WO2016204027A1 - 目的生体物質の解析装置、解析システム、解析方法および解析プログラム - Google Patents
目的生体物質の解析装置、解析システム、解析方法および解析プログラム Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Definitions
- the present invention relates to an analysis apparatus, an analysis system, an analysis method, and an analysis program for a target biological substance.
- Pathological diagnosis refers to the type and nature of the lesion, mainly by examining a part of the tissue collected from the affected area under a microscope in order to determine the treatment policy for the patient's disease and evaluate the effect of the treatment. It is to distinguish.
- Patent Document 1 discloses a technique useful for pathological diagnosis in Patent Document 1.
- a bright-field image representing the morphology of cells in a tissue section and a fluorescence image representing the expression of a specific protein in the same range of the tissue section as fluorescent luminescent spots are acquired and specified from the bright-field image.
- Estimate cell areas including protein expression areas extract fluorescent bright spots from fluorescent images, and calculate feature quantities for each cell area based on the estimated cell nuclei and fluorescent bright spots in each cell area Based on the calculated feature amount, whether each cell region is cancer or not and the expression state of a specific protein can be determined.
- FIG. 15 shows a sample of tissue sections containing 5 types of cell lines of SKOV-3, A549, Calu3, T47D, and MCF7. It is an example of the result of having calculated
- SKOV-3 is a cell line derived from human ovarian cancer cells
- A549 is a cell line derived from human lung cancer cells
- Calu3 is a cell line derived from human lung cancer cells
- T47D is It is a cell line derived from human breast cancer cells
- MCF7 is a cell line derived from human breast cancer cells.
- the “protein quantitative value” on the horizontal axis is the fluorescence value (au) per cell measured by flow cytometry.
- the “number of fluorescent luminescent spots per cell” on the vertical axis from the preparation of the tissue specimen to obtaining a fluorescent image including the fluorescent luminescent spots, it conforms to the description in Patent Documents 1 and 2, This was performed as described in a first embodiment ([staining step] and [imaging step]) described later in this specification.
- an immunostaining agent in which an anti-HER2 antibody that specifically binds to HER2, which is a protein present on the cell surface, is bound to fluorescent substance-integrated nanoparticles is prepared, and immunostained with the immunostaining agent to obtain a fluorescence image Got. Thereafter, the number of fluorescent bright spots per cell was measured using software dedicated to pathological diagnosis.
- black points indicate the average value of the number of fluorescent bright spots, and upper and lower line segments with respect to the black points indicate standard deviations.
- the standard deviation of the number of fluorescent bright spots increases as the protein quantitative value increases, and it can be seen that the diagnosis results (statistics) vary depending on the type of cell line of the tissue section and the diagnostician. .
- the main object of the present invention is to provide an analysis apparatus, analysis system, analysis method, and analysis program for a target biological material that can reduce the analysis time while suppressing the variation of the statistical values by the types of tissue sections and the diagnostician. It is to provide. Further, the object of the present invention includes measurement of bright spots by fluorescent observation of tissue sections in addition to proteins as target biological substances, such as nucleic acids, sugar chains and enzymes.
- Generating means for generating a microscopic image of the stained tissue specimen comprising:
- a microscope for imaging the tissue specimen after staining An analysis device for the target biological material that receives the imaging result of the microscope; A system for analyzing a target biological material is provided.
- Generating a microscopic image of the tissue specimen after staining comprising: Partitioning the microscopic image at a fixed size; Analyzing the stained state of the microscope image for each section; A step of calculating a certain statistical value based on the analysis result of the analysis step; A method for analyzing a target biological material is provided.
- Computer Generating means for generating a microscopic image of the tissue specimen after staining comprising: Partitioning means for partitioning the microscopic image at a certain size; Analysis means for analyzing the staining state of the microscope image for each section, Calculation means for calculating a certain statistical value based on the analysis result of the analysis means; An analysis program of a target biological material for functioning as a target is provided.
- the present invention it is possible to shorten the analysis time while suppressing the variation of the statistical value by the type of tissue section and the diagnostician.
- FIG. 1 It is a figure which shows schematically the process of the analysis method of a target biological material. It is a figure which shows schematic structure of the analysis system of the target biological material. It is a figure which shows an example of a bright field image. It is a figure which shows an example of a fluorescence image. It is a figure which shows an analysis process roughly. It is a figure which shows the example which designates a bright field image. It is a figure which shows the example which designates a background area
- the present embodiment mainly provides an analysis method for analyzing a microscopic image and calculating a certain statistical value.
- Such an analysis method is mainly as shown in FIG. (S1) staining a tissue specimen with a certain stain; (S2) capturing a stained image of the tissue specimen after staining with a fixed microscope; (S3) analyzing the microscope image and calculating a certain statistical value.
- the staining step S1 will be described, and then the imaging step S2 and the analysis step S3 will be described.
- the analysis system 1 of FIG. 2 is used.
- a tissue specimen A containing a target biological material is prepared, and this is subjected to immunostaining and morphological observation staining with a staining agent for immunostaining or a staining agent for morphological observation.
- Target biological substance is a target of immunostaining using a fluorescent label for detection or quantification mainly from the viewpoint of pathological diagnosis, and is expressed in tissue sections.
- Biological substances particularly proteins (antigens), nucleic acids (eg, DNA, RNA, miRNA, etc.).
- Typical target biological materials include biological materials that are expressed in cell membranes of various cancer tissues and can be used as biomarkers.
- the biological material recognition site is a site that specifically binds 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. Therefore, the substance that binds to the target biological substance includes 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. 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.
- examples of the specific nucleic acid gene that has been pointed out to be associated with a disease can include the following, and probes that recognize each specific nucleic acid gene are available as BAC probes: Can be created based on general knowledge. Specific examples of specific nucleic acid genes are as follows. HER2, TOP2A, HER3, EGFR, P53, MET, etc. are mentioned as genes related to cancer growth and molecular target drug response. Furthermore, the following genes are known as various cancer-related genes. Can be mentioned.
- Tyrosine kinase-related genes include ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS (CSF1R), DDR2, EGFR (ERBB1), HER4 (ERBB4), EML4-ALK, IGF1R, EPHA1, INSR, EPHA2, IRR (INSRR) ), EPHA3, KIT, EPHA4, LTK, EPHA5, MER (MERTK), EPHA6, MET, EPHA7, MUSK, EPHA8, NPM1-ALK, EPHB1, PDGFR ⁇ (PDGFRA), EPHB2, PDGFR ⁇ (PDGFRB), EPHEP3, T RON (MST1R), FGFR1, ROS (ROS1), FGFR2, TIE2 (TEK), FGFR3, TRKA (NTRK1), FGFR4, TRKB (NT RK2), FLT1 (VEGFR1), TRKC (NTRK3), and breast cancer-related genes are ATM, BRCA1, BRCA2, BRCA3, CC
- APC As cancer-related genes, APC, MSH6, AXIN2, MYH, BMPR1A, p53, DCC, PMS2, KRAS2 (orKi-ras), PTEN, MLH1, SMAD , MSH2, STK11, MSH6 Lung cancer-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A, RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, MYC.
- genes of the gene include Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1, and KRAS.
- genes include Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb / NONO, and TFEB, and thyroid cancer-related genes include AKAP10, NTRK1, and AKAP. , RET, BRAF, TFG, ELE1, TPM3, H4 / D10S170, TPR and the like.
- Examples of ovarian cancer-related genes include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HRAS, RET, KRAS, and RNASET2.
- Examples of prostate cancer-related genes include AR, KLK3, BRCA2, MYC, CDKN1B, NKX3.1, EZH2, p53, GSTP1, and PTEN.
- Examples of bone tumor-related genes include CDH11, COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.
- the mode of binding between the biological substance recognition site and the fluorescent nanoparticle 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.
- Immunostaining As an immunostaining agent, in order to improve the efficiency of fluorescent labeling and suppress the time lapse leading to the deterioration of fluorescence as much as possible, primary antibodies and fluorescent nanoparticles are used indirectly, that is, other than covalent bonds using antigen-antibody reactions. It is preferable to use a complex linked by these bonds. In order to simplify the staining operation, a complex in which fluorescent nanoparticles are directly bound to a primary antibody or a secondary antibody can also be used as an immunostaining agent.
- immunostaining agents include [primary antibody against target biological substance] ... [antibody against primary antibody (secondary antibody)] to [fluorescent nanoparticles].
- “...” represents binding by an antigen-antibody reaction, and the mode of binding indicated by “ ⁇ ” is not particularly limited.
- a covalent bond, an ionic bond, a hydrogen bond, a coordinate bond, an antigen-antibody bond Examples include biotin avidin reaction, physical adsorption, chemical adsorption, and the like, and a linker molecule may be used as necessary.
- an antibody (IgG) that specifically recognizes and binds a protein as a target biological substance as an antigen can be used.
- an anti-HER2 antibody can be used
- HER3 is the target biological material
- an anti-HER3 antibody can be used.
- an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
- Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferred.
- the type of animal that produces the antibody (immunized animal) is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like as in the past.
- Fluorescent nanoparticles are nano-sized particles that emit fluorescence when irradiated with excitation light, and emit fluorescent light with sufficient intensity to represent the target biological substance as a bright spot one molecule at a time. Possible particles.
- the fluorescent nanoparticles preferably, quantum dots (semiconductor nanoparticles) and fluorescent substance integrated nanoparticles are used.
- Quantum Dots As the quantum dots, semiconductor nanoparticles containing a II-VI group compound, a III-V group compound, or a group IV element are used. Examples thereof include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
- Fluorescent substance integrated nanoparticles are based on organic or inorganic particles, and a plurality of fluorescent substances (for example, the above quantum dots and fluorescent dyes) are encapsulated therein. Nano-sized particles having a structure that is adsorbed and / or adsorbed on its surface. As the fluorescent substance-integrated nanoparticles, it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges, and an electrostatic interaction works. As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles, and the like are used.
- Base material As organic substances, resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin, etc. Resins generally classified as thermoplastic resins such as styrene resins, acrylic resins, acrylonitrile resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers); Examples of other resins such as lactic acid; polysaccharides. Of the matrix, examples of the inorganic substance include silica and glass.
- Quantum dot integrated nanoparticle has a structure in which the quantum dot is included in the matrix and / or adsorbed on the surface thereof.
- the quantum dots may be dispersed inside the matrix and may or may not be chemically bonded to the matrix itself.
- the fluorescent dye integrated nanoparticle has a structure in which a fluorescent dye is included in the matrix and / or adsorbed on the surface thereof.
- fluorescent dyes include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules.
- Alexa Fluor registered trademark, manufactured by Invitrogen
- BODIPY registered trademark, manufactured by Invitrogen
- Cy registered trademark, manufactured by GE Healthcare
- HiLite registered trademark, manufactured by Anaspec
- DyLight registered trademark, manufactured by Thermo Scientific
- ATTO registered trademark, manufactured by ATTO-TEC
- MFP registered trademark, manufactured by Mobitec
- Dye molecules CF (registered trademark, manufactured by Biotium) dye molecules
- DY registered trademark, manufactured by DYOMICICS
- CAL registered trademark, manufactured by BioSearch Technologies
- fluorescent dyes can be exemplified as follows. 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6-carboxy-2' , 4,7,7'-tetrachlorofluorescein, 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5, 6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, sulforhodamine B, sulforhodamine 101, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488,
- Staining method of tissue section An example of a staining method will be described.
- a method for preparing a tissue section to which this staining method can be applied (also simply referred to as “section”, including a section such as a pathological section) is not particularly limited, and a section prepared by a known procedure can be used.
- the 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 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 target biological material 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 A liquid etc. can be used.
- the pH condition is such that a signal is output from a pH range of 2.0 to 13.0 depending on the tissue slice used, and the roughness of the tissue is such that the signal can be evaluated.
- the pH is 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
- an autoclave As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
- the temperature is not particularly limited, but can be performed at room temperature.
- the temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.
- the section after the activation treatment is immersed in a container containing PBS and washed.
- 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 replaced during the immersion.
- Immunostaining step in order to stain the target biological material, an immunostaining solution containing fluorescent nanoparticles having sites that can bind directly or indirectly to the target biological material, Place on section and react with target biological material.
- the immunostaining agent solution used in the immunostaining step may be prepared in advance before this step.
- the conditions for performing the immunostaining step should be adjusted as appropriate so that an appropriate signal can be obtained according to the conventional immunostaining method. Can do.
- 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 or a surfactant such as Tween 20 dropwise.
- tissue specimen after the immunostaining process is preferably subjected to treatment such as immobilization / dehydration, penetration, and encapsulation so as to be suitable for observation.
- the immobilization / dehydration treatment may be performed by immersing the tissue specimen in a fixation treatment solution (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol).
- a fixation treatment solution crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol.
- the tissue specimen after the fixation / dehydration process may be immersed in a penetration liquid (xylene or the like).
- the encapsulating process may be performed by immersing the tissue specimen that has undergone the penetration process in the encapsulating liquid.
- the conditions for performing these treatments for example, the temperature and immersion time when the tissue specimen is immersed in a predetermined treatment solution, can be adjusted as appropriate in accordance with conventional immunostaining methods so as to obtain an appropriate signal. it can.
- Morphological Observation Staining Step In addition to the immunostaining step, morphological observation staining is performed so that the morphology of cells, tissues, organs, and the like can be observed in a bright field.
- the morphological observation staining step can be performed according to a conventional method. For morphological observation of tissue specimens, staining using eosin, in which cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained from red to dark red, is typically used.
- hematoxylin in which cell nuclei, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue, is also used as standard (the method of performing these two stainings simultaneously is hematoxylin and eosin staining). (Known as HE staining)).
- the morphological observation staining step may be performed after the immunostaining step, or may be performed before the immunostaining step.
- the analysis system 1 includes a microscope 10, an analysis device 20, and a display device 30.
- the microscope 10 includes a light source 12 and an imaging camera 14.
- the microscope 10 is connected to an analysis device 20 for controlling these, and the analysis device 20 displays a microscope image (see FIGS. 3 and 5 to 6), a processing result (see FIGS. 7 to 9), and the like.
- a display device 30 is connected.
- Imaging process (1) Acquisition of Bright Field Image
- the microscope 10 is controlled by the analysis device 20, and the tissue specimen A after morphological observation staining is imaged by the imaging camera 14. Thereafter, the analysis device 20 receives the imaging result of the imaging camera 14 and generates a bright field image 100 as shown in FIG. 3A.
- the microscope 10 is controlled by the analysis device 20, and the immunity is obtained from the light source 12 in the same field when the bright field image 100 is acquired.
- the tissue specimen A after staining is irradiated with excitation light, the fluorescent nanoparticles of the immunostaining agent are fluorescently emitted to cause a fluorescent bright spot to appear, and an immunostained image including the fluorescent bright spot is captured by the imaging camera 14.
- the analysis device 20 receives the imaging result of the imaging camera 14 and generates a fluorescent image 200 as shown in FIG. 3B.
- the excitation light can be irradiated by using a filter for excitation light that selectively transmits a predetermined wavelength as required. In this case, at the time of imaging an immunostained image, an immunostained image that includes only target fluorescence and excludes excitation light and other light that are not intended fluorescence and noise can be captured.
- the analysis device 20 performs image processing on the microscope image to calculate a certain statistical value, and displays it on the display device 30.
- the microscopic images are the bright field image 100 and the fluorescence image 200.
- S31 a step of partitioning the microscope image with a certain size;
- S32 analyzing the staining state of the microscope image for each section;
- S33 A process of calculating a certain statistical value based on the analysis result is executed.
- the analysis device 20 includes an execution unit and a storage unit, and the storage unit stores the above-described generation of the bright field image 100 and the fluorescence image 200 and the target biological material for executing the processing of S31 to S33 in FIG.
- An analysis program is stored.
- the execution unit reads out the analysis program for the target biological material, generates the bright field image 100 and the fluorescence image 200, and executes the processing of S31 to S33 in FIG.
- the background region 102 is designated from the bright field image 100 as shown in FIG. 5A.
- the “background region 102” is a region (reference region) that serves as a reference when determining a target section for calculating the number of fluorescent bright spots in the analysis step S32 described later.
- the area where cancer cells do not exist such as quality is specified.
- the same area as the background area 102 of the bright field image 100 is designated as the background area 202 from the fluorescent image 200.
- the background region 202 may be designated as a region where no fluorescent bright spot is observed from the fluorescent image 200 directly. Thereafter, as shown in FIG.
- the fluorescent image 200 is partitioned into the same size as the background region 202 to form a plurality of partitions 204.
- a plurality of sections may be formed in the fluorescent image 200 in advance, and one section may be designated as the background area 202 from among the sections.
- the fluorescent image 200 is subjected to image processing, and the number of fluorescent bright spots is calculated for each of the sections corresponding to the background region 202 and the section 204 in the fluorescent image 200.
- imageJ open source
- the process of calculating the number of fluorescent luminescent spots having a predetermined luminance or higher from the fluorescent image 200 can be performed semi-automatically and rapidly.
- the section 206 having a score less than the number of fluorescent bright spots in the background region 202 and the section 208 having the number of fluorescent bright spots equal to or greater than the number of fluorescent bright spots in the background area 202 are selected, and the section 206 is excluded from the analysis target. Only the section 208 is a section to be analyzed. The selection of the sections 206 and 208 may be based on a value obtained by adding a certain coefficient to the number of fluorescent bright spots in the background region 202.
- an average value of the number of fluorescent bright spots in the section 208 is calculated.
- the average value is an example of a statistical value.
- FIG. 6B shows an example in which five sections 206 are excluded from the analysis target among the 42 sections 204 of 6 ⁇ 7, and the remaining 37 sections 208 are set as the analysis target.
- FIG. 7 is a correlation diagram obtained under the same samples and conditions as in FIG. 15. In FIG. 7, the correlation coefficient was 0.99.
- the size of the background regions 102 and 202 and the compartment 204 is preferably set to 100 ⁇ m 2 which is equivalent to the size of one cell.
- the sizes of the background regions 102 and 202 and the partition 204 may be changed. For example, the size may be reduced to about 1/10 to 1/2 times the size of one cell, or about 2 to 10 times. You may enlarge it.
- the correlation coefficients when the sizes of the background regions 102 and 202 and the partition 204 are reduced and enlarged are as shown in Table 1. From the results shown in Table 1, the size of the background regions 102 and 202 and the compartment 204 should be reduced or enlarged to be equal to the size of one cell, or about 1/5 to 5 times.
- the shape of the background regions 102 and 202 and the section 204 may be changed to a polygonal shape other than the quadrangular shape, a circular shape, a honeycomb shape, or the like.
- the plurality of sections 204 are sorted into the non-analysis target section 206 and the analysis target section 208, and only the section 208 is set as the analysis target. Since the average value of the number of fluorescent bright spots is calculated, the statistical value is calculated uniquely and quickly. Therefore, the analysis time can be shortened while suppressing the variation in the statistical value by the type of tissue section and the diagnostician.
- the median value of the number of fluorescent bright spots may be calculated as a statistical value. That is, in the analysis step 32, all sections 204 are analyzed, and in the calculation step 33, the number of fluorescent bright spots in the section 204 is added and the median of the total number is set as a statistical value. For example, if the total number of fluorescent bright spots in all the sections 204 is 160, the median is 80. Thereafter, when the median value of the number of fluorescent bright spots in the section 204 and the protein quantitative value are plotted in correspondence, a correlation diagram as shown in FIG. 8 can be obtained. FIG. 8 is also a correlation diagram obtained under the same samples and conditions as in FIG. 15, and in FIG. 8, the correlation coefficient was 0.98. In Modification 1, it is not necessary to select the sections 206 and 208, and the background areas 102 and 202 may not be specified in the partitioning step S31.
- a histogram peak value of the number of fluorescent bright spots may be calculated as a statistical value. That is, in the analysis process 32, all the sections 204 are analyzed, and in the calculation process, the number of fluorescent bright spots per section 204 and the ratio of the sections 204 where a certain number of fluorescent bright spots exist to all the sections 204 are obtained. A histogram is generated by calculation, and a class value corresponding to the peak value of the histogram is set as a statistical value for each cell line.
- FIG. 9 is a diagram showing an example of the histogram obtained under the same samples and conditions as in FIG. In FIG.
- the horizontal axis indicates the number of fluorescent bright spots per section 204
- the vertical axis indicates the ratio of the sections 204 in which a certain number of fluorescent bright spots exist with respect to all sections 204.
- the class value corresponding to the peak value (20%) (class 10) ) Is a statistical value.
- A549 is class 13
- Calu3 is class 15
- T47D is class 19
- MCF7 is class 21, and these class values are statistical values.
- the second embodiment is different from the first embodiment in the following points, and is otherwise the same.
- tissue specimen B not containing the target biological material is also prepared, and these are immunostained and stained with a staining agent for immunostaining or a staining agent for morphology observation. Observe and stain.
- the “tissue specimen B not containing the target biological material” is a tissue specimen such as cultured cells that do not express the protein to be detected or quantified.
- tissue specimen B an example in which a tissue specimen of a mouse osteosarcoma cell line LM8 (cultured cell) is used as the tissue specimen B that does not contain the target biological material will be described.
- the imaging step S2 from the tissue sample A after the morphological observation, the bright field image 100 as shown in FIG. 3A and the fluorescent image 200 as shown in FIG. A bright field image 300 as shown in FIG. 10A and a fluorescent image 400 as shown in FIG. 10B are generated.
- the fluorescent bright spot basically does not appear.
- a background region 302 is designated from the bright field image 300 as shown in FIG. 10A.
- the “background region 302” is a region that serves as a reference when determining a target section for calculating the number of fluorescent bright spots in the analysis step S32 described later, and is a region that replaces the background region 102.
- the same area as the background area 302 of the bright field image 300 is designated as the background area 402 from the fluorescent image 400.
- the background region 402 may be directly specified from the fluorescent image 400 without specifying the background region 302 from the bright field image 300.
- the tissue specimen B does not include the target biological material (fluorescence bright spots basically do not appear). May be specified. Thereafter, as shown in FIG. 6A, the fluorescent image 200 is sectioned with the same size as the background region 402 to form a plurality of sections 204.
- the background area 202 in FIG. 5B is replaced with the background area 402 in FIG. 10B, and the same processing as in the first embodiment is executed.
- FIG. 11 is a correlation diagram obtained under the same samples and conditions as in FIG. In FIG. 11, the correlation coefficient is 0.99, and the same accuracy as that obtained in the correlation diagram of FIG. 7 was maintained.
- the size of the background regions 302 and 402 and the compartment 204 is preferably set to 100 ⁇ m 2 which is equivalent to the size of one cell.
- the sizes of the background regions 302 and 402 and the compartment 204 may be changed.
- the size may be reduced to about 1/10 to 1/2 times the size of one cell, or about 2 to 10 times. You may enlarge it.
- the shapes of the background regions 302 and 402 and the partition 204 may be changed to a polygonal shape other than the quadrangular shape, a circular shape, a honeycomb shape, or the like.
- the tissue specimen B that does not include the target biological material is produced, the bright field image 300 and the fluorescence image 400 are generated from the tissue specimen B, the background regions 302 and 402 are designated, Statistical values are calculated based on the background area 402.
- the third embodiment is different from the first embodiment in the following points, and is otherwise the same.
- tissue specimen C of the tissue itself containing the target biological material is prepared as a tissue specimen containing the target biological material, and this is immunostained and observed with a staining agent for immunostaining or a staining agent for morphological observation.
- Stain. “Tissue (self)” refers to a biologically larger structure than cells, such as blood vessels, lymphatic vessels, glomeruli, gland ducts, milk ducts, follicles, and the like.
- tissue specimen of senile plaques alzheimer's disease brain tissue
- staining with an anti-amyloid ⁇ antibody will be described.
- Imaging step S2 from the tissue sample A after morphological observation, the bright field image 100 as shown in FIG. 3A and the fluorescent image 200 as shown in FIG. A bright field image 500 as shown in FIG. 12A and a fluorescent image 600 as shown in FIG. 12B are generated.
- the analysis device 20 performs image processing on the microscopic image to calculate a certain statistical value, and displays it on the display device 30.
- the microscopic images are a bright-field image 500 and a fluorescent image 600.
- a background region 502 is designated from the bright field image 500.
- the “background region 502” is a region serving as a reference when determining the target section for calculating the number of fluorescent bright spots in the analysis step S32 described later, and here, a region free from senile plaques is designated.
- the same area as the background area 502 of the bright field image 500 is designated as the background area 602 from the fluorescent image 600.
- the background region 602 may be designated as a region where the fluorescent bright spot is not directly observed from the fluorescent image 600 without designating the background region 502 from the bright field image 500.
- the fluorescent image 600 is partitioned with the same size as the background region 602 to form a plurality of partitions 604.
- a plurality of sections may be formed in the fluorescent image 600 in advance, and one section may be designated as the background area 602 from among the sections.
- the fluorescent image 600 is subjected to image processing, and the number of fluorescent bright spots is calculated for each of the sections corresponding to the background region 602 and the sections corresponding to the section 604 in the fluorescent image 600.
- a plurality of sections 604 are (i) the number of fluorescent luminescent spots in the background area 602. Are divided into a section 606 having less than the number of fluorescent luminescent spots and a section 608 having the number of fluorescent luminescent spots equal to or greater than the number of fluorescent luminescent spots in the background region 602, the section 606 is excluded from the analysis target, and only the section 608 is selected as the analysis target. To do.
- the selection of the sections 606 and 608 may be based on a value obtained by adding a certain coefficient to the number of fluorescent bright spots in the background region 602.
- the average value of the number of fluorescent bright spots in the section 608 is calculated.
- the average value is an example of a statistical value.
- FIG. 13B shows an example in which five sections 606 are excluded from the analysis target among the 42 sections 604 of 6 ⁇ 7, and the remaining 37 sections 608 are set as the analysis target.
- FIG. 14 is a correlation diagram obtained for five types of Alzheimer specimens. In FIG. 14, the correlation coefficient was 0.99.
- the sizes of the background areas 502 and 602 and the section 604 are preferably set to a size equivalent to the size of one tissue.
- the size of the background regions 502 and 602 and the section 604 is preferably set to 400 ⁇ m 2 which is equivalent to the size of the senile plaque.
- the sizes of the background regions 502 and 602 and the partition 604 may be changed. For example, the size may be reduced to about 1/10 to 1/2 times the size of one tissue, or about 2 to 10 times. You may enlarge it.
- the correlation coefficients when the sizes of the background regions 502 and 602 and the partition 604 are reduced and enlarged are as shown in Table 2. From the results shown in Table 2, the size of the background regions 502 and 602 and the section 604 should be reduced or enlarged to the same size as that of one senile plaque or about 1/5 to 5 times.
- the shapes of the background regions 502 and 602 and the section 604 themselves may be changed to a polygonal shape other than the quadrangular shape, a circular shape, a honeycomb shape, or the like.
- the tissue sample C of the tissue itself containing the target biological material is prepared and the statistical value is calculated based on the tissue sample C. Even if the tissue sample changes from the cell level to the tissue level, The same effect as that of the first embodiment can be obtained.
- the median value of the number of fluorescent bright spots may be calculated as the statistical value, or the fluorescent value may be calculated as the statistical value.
- a histogram peak value of the number of bright spots may be calculated.
- tissue specimens of the tissue itself that does not contain the target biological material is prepared, and a bright field image and a fluorescence image are generated from the tissue specimen and a background region is designated. The statistical value may be calculated based on the background area.
- tissue specimens of tissues themselves that do not contain the target biological substance include tissue specimens such as stroma and skeletal muscle tissues in young brains and other cases in animal experiments such as mice. .
- a PBS dispersion of fluorescent nanoparticles to which a biological material recognition site is bound is placed on a tissue section and reacted with the target biological material.
- the biological material recognition site to be bound to the fluorescent nanoparticles staining corresponding to various biological materials becomes possible.
- the respective fluorescent nanoparticle PBS dispersion liquids may be mixed in advance, or may be separately placed on a tissue section separately.
- 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 tissue section is immersed in a container containing PBS to remove unreacted fluorescent nanoparticles.
- 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 tissue 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. As a result, bright spot measurement data equivalent to the result in the first embodiment could be obtained.
- the first to fourth embodiments described above are not limited to the above, and may be improved as appropriate.
- the number of fluorescent bright spots of the fluorescent images 200 and 600 is calculated in the step of analyzing the staining state of the microscope image.
- the chromaticity of staining of the bright field images 100 and 500, the intensity for each color, the area for each color, and the like may be calculated, and the statistical value may be calculated based on the calculation result.
- a background region a region without cells, or a region without cancer cells such as stroma from the bright field image, fluorescence image or fluorescence image, the operator or the like performs some operation via the operation unit or the like.
- all of the analysis apparatus for the target biological material of the present invention may automatically perform processing and specify.
- the present invention provides an analysis apparatus, an analysis system, an analysis method, and an analysis program for a target biological material that can reduce the analysis time while suppressing variations in the types of tissue sections and statistical values by a diagnostician. Suitable for providing.
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Abstract
Description
特許文献1の技術では、組織切片における細胞の形態を表す明視野画像と、組織切片の同一範囲における特定のタンパク質の発現を蛍光輝点で表す蛍光画像とを取得し、明視野画像から特定のタンパク質の発現領域を包含する細胞領域を推定するとともに、蛍光画像から蛍光輝点を抽出し、推定された各細胞領域に含まれる細胞核および蛍光輝点に基づいて、各細胞領域に関する特徴量を算出し、その算出された特徴量に基づいて各細胞領域が癌であるか否かと特定のタンパク質の発現状況とを判定しうるようになっている。
近年では、組織標本中(明視野画像)で複数の細胞が重畳している場合でも、蛍光画像の蛍光輝点がいずれの細胞に帰属しているかが判別されるようになっており、その結果特定のタンパク質の発現数を正確に定量可能となり、病理診断の精度が向上している(特許文献2、段落0069~0080、0096、図9~図15参照)。
図15中、黒点が蛍光輝点数の平均値を示し、黒点に対する上下の線分が標準偏差を示している。図15に示すとおり、タンパク質定量値が大きくなるほど蛍光輝点数の標準偏差は大きくなっており、組織切片の細胞株の種類や診断者によって診断結果(統計値)にバラツキが生じているのがわかる。
したがって本発明の主な目的は、組織切片の種類や診断者による統計値のバラツキを抑制しながら、解析時間を短縮することができる目的生体物質の解析装置、解析システム、解析方法および解析プログラムを提供することにある。
さらに本発明の目的としては、目的生体物質としてタンパク質のほか、核酸・糖鎖・酵素などを組織切片の蛍光観察により輝点計測することも含まれる。
染色後の組織標本の顕微鏡画像を生成する生成手段と、
前記顕微鏡画像を一定のサイズで区画する区画手段と、
前記顕微鏡画像の染色状態を区画ごとに解析する解析手段と、
前記解析手段の解析結果に基づき一定の統計値を算出する算出手段と、
を備えることを特徴とする目的生体物質の解析装置が提供される。
染色後の組織標本を撮像する顕微鏡と、
前記顕微鏡の撮像結果を受ける前記目的生体物質の解析装置と、
を備えることを特徴とする目的生体物質の解析システムが提供される。
染色後の組織標本の顕微鏡画像を生成する工程と、
前記顕微鏡画像を一定のサイズで区画する工程と、
前記顕微鏡画像の染色状態を区画ごとに解析する工程と、
前記解析工程の解析結果に基づき一定の統計値を算出する工程と、
を備えることを特徴とする目的生体物質の解析方法が提供される。
コンピュータを、
染色後の組織標本の顕微鏡画像を生成する生成手段、
前記顕微鏡画像を一定のサイズで区画する区画手段、
前記顕微鏡画像の染色状態を区画ごとに解析する解析手段、
前記解析手段の解析結果に基づき一定の統計値を算出する算出手段、
として機能させるための目的生体物質の解析プログラムが提供される。
本実施形態では主に、顕微鏡画像を解析し一定の統計値を算出する解析方法を提供する。かかる解析方法は主に、図1に示すとおり、
(S1)一定の染色剤で組織標本を染色する工程と、
(S2)一定の顕微鏡で染色後の組織標本の染色像を撮像する工程と、
(S3)顕微鏡画像を解析し一定の統計値を算出する工程とを、備えている。
染色工程S1では、目的生体物質を含む組織標本Aを作製し、これを免疫染色用の染色剤や形態観察用の染色剤で免疫染色および形態観察染色する。
目的生体物質とは、主に病理診断の観点からの検出または定量のために、蛍光標識体を用いた免疫染色の対象とするものをいい、組織切片に発現している生体物質、特にタンパク質(抗原)、核酸(例えば、DNA、RNA、miRNAなど)である。
典型的な目的生体物質としては、各種の癌組織の細胞膜で発現しており、バイオマーカーとして利用することができる生体物質が挙げられる。
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ダ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。
免疫染色剤としては、蛍光標識の効率を向上させて蛍光の劣化につながる時間経過をなるべく抑えるために、一次抗体および蛍光ナノ粒子が間接的に、つまり抗原抗体反応などを利用した、共有結合以外の結合によって連結される複合体を用いることが好ましい。染色操作を簡便にするため、免疫染色剤として、一次抗体または二次抗体に蛍光ナノ粒子が直結している複合体を用いることもできる。
“…”は抗原抗体反応により結合していることを表し、“~”が示す結合の態様としては特に限定されず、たとえば、共有結合、イオン結合、水素結合、配位結合、抗原抗体結合、ビオチンアビジン反応、物理吸着、化学吸着などが挙げられ、必要に応じてリンカー分子を介していてもよい。
一次抗体には、目的生体物質としてのタンパク質を抗原として特異的に認識して結合する抗体(IgG)を用いることができる。たとえば、HER2を目的生体物質とする場合は抗HER2抗体を、HER3を目的生体物質とする場合は抗HER3抗体を、それぞれ用いることができる。
二次抗体には、一次抗体を抗原として特異的に認識して結合する抗体(IgG)を用いることができる。
一次抗体および二次抗体はいずれも、ポリクローナル抗体であってもよいが、定量の安定性の観点から、モノクローナル抗体が好ましい。抗体を産生する動物(免疫動物)の種類は特に限定されるものではなく、従来と同様、マウス、ラット、モルモット、ウサギ、ヤギ、ヒツジなどから選択すればよい。
蛍光ナノ粒子とは、励起光の照射を受けて蛍光発光するナノサイズの粒子であって、目的生体物質を1分子ずつ輝点として表すのに十分な強度の蛍光を発光しうる粒子である。
蛍光ナノ粒子として、好ましくは量子ドット(半導体ナノ粒子)、蛍光物質集積ナノ粒子が使用される。
量子ドットとしては、II-VI族化合物、III-V族化合物またはIV族元素を含有する半導体ナノ粒子が使用される。たとえば、CdSe、CdS、CdTe、ZnSe、ZnS、ZnTe、InP、InN、InAs、InGaP、GaP、GaAs、Si、Geなどが挙げられる。
蛍光物質集積ナノ粒子は、有機物または無機物でできた粒子を母体とし、複数の蛍光物質(たとえば、上記量子ドット、蛍光色素など)がその中に内包されているおよび/またはその表面に吸着している構造を有する、ナノサイズの粒子である。
蛍光物質集積ナノ粒子としては、母体と蛍光物質とが、互いに反対の電荷を有する置換基または部位を有し、静電的相互作用が働くものであることが好適である。
蛍光物質集積ナノ粒子としては、量子ドット集積ナノ粒子、蛍光色素集積ナノ粒子などが使用される。
母体のうち、有機物としては、メラミン樹脂、尿素樹脂、アニリン樹脂、グアナミン樹脂、フェノール樹脂、キシレン樹脂、フラン樹脂など、一般的に熱硬化性樹脂に分類される樹脂;スチレン樹脂、アクリル樹脂、アクリロニトリル樹脂、AS樹脂(アクリロニトリル-スチレン共重合体)、ASA樹脂(アクリロニトリル-スチレン-アクリル酸メチル共重合体)など、一般的に熱可塑性樹脂に分類される樹脂;ポリ乳酸等のその他の樹脂;多糖を例示することができる。
母体のうち、無機物としては、シリカ、ガラスなどを例示することができる。
量子ドット集積ナノ粒子とは、上記量子ドットが、上記母体の中に内包されている、および/またはその表面に吸着している構造を有する。
量子ドットが母体に内包されている場合、量子ドットは母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。
蛍光色素集積ナノ粒子とは、蛍光色素が、上記母体の中に内包されている、および/またはその表面に吸着している構造を有する。
蛍光色素としては、ローダミン系色素分子、スクアリリウム系色素分子、シアニン系色素分子、芳香環系色素分子、オキサジン系色素分子、カルボピロニン系色素分子、ピロメセン系色素分子などを例示することができる。
蛍光色素としては、Alexa Fluor(登録商標、インビトロジェン社製)系色素分子、BODIPY(登録商標、インビトロジェン社製)系色素分子、Cy(登録商標、GEヘルスケア社製)系色素分子、HiLyte(登録商標、アナスペック社製)系色素分子、DyLight(登録商標、サーモサイエンティフィック社製)系色素分子、ATTO(登録商標、ATTO-TEC社製)系色素分子、MFP(登録商標、Mobitec社製)系色素分子、CF(登録商標、Biotium社製)系色素分子、DY(登録商標、DYOMICS社製)系色素分子、CAL(登録商標、BioSearch Technologies社製)系色素分子などを用いることができる。
なお、蛍光色素が母体に内包されている場合、蛍光色素は母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。
染色方法の一例について説明する。
この染色方法が適用できる組織切片(単に「切片」ともいい、病理切片などの切片も含まれる。)の作製法は特に限定されず、公知の手順により作製されたものを用いることができる。
(5.1.1)脱パラフィン処理
キシレンを入れた容器に、切片を浸漬させ、パラフィン除去する。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。
公知の方法に倣い、目的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のクエン酸緩衝液(pH6.0)、1mMのEDTA溶液(pH8.0)、5%尿素、0.1Mのトリス塩酸緩衝液などを用いることができる。
pH条件は用いる組織切片に応じてpH2.0~13.0の範囲から、シグナルが出て、組織の荒れがシグナルを評価できる程度となる条件で行う。通常はpH6.0~8.0で行うが、特殊な組織切片ではたとえばpH3.0でも行う。
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。
免疫染色工程では、目的生体物質を染色するために、目的生体物質に直接的または間接的に結合しうる部位を有する蛍光ナノ粒子を含む免疫染色剤の溶液を、切片に乗せ、目的生体物質との反応を行う。免疫染色工程に用いる免疫染色剤の溶液については、この工程の前にあらかじめ調製しておけばよい。
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。
上述したような処理を行う前に、BSA含有PBSなど公知のブロッキング剤やTween20などの界面活性剤を滴下することが好ましい。
免疫染色工程を終えた組織標本は、観察に適したものとなるよう、固定化・脱水、透徹、封入などの処理を行うことが好ましい。
これらの処理を行う上での条件、たとえば組織標本を所定の処理液に浸漬する際の温度および浸漬時間は、従来の免疫染色法に準じて、適切なシグナルが得られるよう適宜調整することができる。
免疫染色工程とは別に、明視野において細胞、組織、臓器などの形態を観察することができるようにするための、形態観察染色を行う。
形態観察染色工程は、常法に従って行うことができる。
組織標本の形態観察に関しては、細胞質・間質・各種線維・赤血球・角化細胞が赤~濃赤色に染色される、エオジンを用いた染色が標準的に用いられている。細胞核・石灰部・軟骨組織・細菌・粘液が青藍色~淡青色に染色される、ヘマトキシリンを用いた染色も標準的に用いられている(これら2つの染色を同時に行う方法はヘマトキシリン・エオジン染色(HE染色)として知られている)。
形態観察染色工程は、免疫染色工程の後に行うようにしてもよいし、免疫染色工程の前に行うようにしてもよい。
撮像工程S2および解析工程S3では図2の解析システム1を使用する。
解析システム1は顕微鏡10、解析装置20および表示装置30を備えている。顕微鏡10は光源12および撮像カメラ14を備えている。顕微鏡10にはこれらを制御する解析装置20が接続され、解析装置20には顕微鏡画像(図3、図5~図6参照)や処理結果(図7~図9参照)などを表示するための表示装置30が接続されている。
(1)明視野画像の取得
撮像工程S2では、解析装置20で顕微鏡10を制御し、形態観察染色後の組織標本Aを撮像カメラ14で撮像する。その後、解析装置20で、撮像カメラ14の撮像結果を受け、図3Aのような明視野画像100を生成する。
撮像工程S2では、明視野画像100の取得とは別に、解析装置20で顕微鏡10を制御し、明視野画像100を取得した際の同一視野において、光源12から、免疫染色後の組織標本Aに対し励起光を照射し、免疫染色剤の蛍光ナノ粒子を蛍光発光させて蛍光輝点を出現させ、その蛍光輝点を含む免疫染色像を撮像カメラ14で撮像する。その後、解析装置20で、撮像カメラ14の撮像結果を受け、図3Bのような蛍光画像200を生成する。
なお、励起光の照射は、必要に応じて所定の波長を選択的に透過させる励起光用フィルターを用いることで照射することができる。かかる場合、免疫染色像の撮像のときには、目的とする蛍光のみを含み、目的としない蛍光やノイズとなる励起光およびその他の光を排除した免疫染色像を撮像することができる。
解析工程S3では、解析装置20で、顕微鏡画像を画像処理して一定の統計値を算出し、表示装置30に表示させる。当該顕微鏡画像はここでは明視野画像100および蛍光画像200である。
具体的には、図4に示すとおり、解析装置20において主に、
(S31)顕微鏡画像を一定のサイズで区画する工程と、
(S32)顕微鏡画像の染色状態を区画ごとに解析する工程と、
(S33)その解析結果に基づき一定の統計値を算出する工程と、の処理を実行する。
ここでは、当該実行部がその目的生体物質の解析プログラムを読み出し、明視野画像100および蛍光画像200の生成、ならびに図4のS31~S33の処理を実行するようになっている。
「バックグラウンド領域102」とは、後述の解析工程S32で蛍光輝点数を算出するための対象区画を決定する際の基準となる領域(基準領域)であって、ここでは細胞がない領域、間質など癌細胞が存在しない領域などが指定される。
その後、図5Bに示すとおり、蛍光画像200から、明視野画像100のバックグラウンド領域102と同じ領域を、バックグラウンド領域202として指定する。明視野画像100からバックグラウンド領域102を指定せずに、直接的に蛍光画像200から蛍光輝点が観察されない領域としてバックグラウンド領域202を指定してもよい。
その後、図6Aに示すとおり、蛍光画像200を、バックグラウンド領域202と同じサイズで区画化し、複数の区画204を形成する。あらかじめ蛍光画像200に複数の区画を形成しておき、それら区画のなかから1つの区画をバックグラウンド領域202として指定してもよい。
蛍光画像200の画像処理に用いることができるソフトウエアとしては、たとえば「ImageJ」(オープンソース)が挙げられる。このような画像処理ソフトウエアを利用することにより、蛍光画像200から、所定の輝度以上の蛍光輝点数を算出する処理を、半自動的に、迅速に行うことができる。
数がバックグラウンド領域202の蛍光輝点数未満の区画206と、(ii)蛍光輝点数がバックグラウンド領域202の蛍光輝点数以上の区画208とに選別し、区画206を解析対象から除外し、区画208のみを解析対象の区画とする。区画206、208の選別は、バックグラウンド領域202の蛍光輝点数に一定の係数を加えた値を基準としてもよい。
図7は図15と同様のサンプルおよび条件で求めた相関図であり、図7では相関係数は0.99であった。図7の相関図を得るにあたっては、バックグラウンド領域102、202および区画204のサイズ(面積)を、10μm×10μm=100μm2と設定している。
ただ、バックグラウンド領域102、202および区画204のサイズは変更してもよく、たとえば1つの細胞のサイズに対し1/10~1/2倍程度に縮小させてもよいし、2~10倍程度に拡大してもよい。
図7の相関図を得るにあたり、バックグラウンド領域102、202および区画204のサイズを縮小および拡大させた場合の相関係数は表1のとおりであった。表1の結果から、バックグラウンド領域102、202および区画204のサイズは、1つの細胞のサイズと同等か、または1/5~5倍程度に縮小もしくは拡大するのがよい。
算出工程S33では、統計値として蛍光輝点数の中央値を算出してもよい。
すなわち、解析工程32ではすべての区画204を解析対象とし、算出工程33では区画204の蛍光輝点数を加算しその総数の中央値を統計値とする。たとえば、すべての区画204の蛍光輝点数の総数が160である場合、中央値は80である。
その後、区画204の蛍光輝点数の中央値とタンパク質定量値とを対応付けプロットすると、図8のような相関図を得ることができる。
図8も図15と同様のサンプルおよび条件で求めた相関図であり、図8では相関係数は0.98であった。
なお、変形例1では区画206、208の選別が不要であり、区画化工程S31ではバックグラウンド領域102、202は指定しなくてもよい。
算出工程S33では、統計値として蛍光輝点数のヒストグラムピーク値を算出してもよい。
すなわち、解析工程32ではすべての区画204を解析対象とし、算出工程では、1区画204あたりの蛍光輝点数と、すべての区画204に対する、一定数の蛍光輝点が存在する区画204の割合とを算出してヒストグラムを生成し、細胞株ごとに、ヒストグラムのピーク値に対応する階級値を統計値とする。
図9は図15と同様のサンプルおよび条件で求めた当該ヒストグラムの一例を示す図である。
図9では、横軸が1区画204あたりの蛍光輝点数を示し、縦軸がすべての区画204に対する、一定数の蛍光輝点が存在する区画204の割合を示している。
図9の例では、たとえば、SKOV-3については、10個の蛍光輝点が存在する区画の割合が20%程度と最も多いため、当該ピーク値(20%)に対応する階級値(階級10)を統計値とする。これと同様に、A549については階級13であり、Calu3については階級15であり、T47Dについては階級19であり、MCF7については階級21であり、これら階級値を統計値とする。
その後、区画204の蛍光輝点数のヒストグラムピーク値に対応する階級値とタンパク質定量値とを対応付けプロットすると、一定の相関図を得ることができ、当該一定の相関図では相関係数は0.98であった。
なお、変形例2でも区画206、208の選別が不要であり、区画化工程S31ではバックグラウンド領域102、202は指定しなくてもよい。
第2の実施形態は下記の点で第1の実施形態と異なっており、それ以外は同様となっている。
染色工程S1では、目的生体物質を含む組織標本Aに加えて、目的生体物質を含まない組織標本Bも作製し、これらを免疫染色用の染色剤や形態観察用の染色剤で免疫染色および形態観察染色する。
「目的生体物質を含まない組織標本B」とは、検出または定量対象のタンパク質を発現しない培養細胞などの組織標本である。
第2の実施形態では、目的生体物質を含まない組織標本Bとして、マウス骨肉腫細胞株LM8(培養細胞)の組織標本を使用した例について説明する。
撮像工程S2では、形態観察後の組織標本Aから、図3Aのような明視野画像100と図3Bのような蛍光画像200とを生成したのと同様に、形態観察後の組織標本Bから、図10Aのような明視野画像300と図10Bのような蛍光画像400とを生成する。
なお、組織標本Bは目的生体物質を含まないため、図10Bに示すとおり、蛍光輝点は基本的には出現しない。
区画化工程S31では、はじめに、図10Aに示すとおり、明視野画像300からバックグラウンド領域302を指定する。
「バックグラウンド領域302」とは、後述の解析工程S32で蛍光輝点数を算出するための対象区画を決定する際の基準となる領域であって、バックグラウンド領域102に代わる領域である。
その後、図10Bに示すとおり、蛍光画像400から、明視野画像300のバックグラウンド領域302と同じ領域を、バックグラウンド領域402として指定する。明視野画像300からバックグラウンド領域302を指定せずに、直接的に蛍光画像400からバックグラウンド領域402を指定してもよい。
なお、上記のとおり、組織標本Bは目的生体物質を含まない(蛍光輝点は基本的には出現しない)ため、明視野画像300および蛍光画像400のいずれの領域からバックグラウンド領域302、402を指定してもよい。
その後、図6Aに示すとおり、蛍光画像200を、バックグラウンド領域402と同じサイズで区画化し、複数の区画204を形成する。
図11は図15と同様のサンプルおよび条件で求めた相関図である。図11では相関係数は0.99であり、図7の相関図を得るのと同様の精度が保持されていた。図11の相関図を得るにあたっても、バックグラウンド領域302、402および区画204のサイズ(面積)を、10μm×10μm=100μm2と設定している。
ただ、バックグラウンド領域302、402および区画204のサイズは変更してもよく、たとえば1つの細胞のサイズに対し1/10~1/2倍程度に縮小させてもよいし、2~10倍程度に拡大してもよい。
さらにバックグラウンド領域302、402および区画204の形状自体も、4角形状以外の多角形状、円形状、ハニカム状などに変更してもよい。
第1の実施形態にかかる図7の相関図を得る実験と、第2の実施形態にかかる図11の相関図を得る実験とを、それぞれ8回繰り返し、SKOV-3、A549、Calu3、T47D、MCF7の5種の細胞株についての再現性を検証したところ、図7の相関図を得る実験ではCV値(coefficient of variation)=(標準偏差/平均値)が8%程度であったのに対し、図11の相関図を得る実験ではCV値が6.2%と改善されていた。このことから、目的生体物質を含まない組織標本Bを作製しそこからバックグラウンド領域402を指定することが、統計値のバラツキを抑制するのに特に有用であることがわかった。
第3の実施形態は下記の点で第1の実施形態と異なっており、それ以外は同様となっている。
染色工程S1では、目的生体物質を含む組織標本として、目的生体物質を含む組織自体の組織標本Cを作製し、これを免疫染色用の染色剤や形態観察用の染色剤で免疫染色および形態観察染色する。
「組織(自体)」とは、生物学的に細胞より大きい構造体をいい、たとえば、血管、リンパ管、糸球体、腺管、乳管、卵胞などがある。
第3の実施形態では、組織標本Cとして老人斑(アルツハイマー病の脳の組織)の組織標本を使用し、抗アミロイドβ抗体を用いて染色した例を説明する。
撮像工程S2では、形態観察後の組織標本Aから、図3Aのような明視野画像100と図3Bのような蛍光画像200とを生成したのと同様に、形態観察後の組織標本Cから、図12Aのような明視野画像500と図12Bのような蛍光画像600とを生成する。
解析工程S3では、明視野画像100および蛍光画像200を、明視野画像500および蛍光画像600に置き換え、第1の実施形態にかかる処理と同様の処理を実行する。
「バックグラウンド領域502」とは、後述の解析工程S32で蛍光輝点数を算出するための対象区画を決定する際の基準となる領域であって、ここでは老人斑がない領域が指定される。
その後、図12Bに示すとおり、蛍光画像600から、明視野画像500のバックグラウンド領域502と同じ領域を、バックグラウンド領域602として指定する。明視野画像500からバックグラウンド領域502を指定せずに、直接的に蛍光画像600から蛍光輝点が観察されない領域としてバックグラウンド領域602を指定してもよい。
その後、図13Aに示すとおり、蛍光画像600を、バックグラウンド領域602と同じサイズで区画化し、複数の区画604を形成する。あらかじめ蛍光画像600に複数の区画を形成しておき、それら区画のなかから1つの区画をバックグラウンド領域602として指定してもよい。
図14は5種のアルツハイマー検体について求めた相関図であり、図14では相関係数は0.99であった。図14の相関図を得るにあたっては、バックグラウンド領域502、602および区画604のサイズ(面積)を、20μm×20μm=400μm2と設定している。
ここでは、バックグラウンド領域502、602および区画604のサイズは老人斑のサイズと同等の400μm2と設定するのがよい。
ただ、バックグラウンド領域502、602および区画604のサイズは変更してもよく、たとえば1つの組織のサイズに対し1/10~1/2倍程度に縮小させてもよいし、2~10倍程度に拡大してもよい。
図14の相関図を得るにあたり、バックグラウンド領域502、602および区画604のサイズを縮小および拡大させた場合の相関係数は表2のとおりであった。表2の結果から、バックグラウンド領域502、602および区画604のサイズは、1つの老人斑のサイズと同等か、または1/5~5倍程度に縮小もしくは拡大するのがよい。
第3の実施形態では、第2の実施形態と同様に、目的生体物質を含まない組織自体の組織標本を作製し、当該組織標本から明視野画像および蛍光画像を生成してバックグラウンド領域を指定し、当該バックグラウンド領域に基づき統計値を算出してもよい。たとえば、老人斑に対する、「目的生体物質を含まない組織自体の組織標本」としては、マウスなどの動物実験の場合、若齢の脳やその他には間質、骨格筋組織などの組織標本がある。
第4の実施形態は、第1の実施形態に記載の「(5.2)免疫染色工程」の代わりに、下記「[生体物質認識部位が結合された蛍光ナノ粒子を用いた染色工程]」としたこと以外は第1の実施形態と同様となっている。
生体物質認識部位が結合された蛍光ナノ粒子のPBS分散液を組織切片に載せ、目的とする生体物質と反応させる。蛍光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ナノ粒子を用いる場合には、それぞれの蛍光ナノ粒子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、染色後の組織切片を浸漬させ、未反応蛍光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。その結果、第1の実施形態における結果と同等の輝点計測データを得ることが出来た。
たとえば、第1~第4の実施形態(変形例1、2を含む。)では、顕微鏡画像の染色状態を解析する工程で、蛍光画像200、600の蛍光輝点数を算出している。
これに代えて、明視野画像100、500の染色の色度、色ごとの強度、色ごとの面積などを算出し、その算出結果に基づき統計値を算出してもよい。
10 顕微鏡
12 光源
14 撮像カメラ
20 解析装置
30 表示装置
Claims (14)
- 染色後の組織標本の顕微鏡画像を生成する生成手段と、
前記顕微鏡画像を一定のサイズで区画する区画手段と、
前記顕微鏡画像の染色状態を区画ごとに解析する解析手段と、
前記解析手段の解析結果に基づき一定の統計値を算出する算出手段と、
を備えることを特徴とする目的生体物質の解析装置。 - 請求項1に記載の目的生体物質の解析装置において、
前記区画手段が、前記顕微鏡画像を、1つの細胞もしくは組織のサイズと同等のサイズで、または1つの細胞もしくは組織のサイズの1/5~5倍に縮小もしくは拡大したサイズで区画することを特徴とする目的生体物質の解析装置。 - 請求項1または2に記載の目的生体物質の解析装置において、
前記区画手段が、前記解析手段による解析対象の区画を決定するための基準領域を指定し、
前記解析手段が、前記区画手段により区画された区画のうち、前記基準領域の基準を満たす区画を、解析対象の区画とすることを特徴とする目的生体物質の解析装置。 - 請求項3に記載の目的生体物質の解析装置において、
前記生成手段が、目的生体物質を含まない染色後の組織標本から第2の顕微鏡画像を生成し、
前記区画手段が、前記第2の顕微鏡画像から、前記解析手段による解析対象の区画を決定するための基準領域を指定することを特徴とする目的生体物質の解析装置。 - 請求項3または4に記載の目的生体物質の解析装置において、
前記生成手段が、前記顕微鏡画像として蛍光画像を生成し、
前記解析手段が、前記蛍光画像の蛍光輝点数を前記解析対象の区画ごとに算出し、
前記算出手段が、前記統計値として、前記解析対象の区画の蛍光輝点数の平均値を算出することを特徴とする目的生体物質の解析装置。 - 請求項1または2に記載の目的生体物質の解析装置において、
前記生成手段が、前記顕微鏡画像として蛍光画像を生成し、
前記解析手段が、前記区画手段により区画されたすべての区画を、解析対象の区画とし、
前記算出手段が、前記統計値として、前記解析対象の区画の蛍光輝点数の中央値を算出することを特徴とする目的生体物質の解析装置。 - 請求項1または2に記載の目的生体物質の解析装置において、
前記生成手段が、前記顕微鏡画像として蛍光画像を生成し、
前記解析手段が、前記区画手段により区画されたすべての区画を、解析対象の区画とし、
前記算出手段が、前記統計値として、前記解析対象の区画の蛍光輝点数のヒストグラムピーク値に対応する階級値を算出することを特徴とする目的生体物質の解析装置。 - 請求項3または4に記載の目的生体物質の解析装置において、
前記生成手段が、前記顕微鏡画像として蛍光画像を生成し、
前記算出手段が、前記統計値として、前記解析対象の区画の蛍光輝点数の中央値を算出することを特徴とする目的生体物質の解析装置。 - 請求項3または4に記載の目的生体物質の解析装置において、
前記生成手段が、前記顕微鏡画像として蛍光画像を生成し、
前記算出手段が、前記統計値として、前記解析対象の区画の蛍光輝点数のヒストグラムピーク値に対応する階級値を算出することを特徴とする目的生体物質の解析装置。 - 請求項3、4、5、8または9に記載の目的生体物質の解析装置において、
前記基準領域の指定を自動で行うことを特徴とする目的生体物質の解析装置。 - 請求項1から請求項10までのいずれか一項に記載の目的生体物質の解析装置において、
前記区画が、4角形状、円形状又はハニカム状であることを特徴とする目的生体物質の解析装置。 - 染色後の組織標本を撮像する顕微鏡と、
前記顕微鏡の撮像結果を受ける、請求項1~11のいずれか一項に記載の目的生体物質の解析装置と、
を備えることを特徴とする目的生体物質の解析システム。 - 染色後の組織標本の顕微鏡画像を生成する工程と、
前記顕微鏡画像を一定のサイズで区画する工程と、
前記顕微鏡画像の染色状態を区画ごとに解析する工程と、
前記解析工程の解析結果に基づき一定の統計値を算出する工程と、
を備えることを特徴とする目的生体物質の解析方法。 - コンピュータを、
染色後の組織標本の顕微鏡画像を生成する生成手段、
前記顕微鏡画像を一定のサイズで区画する区画手段、
前記顕微鏡画像の染色状態を区画ごとに解析する解析手段、
前記解析手段の解析結果に基づき一定の統計値を算出する算出手段、
として機能させるための目的生体物質の解析プログラム。
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US15/737,124 US10801959B2 (en) | 2015-06-18 | 2016-06-07 | Objective biological substance analysis device, analysis system, analysis method, and recording medium storing computer readable analysis program |
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