and the like and be connected to an external device via a communication network such as a LAN.
- the image processing apparatus 2A in the present embodiment performs analysis using the bright field image (HE-stained image) and the fluorescence image transmitted from the microscope image acquisition apparatus 1A.
- the bright field image is a microscope image obtained by enlarging and photographing a tissue section stained with a HE (hematoxylin-eosin) staining reagent in a bright field in the microscope image acquisition apparatus 1A.
- Hematoxylin is a blue-violet pigment that stains cell nuclei, bone tissue, part of cartilage tissue, serous components, etc. (basophilic tissue, etc.).
- Eosin is a red to pink pigment that stains the cytoplasm, connective tissue of soft tissues, red blood cells, fibrin, endocrine granules, etc. (eosinophilic tissues, etc.).
- FIG. 3 shows an example of a bright field image obtained by photographing a tissue section subjected to HE staining. As shown in FIG. 3, in the bright field image obtained by photographing the tissue section subjected to HE staining, the morphology of the cells in the tissue section appears. That is, the bright field image is a cell morphology image representing the morphology of cells in the tissue section.
- the cell nucleus appears in a darker color (blue purple) than the surrounding cytoplasm and distinguished from the surrounding, and the morphology of the cell nucleus can be clearly captured.
- the fluorescent image is stained using a staining reagent including nanoparticles (referred to as fluorescent substance-containing nanoparticles) encapsulating a fluorescent substance bound with a biological substance recognition site that specifically binds and / or reacts with a specific biological substance.
- This is a microscope image obtained by irradiating the tissue section with the excitation light of a predetermined wavelength in the microscope image acquisition device 1A to emit fluorescent substance-containing nanoparticles (fluorescence), and enlarging and photographing this fluorescence. . That is, the fluorescence that appears in the fluorescence image indicates the expression of a specific biological material corresponding to the biological material recognition site in the tissue section.
- FIG. 4 shows an example of the fluorescence image.
- a method for acquiring a fluorescent image including a staining reagent (fluorescent substance-containing nanoparticles) used for acquiring the fluorescent image, a method for staining a tissue section with the staining reagent, and the like.
- fluorescent substance examples include fluorescent organic dyes and quantum dots (semiconductor particles). When excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm, it preferably emits visible to near infrared light having a wavelength in the range of 400 to 1100 nm.
- fluorescent organic dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes.
- fluorescent organic dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes.
- examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, cyanine dye molecules, and the like.
- quantum dots containing II-VI group compounds, III-V group compounds, or group IV elements as components ("II-VI group quantum dots”, "III-V group quantum dots”, " Or “Group IV quantum dots”). You may use individually or what mixed multiple types.
- CdSe CdS, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge, but are not limited thereto.
- a quantum dot having the above quantum dot as a core and a shell provided thereon.
- CdSe / ZnS when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS.
- CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO 2 , Si / ZnS, Ge / GeO 2 , Ge / ZnS, and the like can be used, but are not limited thereto.
- the quantum dots those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.
- the fluorescent substance-encapsulating nanoparticles are those in which the fluorescent substance is dispersed inside the nanoparticles, whether the fluorescent substance and the nanoparticles themselves are chemically bonded or not. Good.
- the material constituting the nanoparticles is not particularly limited, and examples thereof include polystyrene, polylactic acid, and silica.
- Fluorescent substance-containing nanoparticles used in the present embodiment can be produced by a known method.
- silica nanoparticles encapsulating a fluorescent organic dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992).
- Various fluorescent organic dye-containing silica nanoparticles can be synthesized by using a desired fluorescent organic dye in place of FITC.
- Silica nanoparticles encapsulating quantum dots can be synthesized with reference to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry, Vol. 33, p. 561 (2009).
- Polystyrene nanoparticles encapsulating a fluorescent organic dye may be copolymerized using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982) or polystyrene described in US Pat. No. 5,326,692 (1992). It can be produced using a method of impregnating nanoparticles with a fluorescent organic dye.
- Polymer nanoparticles encapsulating quantum dots can be prepared using the method of impregnating polystyrene nanoparticles with quantum dots described in Nature Biotechnology, Vol. 19, page 631 (2001).
- the average particle diameter is obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number of particles, and taking each measured value as the area of the circle. As sought. In the present application, the arithmetic average of the particle sizes of 1000 particles is defined as the average particle size. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
- the biological material recognition site is a site that specifically binds and / or reacts with the target biological material.
- the target biological substance is not particularly limited as long as a substance that specifically binds to the target biological substance exists, but typically, protein (peptide), nucleic acid (oligonucleotide, polynucleotide), antibody, etc. Is mentioned. Accordingly, substances that bind to the target biological substance include antibodies that recognize the protein as an antigen, other proteins that specifically bind to the protein, and nucleic acids having a base sequence that hybridizes to the nucleic acid. Is mentioned.
- an anti-HER2 antibody that specifically binds to HER2 which is a protein present on the cell surface
- an anti-ER antibody that specifically binds to an estrogen receptor (ER) present in the cell nucleus and actin that forms a cytoskeleton
- an anti-actin antibody that specifically binds to are preferable.
- the mode of binding between the biological substance recognition site and the fluorescent substance-encapsulating nanoparticles is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, and chemical adsorption.
- a bond having a strong bonding force such as a covalent bond is preferred from the viewpoint of bond stability.
- an organic molecule that connects between the biological substance recognition site and the fluorescent substance-containing nanoparticle.
- a polyethylene glycol chain can be used, and SM (PEG) 12 manufactured by Thermo Scientific can be used.
- a silane coupling agent that is a compound widely used for bonding an inorganic substance and an organic substance can be used.
- This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group.
- silane coupling agent having a polyethylene glycol chain for example, PEG-silane no. SIM6492.7 manufactured by Gelest
- silane coupling agent you may use 2 or more types together.
- a publicly known method can be used for the reaction procedure of the fluorescent organic dye-encapsulated silica nanoparticles and the silane coupling agent.
- the obtained fluorescent organic dye-encapsulated silica nanoparticles are dispersed in pure water, aminopropyltriethoxysilane is added, and the mixture is reacted at room temperature for 12 hours.
- fluorescent organic dye-encapsulated silica nanoparticles whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration.
- the antibody can be bound to the fluorescent organic dye-encapsulated silica nanoparticles via an amide bond.
- a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate, Hydrochloride: manufactured by Pierce (registered trademark)
- EDC 1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate, Hydrochloride: manufactured by Pierce (registered trademark)
- a linker compound having a site that can be directly bonded to the fluorescent organic dye-encapsulated silica nanoparticles modified with organic molecules and a site that can be bonded to the molecular target substance can be used.
- sulfo-SMCC Sulfosuccinimidyl 4 [N-maleimidomethyl] -cyclohexane-1-carboxylate: manufactured by Pierce
- sulfo-SMCC Sulfosuccinimidyl 4 [N-maleimidomethyl] -cyclohexane-1-carboxylate: manufactured by Pierce
- fluorescent substance-encapsulated polystyrene nanoparticles When binding a biological material recognition site to fluorescent substance-encapsulated polystyrene nanoparticles, the same procedure can be applied regardless of whether the fluorescent substance is a fluorescent organic dye or a quantum dot. That is, by impregnating a fluorescent organic dye or quantum dot into polystyrene nanoparticles having a functional group such as an amino group, fluorescent substance-containing polystyrene nanoparticles having a functional group can be obtained, and thereafter using EDC or sulfo-SMCC. Thus, antibody-bound fluorescent substance-encapsulated polystyrene nanoparticles can be produced.
- Antibodies that recognize specific antigens include M. actin, MS actin, SM actin, ACTH, Alk-1, ⁇ 1-antichymotrypsin, ⁇ 1-antitrypsin, AFP, bcl-2, bcl-6, ⁇ -catenin, BCA 225, CA19-9, CA125, calcitonin, calretinin, CD1a, CD3, CD4, CD5, CD8, CD10, CD15, CD20, CD21, CD23, CD30, CD31, CD34, CD43, CD45, CD45R, CD56, CD57, CD61, CD68, CD79a, "CD99, MIC2", CD138, chromogranin, c-KIT, c-MET, collagen type IV, Cox-2, cyclin D1, keratin, cytokeratin (high molecular weight), pankeratin, pankeratin, cytokeratin 5/6, cytokeratin 7, cytokeratin 8, cytokeratin
- the pathological section is immersed in a container containing xylene to remove paraffin.
- the temperature is not particularly limited, but can be performed at room temperature.
- the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
- the pathological section is immersed in a container containing ethanol to remove xylene.
- the temperature is not particularly limited, but can be performed at room temperature.
- the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Further, if necessary, ethanol may be exchanged during the immersion.
- the pathological section is immersed in a container containing water to remove ethanol.
- the temperature is not particularly limited, but can be performed at room temperature.
- the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Moreover, you may exchange water in the middle of immersion as needed.
- the activation process of the target biological substance is performed according to a known method.
- the activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer, etc. are used. be able to.
- 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 can be performed at room temperature. The temperature can be 50-130 ° C. and the time can be 5-30 minutes.
- the section after the activation treatment is immersed in a container containing PBS (Phosphate Buffered Saline) and washed.
- PBS Phosphate Buffered Saline
- the temperature is not particularly limited, but can be performed at room temperature.
- the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion.
- each fluorescent substance-encapsulating nanoparticle PBS dispersion may be mixed in advance or separately placed on a pathological section separately. Also good.
- the temperature is not particularly limited, but can be performed at room temperature.
- the reaction time is preferably 30 minutes or more and 24 hours or less.
- a known blocking agent such as BSA-containing PBS
- the stained section is immersed in a container containing PBS, and unreacted fluorescent substance-containing nanoparticles are removed.
- the temperature is not particularly limited, but can be performed at room temperature.
- the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion.
- a cover glass is placed on the section and sealed. A commercially available encapsulant may be used as necessary.
- staining using a HE dyeing reagent HE dyeing is performed before enclosure with a cover glass.
- Fluorescence image acquisition A wide-field microscope image (fluorescence image) is acquired from the stained pathological section using the microscope image acquisition device 1A.
- an excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent material used for the staining reagent are selected.
- the visual field of the fluorescent image is preferably 3 mm 2 or more, more preferably 30 mm 2 or more, and further preferably 300 mm 2 or more.
- Nanoparticles 1 Cy5 encapsulated silica nanoparticles
- a labeling material A was prepared.
- CdSe / ZnS-encapsulated silica nanoparticles hereinafter referred to as “nanoparticles 2” were produced, and a labeling material B in which an anti-HER2 antibody was bound to the nanoparticles 2 was produced.
- a plurality of fluorescent images are obtained by performing immunostaining using adjacent sections of human breast tissue whose FISH score has been measured in advance using the produced labeling materials A and B and the labeling materials C and D as comparative examples, and changing the visual field.
- the visual field was obtained, and the number of fluorescent bright spots appearing in each fluorescent image was measured to examine the relationship with the FISH score.
- Step (4) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
- SEM scanning electron microscope
- Step (2) Quantum dot-encapsulated silica: synthesis of CdSe / ZnS-encapsulated silica nanoparticles having an emission wavelength of 655 nm
- CdSe / ZnS-encapsulated silica nanoparticles (hereinafter referred to as “nanoparticles 2”) were prepared by the following steps (1) to (5).
- Step (1) 10 ⁇ L of CdSe / ZnS decane dispersion (Invitrogen Qdot655) and 40 ⁇ L of tetraethoxysilane were mixed.
- Step (2) 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.
- Step (3) The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
- Step (4) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
- Step (1) 1 mg of nanoparticles 1 was dispersed in 5 mL of pure water. Next, 100 ⁇ L of an aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours. Step (2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Step (3): Ethanol was added to disperse the sediment, followed by centrifugation again.
- Step (4) The amino group-modified silica nanoparticles obtained in step (3) were adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
- Step (6) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
- Step (7) PBS containing 2 mM of EDTA was added to disperse the precipitate, and then centrifuged again. The washing
- Step (8) When 100 ⁇ g of the anti-HER2 antibody was dissolved in 100 ⁇ L of PBS, 1 M dithiothreitol (DTT) was added and reacted for 30 minutes.
- Step (10) Using the nanoparticle 1 as a starting material, the particle dispersion obtained in step (7) and the reduced anti-HER2 antibody solution obtained in step (9) are mixed in PBS and allowed to react for 1 hour. It was. Step (11): 4 ⁇ L of 10 mM mercaptoethanol was added to stop the reaction. Step (12): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing
- Fluorescent substance-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 1 as a starting material are labeled material A, and phosphor-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 2 as a starting material. This is labeled material B.
- an anti-HER2 antibody was bound to Cy5 to obtain a reduced anti-HER2 antibody solution (labeling material D).
- labeling material D was bound to Cy5 to obtain a reduced anti-HER2 antibody solution.
- labeling material C was prepared by binding an anti-HER2 antibody to CdSe.
- tissue staining using fluorescent substance-containing nanoparticles Using the antibody-binding labeling materials A to D produced by the methods of the following steps (1) to (10), immunostaining was performed using adjacent sections of human breast tissue whose FISH score was measured in advance. As a stained section, a tissue array slide (CB-A712) manufactured by Cosmo Bio was used. Twenty-four sections with a FISH score of 1-9 were used.
- Step (1) The pathological section was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
- Step (2) The pathological section was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
- Step (3) The pathological section was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
- Step (6) The section after autoclaving was immersed in a container containing PBS for 30 minutes.
- Step (7) PBS containing 1% BSA was placed on the tissue and left for 1 hour.
- a plurality of fluorescent images are obtained by changing the field of view (observation area), and the number of fluorescent bright spots (the number of bright spots) is obtained from each fluorescent image using image analysis software.
- the microscope used was an upright microscope Axio Imager M2 manufactured by Carl Zeiss, and the objective lens was set to 20 times, and excitation light having a wavelength of 630 to 670 nm was irradiated to form an image of fluorescence emitted from the tissue section. Then, a fluorescence image (image data) was acquired with a microscope installation camera (monochrome), and the number of bright spots was measured with image analysis software.
- the camera has a pixel size of 6.4 ⁇ m ⁇ 6.4 ⁇ m, a vertical pixel count of 1040, and a horizontal pixel count of 1388 (imaging area 8.9 mm ⁇ 6.7 mm).
- the correlation coefficient R between the measured number of bright spots and the FISH score was calculated in each visual field.
- the FISH score corresponds to the overexpression level of the HER2 gene, and the larger the value of the FISH score, the higher the overexpression level of the HER2 gene.
- FIG. 5 shows the number of bright spots measured from fluorescent images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and FISH when using the labeling material A (Cy5 inclusion labeling material). It is a figure which shows the relationship with a score. The value of R 2 shown in the figure is the square value of the correlation coefficient between the number of bright spots and the FISH score.
- FIG. 6 shows the number of bright spots measured from fluorescent images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and FISH when the labeling material B (CdSe inclusion labeling material) is used. It is a figure which shows the relationship with a score.
- labeling material B CdSe inclusion labeling material
- FIG. 7 shows the number of bright spots measured from fluorescence images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and the FISH score when the labeling material C (CdSe) is used. It is a figure which shows a relationship.
- FIG. 8 shows the number of bright spots measured from fluorescent images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and the FISH score when the labeling material D (Cy5) is used. It is a figure which shows a relationship.
- Table 1 and FIG. 9 show the square value (R 2 ) of the correlation coefficient between the number of bright spots measured from the fluorescence image of each visual field (observation area) and the FISH score for each of the labeling materials A to D.
- the square value (R 2 ) of the correlation coefficient between the number of bright spots and the FISH score is 0. 5387. This value is approximately 0.734 when converted to the correlation coefficient R, and it can be said that there is a strong correlation between the number of bright spots and the FISH score.
- the square value (R 2 ) of the correlation coefficient between the number of bright spots and the FISH score is 0. It was 9887.
- the field of view is 324 mm 2 , it can be said that the correlation is stronger than when the field of view is 32 mm 2 .
- the labeling materials A and B use particles containing a fluorescent substance and are brighter than the labeling materials C and D using a single fluorescent substance, each point of the bright spot is captured from the image. Easy to measure the number of bright spots with high accuracy.
- ⁇ Operation of Pathological Diagnosis Support System 100 (Including Pathological Diagnosis Support Method)>
- staining is performed using a staining reagent including fluorescent substance-encapsulating nanoparticles bound to a biological substance recognition site that recognizes a specific protein (here, Ki67 protein in breast cancer tissue, hereinafter referred to as a specific protein).
- a specific protein here, Ki67 protein in breast cancer tissue, hereinafter referred to as a specific protein.
- the present invention is not limited to this example.
- the operator uses two types of staining reagents, a HE staining reagent and a staining reagent using fluorescent substance-encapsulated nanoparticles bound with a biological substance recognition site that recognizes a specific protein as a fluorescent labeling material. Stain. Thereafter, in the microscope image acquisition apparatus 1A, a bright field image and a fluorescence image are acquired by the procedures (a1) to (a5). (A1) The operator places the tissue sections stained with the HE staining reagent and the staining reagent containing fluorescent substance-containing nanoparticles on the slide, and places the slide on the slide fixing stage of the microscope image acquisition apparatus 1A. .
- (A2) Set to the bright field unit, adjust the imaging magnification and focus, and place the observation target area on the tissue in the field of view.
- (A3) Shooting is performed by the imaging unit to generate bright field image data, and the image data is transmitted to the image processing apparatus 2A.
- (A4) Change the unit to a fluorescent unit.
- (A5) Shooting is performed by the imaging means without changing the field of view and the shooting magnification to generate image data of a fluorescent image, and the image data is transmitted to the image processing apparatus 2A.
- the tissue autofluorescence and eosin emission (background) are 10% (1.1 times). If the fluorescence emission point of the fluorescent substance-encapsulating nanoparticles has the above-mentioned difference in light emission amount, the microscope can be used in any processing system of 8 bits (0 to 255 gradations) and 12 bits (0 to 4095 gradations). It was possible to automatically detect fluorescent luminescent spots from an image (fluorescent image).
- the fluorescent substance-containing nanoparticles In the case of only staining with fluorescent substance-containing nanoparticles, if the fluorescent substance-containing nanoparticles have a difference in light emission amount of 10% (1.1 times) or more with respect to the autofluorescence of the tissue, 8 bits (0 In any processing system of up to 255 gradations) and 12 bits (0 to 4095 gradations), automatic detection of fluorescent bright spots was possible. Therefore, it is preferable to select an excitation light wavelength in the range of 560 to 630 nm in the fluorescent unit. Further, it is preferable to use a fluorescent substance that emits fluorescence having a peak in the range of 580 to 690 nm, more preferably in the range of 600 to 630 nm by the excitation light.
- the difference between the autofluorescence of the tissue including the emission of eosin and the fluorescence from the fluorescent substance-containing nanoparticles is secured. This is because it is possible to ensure that they can be distinguished and recognized (difference of 10% (1.1 times) or more).
- the autofluorescence of the tissue is weak, so the wavelength range of the excitation light is not limited to a general range of 200 nm to 700 nm, and the autofluorescence and fluorescent substance-containing nanoparticles are not particularly limited.
- a difference in fluorescence emission from the light source so that both can be distinguished and recognized (a difference in light quantity between the two is 10% (1.1 times or more)).
- FIG. 10 shows a flowchart of image analysis processing in the image processing apparatus 2A.
- the image analysis processing shown in FIG. 10 is executed in cooperation with the control unit 21 and a program stored in the storage unit 25.
- step S1 when a bright field image is input from the microscope image acquisition device 1A through the communication I / F 24 (step S1), a cell nucleus region is extracted from the bright field image (step S2).
- FIG. 11 shows a detailed flow of the process in step S2.
- the process of step S2 is executed in cooperation with the control unit 21 and the program stored in the storage unit 25.
- step S2 first, a bright-field image is converted into a monochrome image (step S201).
- FIG. 12A shows an example of a monochrome image.
- threshold processing is performed on the monochrome image using a predetermined threshold, and the value of each pixel is binarized (step S202).
- FIG. 12B shows an example of a binary image after threshold processing.
- noise processing is performed (step S203).
- the noise process can be performed by performing a closing process on the binary image.
- the closing process is a process in which the contraction process is performed the same number of times after the expansion process is performed.
- the expansion process is a process of replacing a target pixel with white when at least one pixel in the range of n ⁇ n pixels (n is an integer of 2 or more) from the target pixel is white.
- the contraction process is a process of replacing a target pixel with black when at least one pixel in the range of n ⁇ n pixels from the target pixel contains black.
- FIG. 12C shows an example of an image after noise processing. As shown in FIG. 12C, after noise processing, an image (cell nucleus image) from which cell nuclei are extracted is obtained.
- a labeling process is performed on the image after the noise process, and a label Label_nucleus is assigned to each of the extracted cell nuclei (step S204).
- the labeling process is a process for identifying an object in an image by assigning the same label (number) to connected pixels. By labeling, each cell nucleus can be identified from the image after noise processing and a label can be applied.
- the label Label_nucleus is added to the new cell nucleus as MAX.
- step S3 the observation target cell in the bright field image is determined based on the cell nucleus region extracted in step S2 (step S3).
- step S3 first, for all cell nuclei in the cell nucleus image extracted in step S2, from the cell nucleus image, the area A of the cell nucleus, the average density B of the cell nucleus, the pixel luminance variation ( ⁇ value) C in the region of the cell nucleus, the cell nucleus
- the “cell feature amount” such as the degree of circularity D, the flatness E of the cell nucleus, and the ratio F of the thickness to the nucleus area of the nucleus edge is calculated.
- the area A of the cell nucleus the size of a pixel (pixel) is calculated by measuring a reference length corresponding to the cell nucleus image in advance, and the number of pixels in each cell nucleus extracted in step 2 is integrated.
- the area A of the cell nucleus is determined.
- the average density B of the cell nuclei is determined by obtaining the luminance signal value converted into the gray scale of each pixel (pixel) in the cell nucleus and calculating the average value.
- the pixel luminance variation C is determined by calculating the standard deviation of the luminance signal value of each pixel (pixel) in the cell nucleus.
- the circularity D and the flatness E of the cell nucleus are determined by applying a certain value obtained from the cell nucleus image to the following equations (d) and (e) for each cell nucleus extracted in step 2.
- the thickness ratio F to the nucleus area of the nucleus edge In calculating the thickness ratio F to the nucleus area of the nucleus edge, first, an average value L ave of luminance signal values of an area of 80% from the central part of the cell nucleus is obtained, and about 20% area area of the outer edge part of the cell nucleus, A region where the luminance signal value of a pixel (pixel) is 20% or more lower than L ave was defined as the nucleus of the cell nucleus. Next, the area F of the nucleus edge is obtained, and the ratio F to the nucleus area of the nucleus edge is determined by calculating the ratio with the area of the whole cell nucleus (area A of the cell nucleus).
- a cell including a cell nucleus satisfying one or more of the following conditions (i) to (vi) is determined as an observation target cell in the bright field image from the calculated cell feature amount.
- the cell nucleus area A is included in the top 60% or more, preferably the top 50% or more, more preferably the top 40% or more of all the calculated cell nucleus areas A.
- the average concentration of cell nuclei B is included in the calculated average concentration B of all the cell nuclei in the lower 50% or less, preferably the lower 40% or less, more preferably the upper 30% or less.
- Pixel luminance variation ( ⁇ (Value) C is ⁇ ⁇ 30, preferably ⁇ ⁇ 40, more preferably ⁇ ⁇ 50 in an 8-bit image converted to gray scale
- the circularity D of the cell nucleus is 0.8 or less, preferably 0.6 or less, more preferably 0.4 or less
- the cell nucleus flatness E is 0.6 or less, preferably 0.4 or less, more preferably 0.2 or less
- Thickness ratio F of to nuclear area of i) Kakuen is 3% or more, preferably at least 5%, more preferably 10% or more
- the determination of the observation target cell in the above step 3 is basically automatically performed in cooperation with the program stored in the control unit 21 and the storage unit 25.
- auxiliary work by the observer is performed. May be accompanied.
- the auxiliary work by the observer is, for example, each threshold value of the cell feature amount with respect to the program stored in the storage unit 25 (for example, if the area A is a cell nucleus, the area A of the cell nucleus when determining the observation target cell) Setting of the threshold value for) is stepwise adjusted, and visual confirmation of the determined observation target cell is included.
- each factor of the cell feature amount (the cell nucleus area A to the thickness ratio F to the nucleus area) may be arbitrarily selected and appropriately changed.
- the threshold value in each factor may be arbitrarily selected and appropriately changed.
- another factor different from the above may be used as a factor of the cell feature amount.
- step S5 shows a detailed flow of the process in step S5.
- the process of step S5 is executed in cooperation with the control unit 21 and the program stored in the storage unit 25.
- the R component is extracted from the fluorescence image (step S501).
- Tophat conversion is performed on the image from which the R component has been extracted (step S502).
- the Tophat conversion is a process of subtracting the value of the corresponding pixel of the image obtained by applying the minimum value filter and the maximum value filter to the input image in this order from the value of each pixel of the input image.
- the minimum value filter replaces the value of the target pixel with the minimum value of pixels in the vicinity of the target pixel (for example, 3 ⁇ 3 pixels).
- the maximum value filter replaces the value of the target pixel with the maximum value among the pixels in the vicinity of the target pixel (for example, 3 ⁇ 3 pixels).
- FIG. 14B shows an image obtained by extracting fluorescent luminescent spots obtained after noise removal in the fluorescent luminescent spot candidate image shown in FIG. 14A.
- a labeling process is performed on the image after noise removal, and a label Label_point is assigned to each of the extracted fluorescent luminescent spots (step S504). Label_point is assigned in order from 1. After the labeling process ends, the process proceeds to step S6 in FIG.
- step S6 the images of the bright field image and the fluorescence image are aligned based on the information source that is commonly detected in the bright field image and the fluorescence image.
- the bright field image used for the alignment is the cell nucleus image obtained in step S2 (see FIG. 12C)
- the fluorescent image used for the alignment is the fluorescent bright spot image obtained in step S5 (see FIG. 14B). It is.
- an information source commonly detected in the bright field image and the fluorescence image information recognizable in both the bright field image and the fluorescence image is used.
- staining information of eosin which is a staining material of the tissue section is used. Is used.
- an image feature amount that characterizes the bright field image and the fluorescence image is calculated.
- image feature amount contrast information, edge information, contour information, and the like that can be observed in both the bright field image and the fluorescence image are calculated and used.
- contrast information information on a specific area or color difference or luminance difference between the specific area and the entire image is used, and binarization processing is performed as necessary to emphasize the information itself.
- edge information and the contour information are obtained by performing image processing on each of the bright field image and the fluorescence image.
- a Canny method or an unsharp mask method is preferably used.
- step S6 a cell feature amount is calculated from each eosin staining information of the bright field image and the fluorescence image, and the bright field image and the fluorescence image are aligned based on the cell feature amount.
- contrast information, edge information, and contour information in the bright field image are calculated from the eosin staining information in the bright field image.
- contrast information, edge information, and contour information in the fluorescence image are calculated from the eosin staining information in the fluorescence image.
- contrast information may be compared. Contrast information, edge information, and contour information. All pieces of information may be compared. For example, when comparing contrast information, as shown in FIG. 15, the contrast information in the leftmost specific area 30 and the rightmost specific area 32 is compared between the bright field image and the fluorescent image, Each image is overlaid (positioned) so that the common points (shades) in the specific areas 30 and 32 match. At this time, the bright field image and the fluorescence image may be enlarged or reduced so that the common points in the specific regions 30 and 32 match.
- staining information by a third staining reagent which is different from the staining reagent including the HE staining reagent and the fluorescent substance-containing nanoparticles, is also preferably used as an information source that is commonly detected in the bright field image and the fluorescence image.
- the third staining reagent the fluorescent particles of the fluorescent bright spot source in the fluorescent image (that is, the fluorescent substance-containing nanoparticles) do not overlap with the emission wavelength, and the size (particle diameter) and shape are clearly different.
- the emission wavelength may be different, or all of the emission wavelength, the particle diameter, and the shape may be different.
- fluorescent particles fluorescent particles having a particle size of 1.0 to 5.0 ⁇ m are preferably used.
- particles in which FITC or Alexa Fluor (registered trademark) 488 which is a fluorescent dye is encapsulated in silica are used.
- FITC or Alexa Fluor (registered trademark) 488 which is a fluorescent dye is encapsulated in silica are used.
- image feature amounts such as contrast information, edge information, and contour information are calculated as described above. Based on these image feature amounts, the bright field image and the fluorescence image are accurately aligned.
- step S7 expression of a specific protein in each observation target cell from both images of the bright field image after alignment (cell nucleus image obtained in step S2) and the fluorescence image (fluorescence bright spot image obtained in step S5).
- the situation is determined (step S7). Specifically, in a state where the bright field image and the fluorescence image are overlaid, the number of fluorescent bright spots corresponding to the observation target cell determined in step S3 is calculated. The number of fluorescent bright spots superimposed on the observation target cell indicates the expression state of a specific protein that is an index of cancer malignancy or progression.
- the statistical method used for the determination of malignancy and the treatment plan is not particularly limited.
- a value obtained by dividing the total number of fluorescent bright spots on the observation target cells by the number of observation target cells, or the fluorescent brightness on the observation target cells is used, and the ratio of the number of cells to be observed having a certain number or more of fluorescence points by histogramming the number of fluorescent points on the cells to be observed is also preferable. Used.
- an analysis result screen 231 is generated and displayed on the display unit 23 based on the expression state of the specific protein in each observation target cell after the determination (step S8).
- the expression status of the specific protein is classified into a plurality of stages based on whether or not the number of fluorescent bright spots on the observation target cell exceeds a plurality of predetermined threshold values.
- the areas are classified into areas, low expression areas, and extremely low expression areas.
- an image classified (for example, color-coded) in a different display mode according to the classification result is generated on the bright field image, and is displayed and output on the display unit 23 as the analysis result screen 231.
- FIG. 16 shows an example of the analysis result screen 231.
- the analysis result screen 231 displays a bright field image 231a, a fluorescent bright spot image 231b, and a protein expression status display 231c.
- the protein expression status display 231c a high expression region, a medium expression region, a low expression region, and a very low expression region of a specific protein are displayed in different colors on the bright field image. Because it is displayed in different colors according to the expression status of specific proteins, doctors can efficiently grasp the overexpression and spread of specific proteins that are an indicator of cancer malignancy, and make appropriate treatment plans. It is possible to stand.
- the display method of the analysis result screen 231 is not limited to that shown in FIG.
- the bright field image 231a and the protein expression status display 231c may be switched and displayed in accordance with a switching instruction from the operation unit 22.
- the bright field image 231a, the fluorescent bright spot image 231b, and the protein expression status display 231c displayed on the analysis result screen 231 may be displayed in an enlarged or reduced manner so that they can be easily observed.
- an image obtained by simply superimposing the bright field image 231a and the fluorescent bright spot image 231b may be displayed in accordance with an instruction from the operation unit 22, and in such a case, the specific protein may be displayed based on the superimposed image. It is also possible to visually present the state of the occurrence of the disease to a doctor and prompt judgment.
- the analysis result can be printed out or output to an external device by pressing the print button 231d or the send button 231e.
- the print button 231d is pressed by the operation unit 22
- the data of the analysis result is transmitted by the control unit 21 to a printer (not shown) via a communication network such as the communication I / F 24 or LAN, and the analysis result is printed out.
- the control unit 21 transmits the analysis result data to an external device (for example, PACS (Picture Archiving and Communication System for PACS) via a communication network such as the communication I / F 24 or a LAN). medical application)).
- PACS Picture Archiving and Communication System for PACS
- the observation target cell is determined based on the cell feature amount, and then the images are aligned based on the image feature amount of the bright field image and the fluorescent bright spot image.
- the expression state of the specific protein in the observation target cell is obtained with high accuracy.
- eosin staining information is used as an information source commonly detected in each image, and the image feature amount in each image is calculated from the staining information, and the common points are determined. Since they match, the bright field image and the fluorescence image are accurately aligned.
- the fluorescent bright spot of the fluorescent image is accurately superimposed on the observation target cell of the bright field image determined from the cell feature amount, as a result, the bright field image and the fluorescent image are accurately aligned.
- the expression of a specific protein in the target cell can be accurately quantified.
- the expression status of the specific protein is classified into a plurality of stages based on whether or not the number of fluorescent luminescent spots exceeds a plurality of predetermined thresholds for the observation target cell.
- An image segmented in a manner corresponding to is output as an analysis result. Therefore, the cells to be observed are displayed in different display modes depending on the expression state of the specific protein, and the doctor can efficiently grasp the overexpression and spread of the specific protein that is an index of cancer malignancy. It is possible to make an appropriate treatment plan.
- the Ki67 protein in breast cancer has been mentioned as an example of the specific protein, but the specific protein is not limited thereto.
- the feature quantity that quantitatively indicates the expression level of the specific protein corresponding to the lesion type It can be provided to a doctor.
- an HDD or a semiconductor non-volatile memory is used as a computer-readable medium of the program according to the present invention, but the present invention is not limited to this example.
- a portable recording medium such as a CD-ROM can be applied.
- a carrier wave carrier wave is also applied as a medium for providing program data according to the present invention via a communication line.
- the present invention can be particularly suitably used for accurately quantifying the expression of a specific protein in an observation target cell during pathological diagnosis.
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Abstract
Description
この点、本発明者が特許文献3の技術について検討を重ねたところ、上記の通り、通常、細胞形態画像と蛍光画像全体とを重ね合わせる場合、基本的には位置合わせのズレは大きな問題にならなかったが、細胞核のみを観察対象とする場合には、特に位置合わせが重要となることがわかってきた。すなわち、細胞形態画像から抽出した観察対象細胞(観察すべき細胞核)は非常に小さい場合が多く、そこでの特定のタンパク質の発現を正確に定量するためには、極めて正確に画像を位置合わせする必要があることがわかってきており、観察対象細胞内での特定のタンパク質の発現を正確に定量しうるさらなる改良が望まれる。
組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを入力する入力手段と、
前記細胞形態画像と前記蛍光画像とで共通に検出される情報源に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする位置合わせ手段と、
を備えることを特徴とする画像処理装置が提供される。
前記画像処理装置と、
前記画像処理装置で使用される、前記細胞形態画像と前記蛍光画像とを取得する画像取得装置と、
を備えることを特徴とする病理診断支援システムが提供される。
コンピュータを、
組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを入力する入力手段、
前記細胞形態画像と前記蛍光画像とで共通に検出される情報源に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする位置合わせ手段、
として機能させるための画像処理プログラムが提供される。
組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを用いる病理診断支援方法であって、
前記細胞形態画像と前記蛍光画像とで共通に検出される一定の染色試薬で前記組織切片を染色する工程と、
染色後の前記組織切片から、前記細胞形態画像と前記蛍光画像とを取得する工程と、
前記一定の染色試薬による染色情報に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする工程と、
を有することを特徴とする病理診断支援方法が提供される。
図1に、本実施の形態における病理診断支援システム100の全体構成例を示す。病理診断支援システム100は、所定の染色試薬で染色された人体の組織切片の顕微鏡画像を取得し、取得された顕微鏡画像を解析することにより、観察対象の組織切片における特定の生体物質の発現を定量的に表す特徴量を出力するシステムである。
顕微鏡画像取得装置1Aは、照射手段、結像手段、撮像手段、通信I/F等を備えて構成されている。照射手段は、光源、フィルター等により構成され、スライド固定ステージに載置されたスライド上の組織切片に光を照射する。結像手段は、接眼レンズ、対物レンズ等により構成され、照射した光によりスライド上の組織切片から発せられる透過光、反射光、又は蛍光を結像する。撮像手段は、CCD(Charge Coupled Device)センサー等を備え、結像手段により結像面に結像される像を撮像して顕微鏡画像のデジタル画像データを生成する顕微鏡設置カメラである。通信I/Fは、生成された顕微鏡画像の画像データを画像処理装置2Aに送信する。本実施の形態において、顕微鏡画像取得装置1Aは、明視野観察に適した照射手段及び結像手段を組み合わせた明視野ユニット、蛍光観察に適した照射手段及び結像手段を組み合わせた蛍光ユニットが備えられており、ユニットを切り替えることにより明視野/蛍光を切り替えることが可能である。
図2に、画像処理装置2Aの機能構成例を示す。図2に示すように、画像処理装置2Aは、制御部21、操作部22、表示部23、通信I/F24、記憶部25等を備えて構成され、各部はバス26を介して接続されている。
その他、画像処理装置2Aは、LANアダプターやルーター等を備え、LAN等の通信ネットワークを介して外部機器と接続される構成としてもよい。
明視野画像は、HE(ヘマトキシリン-エオジン)染色試薬を用いて染色された組織切片を、顕微鏡画像取得装置1Aにおいて明視野で拡大結像及び撮影することにより得られる顕微鏡画像である。ヘマトキシリンは青紫色の色素であり、細胞核、骨組織、軟骨組織の一部、漿液成分など(好塩基性の組織等)を染色する。エオジンは赤~ピンク色の色素であり、細胞質、軟部組織の結合組織、赤血球、線維素、内分泌顆粒など(好酸性の組織等)を染色する。図3に、HE染色を行った組織切片を撮影した明視野画像の一例を示す。図3に示すように、HE染色を行った組織切片を撮影した明視野画像においては、組織切片における細胞の形態が表れている。すなわち、明視野画像は組織切片における細胞の形態を表す細胞形態画像である。かかる明視野画像では、細胞核は、周囲の細胞質よりも濃い色(青紫色)で周囲と区別して表れており、細胞核の形態をはっきり捉えることができる。
蛍光画像は、特定の生体物質と特異的に結合及び/又は反応する生体物質認識部位が結合した蛍光物質を内包したナノ粒子(蛍光物質内包ナノ粒子と呼ぶ)を含む染色試薬を用いて染色された組織切片に対し、顕微鏡画像取得装置1Aにおいて所定波長の励起光を照射して蛍光物質内包ナノ粒子を発光(蛍光)させ、この蛍光を拡大結像及び撮影することにより得られる顕微鏡画像である。即ち、蛍光画像に現れる蛍光は、組織切片における、生体物質認識部位に対応する特定の生体物質の発現を示すものである。図4に、蛍光画像の一例を示す。
ここで、蛍光画像の取得方法について、この蛍光画像の取得に際して用いられる染色試薬(蛍光物質内包ナノ粒子)、染色試薬による組織切片の染色方法等も含めて詳細に説明する。
蛍光画像の取得のための染色試薬に用いられる蛍光物質としては、蛍光有機色素及び量子ドット(半導体粒子)を挙げることができる。200~700nmの範囲内の波長の紫外~近赤外光により励起されたときに、400~1100nmの範囲内の波長の可視~近赤外光の発光を示すことが好ましい。
量子ドットは必要に応じて、有機ポリマー等により表面処理が施されているものを用いてもよい。例えば、表面カルボキシ基を有するCdSe/ZnS(インビトロジェン社製)、表面アミノ基を有するCdSe/ZnS(インビトロジェン社製)等が挙げられる。
本実施の形態において蛍光物質内包ナノ粒子とは、蛍光物質がナノ粒子内部に分散されたものをいい、蛍光物質とナノ粒子自体とが化学的に結合していても、結合していなくてもよい。
ナノ粒子を構成する素材は特に限定されるものではなく、ポリスチレン、ポリ乳酸、シリカ等を挙げることができる。
本実施の形態に係る生体物質認識部位とは、目的とする生体物質と特異的に結合及び/又は反応する部位である。目的とする生体物質は、それと特異的に結合する物質が存在するものであれば特に限定されるものではないが、代表的にはタンパク質(ペプチド)および核酸(オリゴヌクレオチド、ポリヌクレオチド)、抗体等が挙げられる。したがって、そのような目的とする生体物質に結合する物質としては、前記タンパク質を抗原として認識する抗体やそれに特異的に結合する他のタンパク質等、および前記核酸にハイブリタイズする塩基配列を有する核酸等が挙げられる。具体的には、細胞表面に存在するタンパク質であるHER2に特異的に結合する抗HER2抗体、細胞核に存在するエストロゲン受容体(ER)に特異的に結合する抗ER抗体、細胞骨格を形成するアクチンに特異的に結合する抗アクチン抗体等があげられる。中でも抗HER2抗体及び抗ER抗体を蛍光物質内包ナノ粒子に結合させたものは、乳癌の投薬選定に用いることができ、好ましい。
以下、組織切片の染色方法について述べる。以下に説明する染色方法は病理切片組織に限定せず、細胞染色にも適用可能である。
また、以下に説明する染色方法が適用できる切片の作製法は特に限定されず、公知の方法により作製されたものを用いることができる。
キシレンを入れた容器に病理切片を浸漬させ、パラフィンを除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、3分以上30分以下であることが好ましい。また、必要により浸漬途中でキシレンを交換してもよい。
次いで、エタノールを入れた容器に病理切片を浸漬させ、キシレンを除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、3分以上30分以下であることが好ましい。また、必要により浸漬途中でエタノールを交換してもよい。
次いで、水を入れた容器に病理切片を浸漬させ、エタノールを除去する。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、3分以上30分以下であることが好ましい。また、必要により浸漬途中で水を交換してもよい。
公知の方法にならい、目的とする生体物質の賦活化処理を行う。賦活化条件に特に定めはないが、賦活液としては、0.01Mクエン酸緩衝液(pH6.0)、1mMEDTA溶液(pH8.0)、5%尿素、0.1Mトリス塩酸緩衝液等を用いることができる。加熱機器は、オートクレーブ、マイクロウェーブ、圧力鍋、ウォーターバス等を用いることができる。温度は特に限定されるものではないが、室温で行うことができる。温度は50-130℃、時間は5-30分で行うことができる。
次いで、PBS(Phosphate Buffered Saline:リン酸緩衝生理食塩水)を入れた容器に、賦活化処理後の切片を浸漬させ、洗浄を行う。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、3分以上30分以下であることが好ましい。また、必要により浸漬途中でPBSを交換してもよい。
生体物質認識部位が結合された蛍光物質内包ナノ粒子のPBS分散液を病理切片に載せ、目的とする生体物質と反応させる。蛍光物質内包ナノ粒子と結合させる生体物質認識部位を変えることにより、さまざまな生体物質に対応した染色が可能となる。数種類の生体物質認識部位が結合された蛍光物質内包ナノ粒子を用いる場合には、それぞれの蛍光物質内包ナノ粒子PBS分散液を予め混合しておいてもよいし、別々に順次病理切片に載せてもよい。
温度は特に限定されるものではないが、室温で行うことができる。反応時間は、30分以上24時間以下であることが好ましい。
蛍光物質内包ナノ粒子による染色を行う前に、BSA含有PBS等、公知のブロッキング剤を滴下することが好ましい。
次いで、PBSを入れた容器に、染色後の切片を浸漬させ、未反応蛍光物質内包ナノ粒子の除去を行う。温度は特に限定されるものではないが、室温で行うことができる。浸漬時間は、3分以上30分以下であることが好ましい。また、必要により浸漬途中でPBSを交換してもよい。カバーガラスを切片に載せ、封入する。必要に応じて市販の封入剤を使用してもよい。
なお、HE染色試薬を用いて染色を行う場合、カバーガラスによる封入前にHE染色を行う。
染色した病理切片に対し顕微鏡画像取得装置1Aを用いて、広視野の顕微鏡画像(蛍光画像)を取得する。顕微鏡画像取得装置1Aにおいて、染色試薬に用いた蛍光物質の吸収極大波長及び蛍光波長に対応した励起光源及び蛍光検出用光学フィルターを選択する。
蛍光画像の視野は、3mm2以上であることが好ましく、30mm2以上であることがさらに好ましく、300mm2以上であることがさらに好ましい。
ここで、本件出願人は、以下に説明するように、一実施例として、Cy5内包シリカナノ粒子(以下、ナノ粒子1という。)を作製し、ナノ粒子1に対して抗HER2抗体を結合させた標識材料Aを作製した。また、CdSe/ZnS内包シリカナノ粒子(以下、ナノ粒子2という)を作製し、ナノ粒子2に対して抗HER2抗体を結合させた標識材料Bを作製した。そして、作製した標識材料A、B及び比較例としての標識材料C、Dを用いて予めFISHスコアを測定したヒト***組織の隣接切片を用いて免疫染色を行って視野を変えて複数の蛍光画像を取得し、各蛍光画像に現れている蛍光輝点の数を計測してFISHスコアとの関連を調べる実験を行った。
(合成例1:蛍光有機色素内包シリカ:Cy5内包シリカナノ粒子の合成)
下記工程(1)~(5)の方法により、Cy5内包シリカナノ粒子(ナノ粒子1)を作製した。
工程(1):Cy5のN-ヒドロキシスクシンイミドエステル誘導体(GEヘルスケア社製)1mg(0.00126mmol)とテトラエトキシシラン400μL(1.796mmol)を混合した。
工程(2):エタノール40mLと14%アンモニア水10mLを混合した。
工程(3):工程(2)で作製した混合液を室温下で撹拌しているところに、工程(1)で調製した混合液を添加した。添加開始から12時間撹拌を行った。
工程(4):反応混合物を10000Gで60分遠心分離を行い、上澄みを除去した。
工程(5):エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を1回ずつ行った。
得られたナノ粒子1を走査型電子顕微鏡(SEM;日立(登録商標)社製S-800型)で観察したところ、平均粒径は110nm、変動係数は12%であった。
下記工程(1)~(5)の方法により、CdSe/ZnS内包シリカナノ粒子(以下、ナノ粒子2という。)を作製した。
工程(1):CdSe/ZnSデカン分散液(インビトロジェン社Qdot655)10μLとテトラエトキシシラン40μLを混合した。
工程(2):エタノール4mLと14%アンモニア水1mLを混合した。
工程(3):工程(2)で作製した混合液を室温下で撹拌しているところに、工程(1)で作製した混合液を添加した。添加開始から12時間撹拌を行った。
工程(4):反応混合物を10000Gで60分遠心分離を行い、上澄みを除去した。
工程(5):エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を1回ずつ行った。
得られたナノ粒子2を走査型電子顕微鏡で観察したところ、平均粒径は130nm、変動係数は13%であった。
下記工程(1)~(12)の方法により、蛍光物質内包シリカナノ粒子に対して抗体を結合させた。ここでは、ナノ粒子1を用いた例を示すが、ナノ粒子2についても同様である。
工程(1):1mgのナノ粒子1を純水5mLに分散させた。次いで、アミノプロピルトリエトキシシラン水分散液100μLを添加し、室温で12時間撹拌した。
工程(2):反応混合物を10000Gで60分遠心分離を行い、上澄みを除去した。
工程(3):エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を1回ずつ行った。
得られたアミノ基修飾したシリカナノ粒子のFT-IR測定を行ったところ、アミノ基に由来する吸収が観測でき、アミノ基修飾されたことが確認できた。
工程(5):工程(4)で調整した溶液に、最終濃度10mMとなるようSM(PEG)12(サーモサイエンティフィック社製、succinimidyl-[(N-maleomidopropionamid)-dodecaethyleneglycol]ester)を混合し、1時間反応させた。
工程(6):反応混合液を10000Gで60分遠心分離を行い、上澄みを除去した
工程(7):EDTAを2mM含有したPBSを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順による洗浄を3回行った。最後に500μLのPBSを用いて再分散させた。
工程(9):反応混合物についてゲルろ過カラムにより過剰のDTTを除去し、還元化抗HER2抗体溶液を得た。
工程(11):10mMメルカプトエタノール4μLを添加し、反応を停止させた。
工程(12):反応混合物を10000Gで60分遠心分離を行い、上澄みを除去した後、EDTAを2mM含有したPBSを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順による洗浄を3回行った。最後に500μLのPBSを用いて再分散させ、抗HER2抗体が結合された蛍光物質内包シリカナノ粒子を得た。
下記工程(1)~(10)の方法により、作製した抗体結合標識材料A~Dを用い、予めFISHスコアを測定したヒト***組織の隣接切片を用いて免疫染色を行った。染色切片はコスモバイオ社製の組織アレイスライド(CB-A712)を用いた。FISHスコアで1~9の24切片を用いた。
工程(2):エタノールを入れた容器に病理切片を30分浸漬させた。途中3回エタノールを交換した。
工程(3):水を入れた容器に病理切片を30分浸漬させた。途中3回水を交換した。
工程(4):10mMクエン酸緩衝液(pH6.0)に病理切片を30分浸漬させた。
工程(5):121度で10分オートクレーブ処理を行った。
工程(6):PBSを入れた容器に、オートクレーブ処理後の切片を30分浸漬させた。
工程(7):1%BSA含有PBSを組織に載せて、1時間放置した。
工程(8):1%BSA含有PBSで0.05nMに希釈した抗HER2抗体が結合された標識材料A~Dを、各組織切片に載せて3時間放置した。
工程(9):PBSを入れた容器に、染色後の切片をそれぞれ30分浸漬させた。
工程(10):Merck Chemicals社製Aquatexを滴下後、カバーガラスを載せ封入した。
各標識材料A~Dを用いて染色した組織切片について、視野(観察面積)を変えて複数の蛍光画像を取得し、画像解析ソフトにより、各蛍光画像から蛍光輝点の数(輝点数)を計測した。
なお、顕微鏡は、カールツアイス社製正立顕微鏡Axio Imager M2を用い、対物レンズを20倍に設定し、630~670nmの波長を有する励起光を照射して、組織切片から発せられる蛍光を結像し、顕微鏡設置カメラ(モノクロ)により蛍光画像(画像データ)を取得し、画像解析ソフトにより輝点数を計測した。なお、上記カメラは画素サイズ6.4μm×6.4μm、縦画素数1040個、横画素数1388個(撮像領域8.9mm×6.7mm)を有している。
また、各標識材料A~Dについて、各視野において、計測された輝点数とFISHスコアとの相関係数Rを算出した。FISHスコアは、HER2遺伝子の過剰発現レベルと対応しており、FISHスコアの値が大きいほど、HER2遺伝子の過剰発現レベルが高いことを示している。
以下、病理診断支援システム100において、上記説明した蛍光画像及び明視野画像を取得して解析を行う動作について説明する。ここでは、特定のタンパク質(ここでは、乳癌組織におけるKi67タンパクとする。以下、特定タンパクと呼ぶ。)を認識する生体物質認識部位が結合した蛍光物質内包ナノ粒子を含む染色試薬を用いて染色された組織切片を観察対象とする場合を例にとり説明するが、これに限定されるものではない。
その後、顕微鏡画像取得装置1Aにおいて、(a1)~(a5)の手順により明視野画像及び蛍光画像が取得される。
(a1)操作者は、HE染色試薬と蛍光物質内包ナノ粒子を含む染色試薬とによりそれぞれ染色された組織切片をスライドに載置し、そのスライドを顕微鏡画像取得装置1Aのスライド固定ステージに設置する。
(a2)明視野ユニットに設定し、撮影倍率、ピントの調整を行い、組織上の観察対象の領域を視野に納める。
(a3)撮像手段で撮影を行って明視野画像の画像データを生成し、画像処理装置2Aに画像データを送信する。
(a4)ユニットを蛍光ユニットに変更する。
(a5)視野及び撮影倍率を変えずに撮像手段で撮影を行って蛍光画像の画像データを生成し、画像処理装置2Aに画像データを送信する。
なお、HE染色を同時に行わない場合においては、組織の自家蛍光が微弱なため励起光の波長の範囲は、一般的な200nm~700nmの範囲で特に限定せずとも自家蛍光と蛍光物質内包ナノ粒子からの蛍光の発光差を確保して両者を区別して認識可能とする(両者の光量差10%(1.1倍)以上)を確保することができる。
図10に、画像処理装置2Aにおける画像解析処理のフローチャートを示す。図10に示す画像解析処理は、制御部21と記憶部25に記憶されているプログラムとの協働により実行される。
図11に、ステップS2における処理の詳細フローを示す。ステップS2の処理は、制御部21と記憶部25に記憶されているプログラムとの協働により実行される。
次いで、モノクロ画像に対し予め定められた閾値を用いて閾値処理が施され、各画素の値が2値化される(ステップS202)。図12Bに、閾値処理後の二値画像の一例を示す。
なお、後述する蛍光輝点の抽出におけるラベルの番号と区別するため、コンピュータの保持できる最大値をMAXとし、現在までに行ったラベリング回数をLabel_tempとすると、新たな細胞核にはラベルLabel_nucleusとして、MAX-Label_tempが付与される。例えば、101個目の細胞核にラベルを付与する場合、Label_temp=100であるので、MAX=65536とすると、Label_nucleusとして65436が付与される。ラベリング処理後、処理は図10のステップS3に移行する。
細胞核の面積Aについては、予め細胞核画像に対応した基準となる長さを測定することで画素(ピクセル)の大きさを算出し、ステップ2で抽出された各細胞核内の画素数を積算することにより、細胞核の面積Aが決定される。
細胞核の平均濃度Bは、細胞核内の各画素(ピクセル)のグレイスケールに変換した輝度信号値を求め、その平均値を算出することにより決定される。
ピクセル輝度バラツキCは、細胞核内の各画素(ピクセル)の輝度信号値の標準偏差を算出することにより決定される。
細胞核の円形度D及び扁平率Eは、ステップ2で抽出された各細胞核について、細胞核画像から得られる一定の値を、下記式(d)、(e)に当てはめることで決定される。
(円形度D)=4πS/L2 … (d)
(扁平率F)=(a-b)/a … (e)
ただし、式(d)中、「S」は細胞核の面積(細胞核の面積A)を、「L」は細胞核の外周長をそれぞれ表す。式(e)中、「a」は長半径を、「b」は短半径をそれぞれ表す。
核縁の核面積に対する太さの割合Fの算出にあたっては、まず、細胞核の中心部から80%の面積の輝度信号値の平均値Laveを求め、細胞核外縁部の20%の面積領域について、画素(ピクセル)の輝度信号値がLaveに対し20%以上低い値を有する領域を細胞核の核縁とした。次に、その核縁の面積を求め、細胞核全体の面積(細胞核の面積A)との比を算出することにより、核縁の核面積に対する太さの割合Fが決定される。
(i)細胞核の面積Aが、算出されたすべての細胞核の面積Aのうち、上位60%以上、好ましくは上位50%以上、更に好ましくは上位40%以上に含まれる
(ii)細胞核の平均濃度Bが、算出されたすべての細胞核の平均濃度Bのうち、下位50%以下、好ましくは下位40%以下、更に好ましくは上位30%以下に含まれる
(iii)細胞核領域内のピクセル輝度バラツキ(σ値)Cが、細胞核画像を、8ビット画像でかつグレイスケールに変換した画像において、σ≧30、好ましくはσ≧40、更に好ましくはσ≧50である
(iv)細胞核の円形度Dが、0.8以下、好ましくは0.6以下、更に好ましくは0.4以下である
(v)細胞核の扁平率Eが、0.6以下、好ましくは0.4以下、更に好ましくは0.2以下である
(vi)核縁の核面積に対する太さの割合Fが、3%以上、好ましくは5%以上、更に好ましくは10%以上である
なお、上記(i)~(vi)の条件、すなわち細胞特徴量の各因子(細胞核の面積A~核縁の核面積に対する太さの割合F)は任意に選択され適宜変更されてもよいし、各因子中の閾値も任意に選択され適宜変更されてもよい。もちろん、細胞特徴量の因子として上記とは異なる別の因子が使用されてよい。
図13に、ステップS5における処理の詳細フローを示す。ステップS5の処理は、制御部21と記憶部25に記憶されているプログラムとの協働により実行される。
次いで、R成分が抽出された画像にTophat変換が施される(ステップS502)。Tophat変換は、入力画像の各画素の値から、入力画像に最小値フィルター及び最大値フィルターをこの順でかけた画像の、対応する画素の値を減算する処理である。最小値フィルターは、注目画素の近傍の画素(例えば、3×3画素)のうちの最小値で注目画素の値を置き換えるものである。最大値フィルターは、注目画素の近傍の画素(例えば、3×3画素)のうちの最大値で注目画素の値を置き換えるものである。Tophat変換により、濃淡プロファイル上の小突起(近傍の画素に比べて輝度の高い領域)を抽出することができる。これにより、蛍光輝点候補画像を得ることができる。図14Aに、蛍光輝点候補画像の一例を示す。
当該位置合わせに用いられる明視野画像はステップS2で得られた細胞核画像(図12C参照)であり、当該位置合わせに用いられる蛍光画像はステップS5で得られた蛍光輝点画像(図14B参照)である。
明視野画像と蛍光画像とで共通に検出される情報源としては、明視野画像及び蛍光画像の両方で認識可能な情報が用いられ、本実施形態では組織切片の染色材料であるエオジンの染色情報が用いられる。
エオジンの染色情報からは、明視野画像及び蛍光画像の各画像においてその画像を特徴付ける画像特徴量が算出される。当該画像特徴量としては、明視野画像及び蛍光画像の両方で観察できるコントラスト情報やエッジ情報、輪郭情報等が算出され用いられる。
コントラスト情報としては、特定領域やその特定領域と画像全体との間の色差や輝度差の情報が用いられ、必要に応じ二値化処理が施され、情報そのものが強調される。
エッジ情報及び輪郭情報は、明視野画像及び蛍光画像の各画像を画像処理することより得られる。画像処理方法としては、キャニー法やアンシャープマスク法が好ましく用いられる。
詳しくは、明視野画像のエオジンの染色情報から、明視野画像におけるコントラスト情報、エッジ情報及び輪郭情報が算出される。これと同様に、蛍光画像のエオジンの染色情報からも、蛍光画像におけるコントラスト情報、エッジ情報及び輪郭情報が算出される。そしてこれら情報同士が比較され、その共通点を合致させることにより、明視野画像と蛍光画像との位置合わせが行われる。コントラスト情報、エッジ情報及び輪郭情報の比較では、これら情報のうち、少なくとも1つの情報同士が比較されればよく、例えばコントラスト情報同士のみが比較されてもよいし、コントラスト情報、エッジ情報及び輪郭情報のすべての情報同士が比較されてもよい。
例えば、コントラスト情報同士を比較する場合には、図15に示すように、明視野画像と蛍光画像とで、左端側の特定領域30と右端側の特定領域32とにおけるコントラスト情報を対比させ、それら特定領域30、32における各共通点(濃淡)が合致するよう、各画像が重ね合わされる(位置合わせされる)。このとき、明視野画像と蛍光画像は、特定領域30、32における各共通点が合致するよう、画像自体が拡大又は縮小されてもよい。
第3の染色試薬としては、蛍光画像中の蛍光輝点源の蛍光粒子(すなわち上記蛍光物質内包ナノ粒子)と発光波長が重ならない蛍光粒子や、明らかに大きさ(粒子径)や形状の異なる、可視光及び蛍光の両方で観察される蛍光粒子を含む染色試薬が挙げられる。当該蛍光粒子は発光波長、粒子径及び形状の少なくとも1つが異なっていればよく、例えば発光波長のみが異なっていてもよいし、発光波長、粒子径及び形状のすべてが異なっていてもよい。
具体的に当該蛍光粒子としては、粒子径が1.0~5.0μmの蛍光粒子が好ましく用いられ、例えば、蛍光色素であるFITCやAlexa Fluor(登録商標)488をシリカに内包させた粒子が挙げられる。
第3の染色試薬による染色情報を情報源とした場合も、明視野画像と蛍光画像とを位置合わせするときは、上記と同様、コントラスト情報、エッジ情報及び輪郭情報等の画像特徴量が算出され、それら画像特徴量に基づき、明視野画像と蛍光画像とが正確に位置合わせされる。
詳しくは、明視野画像と蛍光画像とを重ね合わせた状態において、ステップS3で決定した観察対象細胞に対応する部分の蛍光輝点数が算出される。観察対象細胞上に重なる蛍光輝点数は、癌の悪性度や進行度の指標となる特定タンパクの発現状況を示す。
悪性度の判定や治療計画に用いる統計方法としては、特に限定されないが、例えば、観察対象細胞上の蛍光輝点数の総計値を観察対象細胞数で除した値や、観察対象細胞上の蛍光輝点数の総計値を観察対象細胞の総面積で除した値が用いられ、また観察対象細胞上の蛍光輝点数をヒストグラム化して一定数以上の蛍光輝点を有する観察対象細胞数の比率等も好ましく用いられる。
例えば、ステップS8では、観察対象細胞上の蛍光輝点数が予め定められた複数の閾値を超えるか否かに基づいて、特定タンパクの発現状況が複数の段階に分類され、高発現領域、中発現領域、低発現領域、極低発現領域といった態様で区分けされる。その後、明視野画像上に、分類結果に応じて異なる表示態様で区分け(例えば、色分け)された画像が生成され、解析結果画面231として表示部23に表示出力される。
タンパク発現状況表示231cには、明視野画像上に、特定タンパクの高発現領域、中発現領域、低発現領域、極低発現領域が色分けして表示されている。特定タンパクの発現状況に応じて色分けして表示されるので、医師は、癌の悪性度の指標となる特定タンパクの過剰発現、その広がりを効率よく把握することが可能となり、適切な治療計画を立てることが可能となる。
例えば、タンパク発現状況表示231cのみを表示することとしてもよい。また、操作部22からの切り替え指示に応じて明視野画像231aとタンパク発現状況表示231cを切り替え表示することとしてもよい。また、解析結果画面231に表示する明視野画像231a、蛍光輝点画像231b、タンパク発現状況表示231cは、観察しやすいように何れか又は全てを拡大縮小して表示することとしてもよい。
さらに、操作部22からの指示に応じて、明視野画像231aと蛍光輝点画像231bとを単に重ね合わせた画像を表示することとしてもよく、かかる場合にはその重ね合わせた画像により、特定タンパクの発現状況を医師に視覚的に提示し、判断を促すこともできる。
操作部22により印刷ボタン231dが押下されると、制御部21により解析結果のデータが通信I/F24やLAN等の通信ネットワークを介して図示しないプリンタに送信され、解析結果が印刷出力される。また、操作部22により送信ボタン231eが押下されると、制御部21により解析結果のデータが通信I/F24やLAN等の通信ネットワークを介して外部機器(例えば、PACS(Picture Archiving and Communication System for medical application))に送信される。
特に、明視野画像と蛍光画像との位置合わせでは、各画像で共通に検出される情報源としてエオジンの染色情報を用いて、かかる染色情報から各画像における画像特徴量を算出しその共通点を一致させるから、明視野画像と蛍光画像とが正確に位置合わせされる。かかる場合、細胞特徴量から決定された明視野画像の観察対象細胞に対し蛍光画像の蛍光輝点が正確に重ねられるから、結果的に明視野画像と蛍光画像との正確な位置合わせにより、観察対象細胞内での特定タンパクの発現を正確に定量することができる。
2A 画像処理装置
3A ケーブル
21 制御部
22 操作部
23 表示部
24 通信I/F
25 記憶部
26 バス
100 病理診断支援システム
Claims (8)
- 組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを入力する入力手段と、
前記細胞形態画像と前記蛍光画像とで共通に検出される情報源に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする位置合わせ手段と、
を有することを特徴とする画像処理装置。 - 請求項1に記載の画像処理装置において、
前記情報源が一定の染色試薬による染色情報であり、
前記位置合わせ手段が、前記細胞形態画像と前記蛍光画像との前記各染色情報から、各画像における画像特徴量を算出し、それら画像特徴量に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせすることを特徴とする画像処理装置。 - 請求項2に記載の画像処理装置において、
前記情報源が前記一定の染色試薬とは異なる他の染色試薬による他の染色情報であり、
前記位置合わせ手段が、前記細胞形態画像と前記蛍光画像との前記各他の染色情報から、各画像における画像特徴量を算出し、それら画像特徴量に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせすることを特徴とする画像処理装置。 - 請求項3に記載の画像処理装置において、
前記他の染色試薬には、前記蛍光画像中の蛍光輝点源の蛍光粒子に対し、発光波長、粒子径及び形状の少なくとも1つが異なる他の蛍光粒子が含まれていることを特徴とする画像処理装置。 - 請求項1~4のいずれか一項に記載の画像処理装置において、
前記形態観察画像から、細胞核の面積、細胞核の平均濃度、細胞核の輝度バラツキ、細胞核の円形度、細胞核の扁平率及び細胞核の核縁の核面積に対する太さのうち、少なくとも1つの因子を含む細胞特徴量を算出する算出手段と、
前記細胞特徴量に基づき、観察対象となる観察対象細胞を決定する決定手段と、
前記観察対象細胞に対応する位置合わせ後の前記蛍光画像の蛍光輝点数に基づき、前記観察対象細胞における前記特定タンパクの発現状況を判定する判定手段と、
を有することを特徴とする画像処理装置。 - 請求項1~5のいずれか一項に記載の画像処理装置と、
前記画像処理装置で使用される、前記細胞形態画像と前記蛍光画像とを取得する画像取得装置と、
を備えることを特徴とする病理診断支援システム。 - コンピュータを、
組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを入力する入力手段、
前記細胞形態画像と前記蛍光画像とで共通に検出される情報源に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする位置合わせ手段、
として機能させるための画像処理プログラム。 - 組織切片における細胞の形態を表す細胞形態画像と、前記組織切片の同一範囲における特定タンパクの発現を蛍光輝点で表す蛍光画像とを用いる病理診断支援方法であって、
前記細胞形態画像と前記蛍光画像とで共通に検出される一定の染色試薬で前記組織切片を染色する工程と、
染色後の前記組織切片から、前記細胞形態画像と前記蛍光画像とを取得する工程と、
前記一定の染色試薬による染色情報に基づき、前記細胞形態画像と前記蛍光画像とを位置合わせする工程と、
を有することを特徴とする病理診断支援方法。
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