WO2015145644A1 - 画像処理装置および画像処理プログラム - Google Patents
画像処理装置および画像処理プログラム Download PDFInfo
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- WO2015145644A1 WO2015145644A1 PCT/JP2014/058725 JP2014058725W WO2015145644A1 WO 2015145644 A1 WO2015145644 A1 WO 2015145644A1 JP 2014058725 W JP2014058725 W JP 2014058725W WO 2015145644 A1 WO2015145644 A1 WO 2015145644A1
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Definitions
- the present invention relates to an image processing apparatus and an image processing program, and more particularly to image processing used for pathological diagnosis.
- a cell membrane is stained with a certain phosphor, and a biological substance existing on the cell membrane is stained with another phosphor different from that to measure the number of bright spots of the phosphor on the cell membrane.
- the expression level of the biological substance in the cell membrane is evaluated (see paragraphs 0012 to 0013, 0060 to 0066, 0107, etc.).
- tissue sections are usually stained with hematoxylin (H stain) or hematoxylin-eosin (HE stain), and the technique for staining cell membranes as in Patent Document 1 is special. It takes time and effort.
- the tissue section is stained with HE to determine the expression level of the biological material (see paragraphs 0019, 0029, 0085, etc.).
- a tissue section is HE-stained to obtain a bright field image
- a cell nucleus region is extracted from the bright field image
- a predetermined range is estimated as a cell region from the center of gravity of the extracted cell nucleus region.
- the expression level of the biological substance in the cell region is determined based on the cell nucleus and the number of fluorescent bright spots contained in the cell region (see paragraphs 0092 to 0104, FIGS. 20A, 0128, etc.).
- a circular region having a predetermined radius from the center of gravity of the cell nucleus 202 of the cell 200 is estimated as the cell region 210, and the fluorescent bright spots (204a, 204c, 224a and 224b), the fluorescent spots 224a and 224b should belong to the cell nucleus 222 of the cell 220, and the neighboring areas different from the true cell 220 to which the fluorescent spots 224a and 224b should belong.
- Cell (fake cell) 200 Conversely, when a tissue section after HE staining is placed on a slide and observed with a microscope, as shown in FIG.
- a main object of the present invention is to provide an image processing apparatus and an image processing program capable of accurately assigning a fluorescent bright spot to a true cell.
- the fluorescent bright spot since the cell nucleus to which the fluorescent bright spot belongs should be specified based on the actual distance between the cell nucleus and the fluorescent bright spot, the fluorescent bright spot can be accurately assigned to the true cell.
- FIG. 1 is a diagram schematically showing a configuration of a pathological diagnosis support system. It is a block diagram which shows roughly the functional structure of an image processing apparatus. It is a flowchart which shows roughly the flow of the image analysis process concerning 1st Embodiment. It is a flowchart which shows roughly the flow of the extraction process of a cell nucleus. It is a flowchart which shows roughly the flow of the extraction process of a fluorescent luminescent spot. It is a figure which illustrates roughly the calculation of the distance of the surface of a cell nucleus, and a fluorescent luminescent spot. It is a figure which shows roughly the cell nucleus of the shortest distance from a fluorescent luminescent point.
- FIG. 10 is a flowchart showing a modification of the image analysis processing according to the first to fourth embodiments. It is a figure which illustrates the problem of a prior art schematically. It is a figure which roughly compares and demonstrates a prior art and embodiment of this invention. It is a figure which illustrates a prior art schematically. It is a figure which illustrates the problem of a prior art schematically. It is a figure which roughly compares and demonstrates a prior art and embodiment of this invention.
- FIG. 1 shows an example of the overall configuration of the pathological diagnosis support system 10.
- the pathological diagnosis support system 10 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 10 is configured by connecting a microscope image acquisition device 1A and an image processing device 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 apparatus 1A and the image processing apparatus 2A is not particularly limited.
- the microscope image acquisition device 1A and the image processing device 2A may be connected via a LAN (Local Area Network) or may 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 the tissue section on the slide placed on the slide fixing stage with light.
- the image forming 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 image pickup means is a microscope-installed camera that includes a CCD (Charge Coupled Device) sensor and the like, picks up an image formed on the image forming surface by the image forming 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.
- 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 apparatus 2A calculates the expression distribution of a specific biological material in the tissue section to be observed by analyzing the microscope image transmitted from the microscope image acquisition apparatus 1A.
- FIG. 2 shows a functional configuration example of the image processing apparatus 2A.
- 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, 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. 3) in cooperation with the image processing program stored in the storage unit 25, and includes a first extraction unit, a second extraction unit, an attribution unit, A function as an exclusion means is realized.
- 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 includes a key press signal pressed by the keyboard and an operation signal by the mouse. Is output to the control unit 21 as an input signal.
- the display unit 23 includes 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.
- a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display)
- LCD Liquid Crystal Display
- 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 an input unit for a bright field image and a fluorescence image.
- the storage unit 25 is configured by, for example, an HDD (Hard Disk Drive), a semiconductor nonvolatile memory, or the like. As described above, the storage unit 25 stores various programs and various data.
- 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 performs analysis using the bright field image and the fluorescence image transmitted from the microscope image acquisition apparatus 1A.
- “Bright field image” refers to a tissue section stained with a hematoxylin staining reagent (H staining reagent) and a hematoxylin-eosin staining reagent (HE staining reagent) in the bright field in the microscope image acquisition apparatus 1A. It is a microscope image obtained by imaging
- Hematoxylin (H) is a blue-violet pigment that stains cell nuclei, bone tissue, part of cartilage tissue, serous components, etc.
- E is a red to pink pigment that stains cytoplasm, connective tissue of soft tissues, erythrocytes, fibrin, endocrine granules (acidophilic tissues, etc.).
- Fluorescence image means that tissue sections stained with a fluorescent staining reagent are irradiated with excitation light of a predetermined wavelength in the microscope image acquisition apparatus 1A to emit fluorescence, and this fluorescence is enlarged and imaged and photographed. It is the microscope image obtained by this.
- Fluorescent staining reagent is a staining reagent containing fluorescent substance-encapsulating nanoparticles bound with a biological substance recognition site that specifically binds and / or reacts with a specific biological substance.
- Fluorescent substance-encapsulating nanoparticles It is a nanoparticle containing a fluorescent substance. Fluorescence that appears in the fluorescence image is emitted when the fluorescent substance-containing nanoparticles (fluorescent substance) of the fluorescent staining reagent are excited, and indicates the expression of a specific biological substance corresponding to the biological substance recognition site in the tissue section It is.
- 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, and cyanine dye molecules.
- Quantum dots include II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements as components ("II-VI group quantum dots”, "III-V group quantum dots”, “ Or “Group IV quantum dots”). These quantum dots may be used alone or in combination of a plurality of types. Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
- a quantum dot having the above quantum dot as a core and a shell provided thereon.
- the core is CdSe and the shell is ZnS
- 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.
- Quantum dots may be subjected to surface treatment with an organic polymer or the like as necessary.
- organic polymer or the like 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.
- Fluorescent substance-encapsulated nanoparticles are nanoparticles encapsulating a fluorescent substance as described above, and more specifically, those in which a fluorescent substance is dispersed inside the nanoparticle.
- the substance and the nanoparticle itself may be chemically bonded or may not be bonded.
- the material constituting the nanoparticles is not particularly limited, and examples thereof include silica, polystyrene, polylactic acid, and melamine.
- the fluorescent substance-containing nanoparticles 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.
- the polymer nanoparticles encapsulating the quantum dots can be prepared by using the method of impregnating the quantum nanoparticles into polystyrene nanoparticles 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. It is the value calculated as. In this embodiment, the arithmetic average of the particle diameters of 1000 particles is defined as the average particle diameter.
- the coefficient of variation is also a value calculated from the particle size distribution of 1000 particles.
- Bio material recognition site is a site that specifically binds and / or reacts with a specific biological material.
- the specific biological substance is not particularly limited as long as a substance that specifically binds to the specific biological substance exists, but typically includes proteins (peptides) and nucleic acids (oligonucleotides, polynucleotides). It is done. Therefore, examples of the biological substance recognition site include an antibody that recognizes the protein as an antigen, another protein that specifically binds to the protein, and a nucleic acid having a base sequence that hybridizes to the nucleic acid.
- Specific biological substance recognition sites include anti-HER2 antibody that specifically binds to HER2, which is a protein present on the cell surface, anti-ER antibody that specifically binds to estrogen receptor (ER) present in the cell nucleus, cells An anti-actin antibody that specifically binds to actin forming the skeleton is exemplified.
- anti-HER2 antibody and anti-ER antibody combined with fluorescent substance-encapsulating nanoparticles can be used for breast cancer medication selection and 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 preferable from the viewpoint of bond stability.
- 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), etc. Is mentioned.
- silane coupling agent you may use 2 or more types together.
- a known procedure 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: Pierce (registered trademark)
- EDC 1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate Hydrochloride: 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.
- biological substance recognition sites include antibodies that recognize specific antigens: 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 (high molecular weight), Pankeratin, Pankeratin (high molecular weight
- HBc antigen HBs antigen
- hepatocyte specific antigen HER2
- HSV -I, HSV -II, HHV-8 IgA, IgG, IgM, IGF-1R, inhibin, insulin, kappa L chain, Ki67, lambda L chain, LH, lysozyme, macrophage, melan A, MLH-1, MSH-2, myeloperoxidase, myogenin, myoglobin, myosin, neurofilament, NSE, p27 (Kip1), p53, p53, P63, PAX 5, PLAP, pneumosis Tis Carini, podoplanin (D2-40), PGR, prolactin, PSA, prostatic acid phosphatase, Renal Cell Carcinoma, S100, somatostatin, spectrin, synaptophysin, TAG-72, TdT, thyroglobulin, T
- the following staining method is not limited to a pathological tissue section, but can also be applied to cultured cells.
- the method for preparing the tissue section is not particularly limited, and a tissue slice prepared by a known method can be used.
- a tissue 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 tissue 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. If necessary, ethanol may be exchanged during the immersion.
- the tissue 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. If necessary, water may be exchanged during the immersion.
- the activation process of the biological material of a tissue section 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.
- An autoclave, a microwave, a pressure cooker, a water bath, etc. can be used for a heating apparatus.
- 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 tissue 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, PBS may be exchanged during immersion.
- each fluorescent substance-encapsulated nanoparticle PBS dispersion may be mixed in advance, or sequentially separately. It may be placed on a tissue section.
- 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
- a fluorescent staining reagent it is preferable to drop a known blocking agent such as BSA-containing PBS before staining with a fluorescent staining reagent.
- the stained tissue 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.
- PBS may be exchanged during immersion.
- a cover glass is placed on the tissue section and sealed.
- a commercially available encapsulant may be used as necessary. Note that HE staining using an HE staining reagent is performed before sealing with a cover glass.
- a microscope image (fluorescence image) with a wide field of view is acquired from the stained tissue section using the microscope image acquisition device 1A.
- an excitation light source and an optical filter for fluorescence detection corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent material used for the fluorescent 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.
- the operator stains the same tissue section using two types of staining reagents, an HE staining reagent and a fluorescent staining reagent (fluorescent substance-encapsulating nanoparticles bound with an anti-HER2 antibody). Thereafter, the bright field image and the fluorescence image are acquired by the procedure (a1) to (a5) using the microscope image acquisition apparatus 1A.
- A1 The operator places the tissue sections stained with the HE staining reagent and the fluorescent staining reagent on the slide, and places the slide on the slide fixing stage of the microscope image acquisition apparatus 1A.
- A2 The unit is set as a bright field unit, the imaging magnification and the focus are adjusted, and the region to be observed on the tissue section is placed 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 the fluorescent image, and the image data is transmitted to the image processing apparatus 2A.
- FIG. 3 shows a flowchart of image analysis processing in the image processing apparatus 2A.
- the image analysis processing shown in FIG. 3 is executed in cooperation with the control unit 21 and the image processing program stored in the storage unit 25, and the control unit 21 executes the following processing according to the image processing program.
- step S10 when a bright field image and a fluorescence image are input from the microscope image acquisition apparatus 1A through the communication I / F 24 (step S10), cell nuclei are extracted from the bright field image (step S20).
- step S20 as shown in FIG. 4, the bright-field image is converted into a monochrome image (step S201), and threshold processing is performed on the monochrome image with a predetermined threshold to binarize each pixel value (step S202). ) Noise processing is executed on the binary image (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.
- step S30 a color component corresponding to the wavelength of the fluorescent bright spot is extracted from the fluorescent image (step S301), and threshold processing is performed on the fluorescent image after the color component extraction to generate a binary image.
- step S301 for example, when the emission wavelength of the fluorescent particles is 550 nm, only the fluorescent bright spot having the wavelength component is extracted as an image.
- steps S301 to S302 an image (fluorescent luminescent spot image) from which fluorescent luminescent spots are extracted can be generated. Note that noise removal processing such as cell autofluorescence and other unnecessary signal components may be performed before the threshold processing in step S302.
- the order of step S20 and step S30 may also be changed.
- step S40 the process returns to the process of FIG. 3 and the addition process of the cell nucleus image and the fluorescent luminescent spot image is executed, and the cell nucleus image and the fluorescent luminescent spot image are superimposed (step S40).
- the distance between the cell nucleus and the fluorescent bright spot is calculated on the superimposed image after the addition processing (step S50), and based on the distance, the cell nucleus to which the fluorescent bright spot belongs is specified and the fluorescent bright spot is determined. Assign to the cell nucleus (step S60).
- step S50 the distance from the fluorescent bright spot 30 to the surface of the cell nucleus 40, 42, 44 is calculated. Such distance calculation is performed for all the pixels of the cell nuclei 40, 42, and 44.
- step S60 as shown in FIG. 6B, among the calculated distances, the shortest distance from the fluorescent bright spot 30 to the surface of the cell nucleus 40, 42, 44 is calculated, and the cell nucleus 42 with the shortest distance from the fluorescent bright spot 30 is specified.
- the fluorescent bright spot 30 is assigned to the cell nucleus 42.
- FIG. 7 is a diagram illustrating an example of a state in which fluorescent luminescent spots are assigned to cell nuclei.
- the fluorescent luminescent spot is accurately assigned to a true cell. It can be attributed to an accurate pathological diagnosis. Even in comparison with the technique of Patent Document 2, the technique of Patent Document 2 needs to estimate the cell region 210 as shown in FIG. 18A, and if the range of the cell region 210 is excessively widened, the fluorescence bright spot is false.
- 18B according to the first embodiment, as shown in FIG. 18B, the cell region estimation process itself is not necessary, and the fluorescent bright spots 204a to 204d are also fluorescent bright spots 224a and 224b.
- the fluorescent bright spots 224a and 224b can be prevented from belonging to the false cells 200.
- the fluorescent bright spot 244 can be attributed to the fake cell 230 by the cross section of the cells 230, 240, whereas according to the first embodiment, FIG. As shown, the fluorescent bright spot 244 can be attributed to the true cell 240 regardless of the cross section of the cells 230, 240.
- the second embodiment is different from the first embodiment mainly in the following points, and is otherwise the same as the first embodiment.
- the distance calculated in step S50 is corrected based on the morphology of the cell nucleus (step S52).
- the distance 64 to the surface may be shorter than the distance 54.
- step S52 the area of the cell nucleus 52 and the area of the cell nucleus 62 are calculated, and the distance 54 and the distance 64 are corrected based on these areas. Specifically, the distance 54 and the distance 64 are corrected according to the correction expressions (1) and (2).
- step S60 the fluorescent bright spot 30 can be attributed to the cell nucleus 52 on the side having the smaller distance among the distance 54a and the distance 64a.
- the cell 70 (cell nucleus 72) is crushed and partially expanded or contracted at the time of preparation or staining of a tissue section, and the distance 74 from the fluorescent bright spot 30 a to the surface of the cell nucleus 72, The distance 76 from the fluorescent bright spot 30b to the surface of the cell nucleus 72 may be significantly different.
- the flatness of the cell nucleus 72 is calculated from the width W and height H of the cell nucleus 72, and the distance 74 and the distance 76 are corrected based on the flatness. Specifically, the distance 74 and the distance 76 are corrected according to the correction equations (3) and (4).
- step S60 the fluorescent bright spot 30a and the fluorescent bright spot 30b can be attributed to the same cell nucleus 70 with reference to the distance 74a and the distance 76a.
- the third embodiment differs from the first embodiment mainly in the following points, and is otherwise the same as the first embodiment.
- a threshold is set for the distance between the cell nucleus and the fluorescent luminescent spot (step S42), and the cell nuclei to which the fluorescent luminescent spot should belong are limited (step S44).
- step S50 the distance between the surface of the cell nucleus 40, 42, 44 and the fluorescent bright spot 30 is calculated for all pixels of other cell nuclei including the cell nucleus 40, 42, 44. Then, the calculation amount of the distance from the fluorescent bright spot 30 to the surface of the cell nucleus becomes enormous, and the processing can be delayed.
- step S42 as shown in FIG. 12B, a threshold value (radial distance 46) is set in a certain circular range from the fluorescent bright spot 30, and in step S44, the cell nucleus to be subjected to the distance calculation in step S50 is set to the radial distance 46.
- the cell nuclei 40, 42, 44 are limited to some or all of them, and these cell nuclei 40, 42, 44 are set as distance calculation targets in step S 50.
- step S42 a square frame including cell nuclei is assumed and a threshold (total distance 48) is set to the total distance between the four vertices of the frame and the fluorescent luminescent spot.
- step S44 FIG. As shown in FIG. 4, for each cell nucleus 40, 42, 44, the total distance between the vertex of the frame of the cell nucleus 40, 42, 44 and the fluorescent luminescent point 30 is calculated.
- the cell nucleus 44 may be limited to the cell distance 44 within the limit distance 48, and the cell nucleus 44 may be a distance calculation target in step S50.
- step S44 for the cell nuclei 40, 42 and 44, assuming a rectangular frame including them, the total distance between the four vertices of the frame and the fluorescent bright spot 30 is calculated. When the distance exceeds the limit distance 48, the calculation is stopped, and the cell nuclei 40 and 42 for which the calculation has been made are excluded from the distance calculation targets in step S50.
- step S50 only cell nuclei having a fixed distance relationship from the fluorescent bright spot are subject to distance calculation, and the calculation of the distance between the surface of the cell nucleus and the fluorescent bright spot can be speeded up.
- the radial distance 46 and the total distance 48 as the threshold values set in step S42 can be appropriately set based on the area of the cell nucleus, the area of the cell membrane, the N / C ratio (Nuclear-cytoplasm ratio), and the like. As shown in FIG. 13, the “N / C ratio” is the ratio of the area occupied by the cell nucleus 82 in the cytoplasm 80, and the range of the value is determined depending on the cell type and state.
- the rectangular frame assumed in steps S42 and S44 may have any shape as long as it can include a cell nucleus.
- the fourth embodiment is different from the first embodiment mainly in the following points, and is otherwise the same as the first embodiment.
- the fluorescent spots after belonging are excluded from the fluorescent spots of the object to be assigned (step S62).
- the fluorescent luminescent spots 30a to 30k correctly belong to the cell nucleus 92 of the cell 90, while the fluorescent luminescent spot 30x It may be mistakenly assigned to the cell nucleus 92 of the cell 90.
- the average distance 104 between the surface of the cell nucleus 92 and the fluorescent luminescent spots 30a to 30k, 30x after the assignment is calculated, and the distance from the surface of the cell nucleus 92 is calculated from the average distance 104.
- the fluorescent luminescent spots 30x that are remarkably separated are excluded from the fluorescent luminescent spots to be assigned.
- a straight line is assumed between the two fluorescent light spots 30a to 30k and 30x, such as the fluorescent light spots 30a ⁇ 30b, 30b ⁇ 30c, 30c ⁇ ... ⁇ 30k, 30k ⁇ 30x, 30x ⁇ 30a.
- the fluorescent luminescent spots 30x constituting the straight line may be excluded from the fluorescent luminescent spots to be assigned.
- step S62 the accuracy of assigning the fluorescent luminescent spot to the cell nucleus can be improved, and the fluorescent luminescent spot can be more accurately assigned to the true cell.
- the cell nucleus 102 falls off due to the cross section of the cell 100, and the fluorescent bright spot 30y of the cell 100 cannot be attributed to the cell nucleus 102. It can be attributed to 120.
- the N / C ratio (see FIG. 13) is calculated for each of the cells 100, 110 to 120 once assigned with the fluorescent bright spot in step S60, and the N / C ratio is constant (for example, 70). %),
- the fluorescent bright spot 30y attributed to the cell 100 is excluded from the fluorescent bright spots to be assigned. Even in such a case, in step S62, it is possible to improve the accuracy of assigning the fluorescent bright spot to the cell nucleus, and to assign the fluorescent bright spot to the true cell more accurately.
- step S52 is performed after step S50 (second embodiment), and steps S42 and S44 are performed before step S50.
- Step S62 (fourth embodiment) may be executed after step S60.
- any three of the first to fourth embodiments may be combined, or any two of them may be combined.
- the HER2 protein in breast cancer is mentioned as an example of the specific biological material, but is not limited thereto.
- the target of pathological diagnosis is a tissue section collected from a human body.
- the tissue includes a cultured tissue, and instead of the tissue, cells separated from the tissue or cultured It is also possible to use cells.
- an HDD or a semiconductor non-volatile memory is used as a computer-readable medium of the image processing 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
- the detailed configuration and detailed operation of each device constituting the pathological diagnosis support system 10 can be changed as appropriate without departing from the spirit of the invention.
- the present invention can be suitably used for image processing for pathological diagnosis.
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Abstract
Description
具体的に特許文献2の技術では、組織切片をHE染色して明視野画像を取得し、明視野画像から細胞核の領域を抽出し、抽出した細胞核の領域の重心から所定範囲を細胞領域と推定し、その細胞領域に含まれる細胞核および蛍光輝点数に基づき、細胞領域における生体物質の発現レベルを判定している(段落0092~0104、図20A、0128などご参照)。
また逆に、HE染色後の組織切片をスライドに載置して顕微鏡観察する場合、図19Aに示すとおり、細胞230、240ごとに断面が異なりその断面によって細胞核232、242の大きさが異なるため、図19Bに示すとおり、蛍光輝点244が細胞240の細胞核242に帰属すべきところ、蛍光輝点244が帰属すべき真の細胞240とは異なる隣の細胞(偽の細胞)230に帰属することもある。
したがって、本発明の主な目的は、蛍光輝点を真の細胞に正確に帰属させることができる画像処理装置および画像処理プログラムを提供することにある。
細胞核が染色された組織切片の明視野画像と、特定の生体物質が蛍光染色試薬で染色された前記組織切片の蛍光画像とを入力する入力手段と、
前記細胞核画像から細胞核を抽出する第1の抽出手段と、
前記蛍光画像から蛍光輝点を抽出する第2の抽出手段と、
細胞核と蛍光輝点との距離に基づき、蛍光輝点が帰属すべき細胞核を特定して蛍光輝点を当該細胞核に帰属させる帰属手段と、
を備えることを特徴とする画像処理装置が提供される。
コンピュータに、
細胞核が染色された組織切片の明視野画像と、特定の生体物質が蛍光染色試薬で染色された前記組織切片の蛍光画像とを入力する入力手段と、
前記細胞核画像から細胞核を抽出する第1の抽出手段と、
前記蛍光画像から蛍光輝点を抽出する第2の抽出手段と、
細胞核と蛍光輝点との距離に基づき、蛍光輝点が帰属すべき細胞核を特定して蛍光輝点を当該細胞核に帰属させる帰属手段と、
として機能させることを特徴とする画像処理プログラムが提供される。
<病理診断支援システム10の構成>
図1に、病理診断支援システム10の全体構成例を示す。
病理診断支援システム10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と画像処理装置2Aとの接続方式は特に限定されない。たとえば、顕微鏡画像取得装置1Aと画像処理装置2AはLAN(Local Area Network)により接続されることとしてもよいし、無線により接続される構成としてもよい。
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などを備えて構成されている。照射手段は、光源、フィルターなどにより構成され、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、対物レンズなどにより構成され、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、または蛍光を結像する。撮像手段は、CCD(Charge Coupled Device)センサーなどを備え、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。
顕微鏡画像取得装置1Aでは、明視野観察に適した照射手段およ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およ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などを備えて構成され、各部はバス26を介して接続されている。
たとえば、制御部21は、記憶部25に記憶されている画像処理プログラムとの協働により画像解析処理(図3参照)を実行し、第1の抽出手段、第2の抽出手段、帰属手段、除外手段としての機能を実現する。
その他、画像処理装置2Aは、LANアダプターやルーターなどを備え、LANなど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」とは、ヘマトキシリン染色試薬(H染色試薬)、ヘマトキシリン-エオジン染色試薬(HE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および撮影することにより得られる顕微鏡画像であって、当該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。ヘマトキシリン(H)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(好塩基性の組織など)を染色する。エオジン(E)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(好酸性の組織など)を染色する。
「蛍光画像」とは、蛍光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発光させ、この蛍光を拡大結像および撮影することにより得られる顕微鏡画像である。
「蛍光染色試薬」とは、特定の生体物質と特異的に結合および/または反応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であり、「蛍光物質内包ナノ粒子」とは蛍光物質を内包したナノ粒子である。
蛍光画像に現れる蛍光は、蛍光染色試薬の蛍光物質内包ナノ粒子(蛍光物質)が励起して発光したものであり、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。
蛍光染色試薬や当該蛍光染色試薬を用いた組織切片の染色方法について説明する。
蛍光染色試薬に用いられる蛍光物質としては、蛍光有機色素および量子ドット(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。
具体的には、5-カルボキシ-フルオレセイン、6-カルボキシ-フルオレセイン、5,6-ジカルボキシ-フルオレセイン、6-カルボキシ-2’,4,4’,5’,7,7’-ヘキサクロロフルオレセイン、6-カルボキシ-2’,4,7,7’-テトラクロロフルオレセイン、6-カルボキシ-4’,5’-ジクロロ-2’,7’-ジメトキシフルオレセイン、ナフトフルオレセイン、5-カルボキシ-ローダミン、6-カルボキシ-ローダミン、5,6-ジカルボキシ-ローダミン、ローダミン 6G、テトラメチルローダミン、X-ローダミン、およびAlexa Fluor 350、Alexa Fluor 405、Alexa Fluor 430、Alexa Fluor 488、Alexa Fluor 500、Alexa Fluor 514、Alexa Fluor 532、Alexa Fluor 546、Alexa Fluor 555、Alexa Fluor 568、Alexa Fluor 594、Alexa Fluor 610、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、Alexa Fluor 680、Alexa Fluor 700、Alexa Fluor 750、BODIPY FL、BODIPY TMR、BODIPY 493/503、BODIPY 530/550、BODIPY 558/568、BODIPY 564/570、BODIPY 576/589、BODIPY 581/591、BODIPY 630/650、BODIPY 650/665(以上インビトロジェン社製)、メトキシクマリン、エオジン、NBD、ピレン、Cy5、Cy5.5、Cy7などを挙げることができる。これら蛍光有機色素は単独で使用されてもよいし、複数種を混合して使用されてもよい。
具体的には、CdSe、CdS、CdTe、ZnSe、ZnS、ZnTe、InP、InN、InAs、InGaP、GaP、GaAs、Si、Geが挙げられるが、これらに限定されない。
たとえば、CdSe/ZnS、CdS/ZnS、InP/ZnS、InGaP/ZnS、Si/SiO2、Si/ZnS、Ge/GeO2、Ge/ZnSなどを用いることができるが、これらに限定されない。
「蛍光物質内包ナノ粒子」とは、上記のとおり蛍光物質を内包したナノ粒子であって、詳しくは蛍光物質をナノ粒子の内部に分散させたものをいい、蛍光物質とナノ粒子自体とが化学的に結合していてもよいし、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、シリカ、ポリスチレン、ポリ乳酸、メラミンなどを挙げることができる。
たとえば、蛍光有機色素を内包したシリカナノ粒子は、ラングミュア 8巻 2921ページ(1992)に記載されているFITC内包シリカ粒子の合成を参考に合成することができる。FITCの代わりに所望の蛍光有機色素を用いることで種々の蛍光有機色素内包シリカナノ粒子を合成することができる。
量子ドットを内包したシリカナノ粒子は、ニュー・ジャーナル・オブ・ケミストリー 33巻 561ページ(2009)に記載されているCdTe内包シリカナノ粒子の合成を参考に合成することができる。
蛍光有機色素を内包したポリスチレンナノ粒子は、米国特許4326008(1982)に記載されている重合性官能基をもつ有機色素を用いた共重合法や、米国特許5326692(1992)に記載されているポリスチレンナノ粒子への蛍光有機色素の含浸法を用いて作製することができる。
量子ドットを内包したポリマーナノ粒子は、ネイチャー・バイオテクノロジー19巻631ページ(2001)に記載されているポリスチレンナノ粒子への量子ドットの含浸法を用いて作製することができる。
平均粒径は、走査型電子顕微鏡(SEM)を用いて電子顕微鏡写真を撮影し十分な数の粒子について断面積を計測し、各計測値を円の面積としたときの円の直径を粒径として求めた値である。本実施形態では、1000個の粒子の粒径の算術平均を平均粒径とする。変動係数も、1000個の粒子の粒径分布から算出した値とする。
「生体物質認識部位」とは、特定の生体物質と特異的に結合および/または反応する部位である。
特定の生体物質としては、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)などが挙げられる。
したがって、生体物質認識部位としては、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など、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などが挙げられる。
具体的な生体物質認識部位としては、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などが挙げられる。
中でも、抗HER2抗体および抗ER抗体を蛍光物質内包ナノ粒子に結合させたもの(蛍光染色試薬)は、乳癌の投薬選定に用いることができ、好ましい。
たとえば、無機物と有機物を結合させるために広く用いられている化合物であるシランカップリング剤を用いることができる。このシランカップリング剤は、分子の一端に加水分解でシラノール基を与えるアルコキシシリル基を有し、他端に、カルボキシル基、アミノ基、エポキシ基、アルデヒド基などの官能基を有する化合物であり、上記シラノール基の酸素原子を介して無機物と結合する。
具体的には、メルカプトプロピルトリエトキシシラン、グリシドキシプロピルトリエトキシシラン、アミノプロピルトリエトキシシラン、ポリエチレングリコール鎖をもつシランカップリング剤(たとえば、Gelest社製PEG-silane no.SIM6492.7)などが挙げられる。
シランカップリング剤を用いる場合、2種以上を併用してもよい。
たとえば、得られた蛍光有機色素内包シリカナノ粒子を純水中に分散させ、アミノプロピルトリエトキシシランを添加し、室温で12時間反応させる。反応終了後、遠心分離またはろ過により表面がアミノプロピル基で修飾された蛍光有機色素内包シリカナノ粒子を得ることができる。続いてアミノ基と抗体中のカルボキシル基とを反応させることで、アミド結合を介し抗体を蛍光有機色素内包シリカナノ粒子と結合させることができる。必要に応じて、EDC(1-Ethyl-3-[3-Dimethylaminopropyl]carbodiimide Hydrochloride:Pierce(登録商標)社製)のような縮合剤を用いることもできる。
下記染色方法は病理組織切片に限定せず、培養細胞にも適用可能である。
組織切片の作製方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組織切片を浸漬させ、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。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組織切片を浸漬させ、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。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、水を入れた容器に組織切片を浸漬させ、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。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、組織切片の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トリス塩酸緩衝液など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、ウォーターバスなど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、賦活化処理後の組織切片を浸漬させ、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。必要により浸漬途中でPBSを交換してもよい。
蛍光染色試薬のPBS分散液を組織切片に載せ、組織切片の生体物質と反応させる。
蛍光染色試薬の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。蛍光染色試薬として、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、別々に順次組織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。
蛍光染色試薬による染色を行う前に、BSA含有PBSなど、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、染色後の組織切片を浸漬させ、未反応の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。必要により浸漬途中でPBSを交換してもよい。カバーガラスを組織切片に載せ、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染色試薬を用いるHE染色はカバーガラスによる封入前に行う。
染色した組織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、蛍光染色試薬に用いた蛍光物質の吸収極大波長および蛍光波長に対応した励起光源と蛍光検出用光学フィルターとを選択する。
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。
以下、病理診断支援システム10において、上記説明した明視野画像および蛍光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、特定の生体物質として乳癌組織におけるHER2タンパクを含む組織切片を観察対象とする場合を例にとり説明する。
その後、顕微鏡画像取得装置1Aを用いて、(a1)~(a5)の手順により明視野画像および蛍光画像を取得する。
(a1)操作者は、HE染色試薬と蛍光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)ユニットを明視野ユニットに設定し、撮影倍率、ピントの調整を行い、組織切片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)視野およ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。
図3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3に示す画像解析処理は、制御部21と記憶部25に記憶されている画像処理プログラムとの協働により実行され、制御部21はその画像処理プログラムにしたがって下記の処理を実行する。
ステップS20では、図4に示すとおり、明視野画像をモノクロ画像に変換し(ステップS201)、モノクロ画像に対し予め定められた閾値で閾値処理を施して各画素の値を二値化し(ステップS202)、二値画像にノイズ処理を実行する(ステップS203)。
ステップS201~S203により、細胞核が抽出された画像(細胞核画像)を生成することができる。
ステップS30では、図5に示すとおり、蛍光画像から蛍光輝点の波長に応じた色成分を抽出し(ステップS301)、色成分抽出後の蛍光画像に閾値処理を施して二値画像を生成する(ステップS302)。
ステップS301では、たとえば、蛍光粒子の発光波長が550nmである場合には、その波長成分を有する蛍光輝点のみが画像として抽出される。
ステップS301~S302により、蛍光輝点が抽出された画像(蛍光輝点画像)を生成することができる。
なお、ステップS302の閾値処理の前に、細胞自家蛍光や他の不要信号成分などのノイズ除去処理が施されてもよい。
ステップS20とステップS30との順序も、入れ替えてもよい。
ステップS60では、図6Bに示すとおり、計算した距離のうち、蛍光輝点30から細胞核40、42、44の表面までの最短距離を算出し、蛍光輝点30から最短距離の細胞核42を特定して蛍光輝点30を細胞核42に帰属させる。
図7は、蛍光輝点を細胞核に帰属させた状態の一例を示す図である。
特許文献2の技術と比較しても、特許文献2の技術では、図18Aに示すとおり、細胞領域210を推定する必要があるし、細胞領域210の範囲を過大に広げると蛍光輝点が偽の細胞に帰属しうるのに対し、第1の実施形態によれば、図18Bに示すとおり、細胞領域の推定処理そのものが不要であるし、蛍光輝点204a~204dも蛍光輝点224a、224bもそれぞれ真の細胞200、220に帰属し、蛍光輝点224a、224bが偽の細胞200に帰属するのを防止することができる。
さらに特許文献2の技術では、図19Bに示すとおり、細胞230、240の断面によって蛍光輝点244が偽の細胞230に帰属しうるのに対し、第1の実施形態によれば、図19Cに示すとおり、細胞230、240の断面によらずに蛍光輝点244を真の細胞240に帰属させることができる。
第2の実施形態は主に下記の点で第1の実施形態と異なり、それ以外は第1の実施形態と同様である。
図8に示すとおり、ステップS50の後に、細胞核の形態に基づき、ステップS50で計算した距離を補正する(ステップS52)。
かかる場合、ステップS52では、細胞核52の面積と細胞核62の面積とを算出してそれら面積に基づき、距離54と距離64とを補正する。
詳しくは距離54と距離64とを補正式(1)、(2)にしたがって補正する。
距離54a=距離54/√(細胞核52の面積) … (1)
距離64a=距離64/√(細胞核62の面積) … (2)
補正式(1)、(2)によれば、距離54と距離64との大小関係が、細胞核52の面積と細胞核62の面積とに反比例して逆転する。
その結果、ステップS60では、蛍光輝点30を、距離54aと距離64aとのうち、当該距離の小さい側の細胞核52に帰属させることができる。
かかる場合、ステップS52では、細胞核72の幅Wと高さHから細胞核72の扁平率を算出してその扁平率に基づき、距離74と距離76とを補正する。
詳しくは距離74と距離76とを補正式(3)、(4)にしたがって補正する。
距離74a=距離74×(高さH/幅W) … (3)
距離76a=距離76×(幅W/高さH) … (4)
補正式(3)、(4)によれば、距離74aと距離76aとで距離差が緩和され、距離74aと距離76aとをほぼ同等に扱うことができる。
その結果、ステップS60では、距離74aと距離76aとを基準として、蛍光輝点30aも蛍光輝点30bも、同一の細胞核70に帰属させることができる。
第3の実施形態は主に下記の点で第1の実施形態と異なり、それ以外は第1の実施形態と同様である。
図11に示すとおり、ステップS50の前に、細胞核と蛍光輝点との距離に閾値を設定し(ステップS42)、蛍光輝点が帰属すべき細胞核を制限する(ステップS44)。
かかる場合、ステップS42では、図12Bに示すとおり、蛍光輝点30から一定の円形範囲に閾値(半径距離46)を設定し、ステップS44では、ステップS50の距離計算対象の細胞核を、半径距離46のなかに一部または全部存在する細胞核40、42、44に制限し、これら細胞核40、42、44をステップS50の距離計算対象とする。
特にステップS44では、細胞核40、42、44に対し、これらを包含する四角枠を仮定してその枠の4つの頂点と蛍光輝点30との合計距離を計算するが、その計算途中で、合計距離が制限距離48を超えたときはその計算を中止し、当該計算がなされた細胞核40、42をステップS50の距離計算対象から除外する。
「N/C比」とは、図13に示すとおり、細胞質80における細胞核82の占める面積の割合であり、細胞の種類や状態によってその値の範囲が定まっているものである。
ステップS42、S44で仮定する四角枠は、細胞核を包含しうるものであれば、いかなる形状を呈していてもよい。
第4の実施形態は主に下記の点で第1の実施形態と異なり、それ以外は第1の実施形態と同様である。
図14に示すとおり、ステップS60の後に、帰属後の蛍光輝点を、被帰属対象の蛍光輝点から除外する(ステップS62)。
かかる場合、ステップS62では、図15に示すとおり、細胞核92の表面と帰属後の蛍光輝点30a~30k、30xとの平均距離104を算出し、細胞核92の表面との距離が平均距離104から顕著に離れた蛍光輝点30xを、被帰属対象の蛍光輝点から除外する。
これに代えて、蛍光輝点30a→30b、30b→30c、30c→…→30k、30k→30x、30x→30aというように、蛍光輝点30a~30k、30xの2点間で直線を仮定し、隣り合う直線同士の傾きまたは角度を算出し、直線同士の傾きまたは角度を、蛍光輝点30a→30b→…→30k→30x→30aの配置順に比較した場合において、急激な数値変化があったときに、その直線を構成する蛍光輝点30xを、被帰属対象の蛍光輝点から除外してもよい。
その結果、ステップS62では、蛍光輝点の細胞核への帰属精度を向上させることができ、より正確に蛍光輝点を真の細胞に帰属させることができる。
かかる場合、ステップS60でいったん蛍光輝点を帰属させた細胞100、110~120ごとにN/C比(図13参照)を算出し、そのN/C比が一定のN/C比(たとえば70%)未満である場合、その細胞100に帰属させた蛍光輝点30yを、被帰属対象の蛍光輝点から除外する。
かかる場合でも、ステップS62では、蛍光輝点の細胞核への帰属精度を向上させることができ、より正確に蛍光輝点を真の細胞に帰属させることができる。
もちろん、第1~第4の実施形態のうち、いずれか3つの形態を組み合わせてもよいし、いずれか2つの形態を組み合わせてもよい。
第1~第4の実施形態では、病理診断の対象を人体から採取した組織切片としたが、当該組織には培養組織も含まれるし、当該組織に代えて、当該組織から分離した細胞や培養細胞を使用することも可能である。
その他、病理診断支援システム10を構成する各装置の細部構成および細部動作に関しても、発明の趣旨を逸脱することのない範囲で適宜変更可能である。
2A 画像処理装置
3A ケーブル
10 病理診断支援システム
21 制御部
22 操作部
23 表示部
24 通信I/F
25 記憶部
26 バス
30、30a~30k、30x、30y 蛍光輝点
40、42、44 細胞核
46 半径距離
50 細胞
52 細胞核
54 距離
60 細胞
62 細胞核
64 距離
70 細胞
72 細胞核
74、76 距離
80 細胞質
82 細胞核
90 細胞
92 細胞核
100 細胞
102 細胞核
110、112、114、116、118、120 細胞
Claims (10)
- 細胞核が染色された組織切片の明視野画像と、特定の生体物質が蛍光染色試薬で染色された前記組織切片の蛍光画像とを入力する入力手段と、
前記細胞核画像から細胞核を抽出する第1の抽出手段と、
前記蛍光画像から蛍光輝点を抽出する第2の抽出手段と、
細胞核と蛍光輝点との距離に基づき、蛍光輝点が帰属すべき細胞核を特定して蛍光輝点を当該細胞核に帰属させる帰属手段と、
を備え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、
前記帰属手段が、細胞核の表面と蛍光輝点との最短距離を算出し、蛍光輝点を最短距離の細胞核に帰属させ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、
前記帰属手段が、細胞核の表面と蛍光輝点との距離を細胞核の形態に基づき補正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、
前記帰属手段が、細胞核の面積を算出してその面積に基づき前記距離を補正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、
前記帰属手段が、細胞核の扁平率を算出してその扁平率に基づき前記距離を補正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、
前記帰属手段が、細胞核と蛍光輝点との距離に閾値を設定し、蛍光輝点が帰属すべき細胞核を制限することを特徴とする画像処理装置。 - 請求項6に記載の画像処理装置において、
前記帰属手段が、前記閾値を、細胞核の面積または細胞のN/C比に基づき設定することを特徴とする画像処理装置。 - 請求項6に記載の画像処理装置において、
前記帰属手段が、前記閾値を、細胞核を包含する一定の枠を仮定してその枠の頂点と蛍光輝点との距離に基づき設定することを特徴とする画像処理装置。 - 請求項1~8のいずれか一項に記載の画像処理装置において、
細胞核の表面と帰属後の蛍光輝点との平均距離、帰属後の蛍光輝点間の直線同士の傾きもしくは角度、または蛍光輝点を帰属させた細胞のN/C比に基づき、帰属後の蛍光輝点を、被帰属対象の蛍光輝点から除外する除外手段を備えることを特徴とする画像処理装置。 - コンピュータに、
細胞核が染色された組織切片の明視野画像と、特定の生体物質が蛍光染色試薬で染色された前記組織切片の蛍光画像とを入力する入力手段と、
前記細胞核画像から細胞核を抽出する第1の抽出手段と、
前記蛍光画像から蛍光輝点を抽出する第2の抽出手段と、
細胞核と蛍光輝点との距離に基づき、蛍光輝点が帰属すべき細胞核を特定して蛍光輝点を当該細胞核に帰属させる帰属手段と、
として機能させることを特徴とする画像処理プログラム。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP14885851.7A EP3124956B1 (en) | 2014-03-27 | 2014-03-27 | Image processing device and image processing program |
PCT/JP2014/058725 WO2015145644A1 (ja) | 2014-03-27 | 2014-03-27 | 画像処理装置および画像処理プログラム |
JP2015506957A JP5768948B1 (ja) | 2014-03-27 | 2014-03-27 | 画像処理装置および画像処理プログラム |
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EP3124956A4 (en) | 2017-09-13 |
EP3124956A1 (en) | 2017-02-01 |
JP5768948B1 (ja) | 2015-08-26 |
US20160275673A1 (en) | 2016-09-22 |
US9558393B2 (en) | 2017-01-31 |
EP3124956B1 (en) | 2018-12-26 |
JPWO2015145644A1 (ja) | 2017-04-13 |
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