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WO2022196203A1 - Procédé de formation d'image - Google Patents

Procédé de formation d'image Download PDF

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Publication number
WO2022196203A1
WO2022196203A1 PCT/JP2022/005553 JP2022005553W WO2022196203A1 WO 2022196203 A1 WO2022196203 A1 WO 2022196203A1 JP 2022005553 W JP2022005553 W JP 2022005553W WO 2022196203 A1 WO2022196203 A1 WO 2022196203A1
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WIPO (PCT)
Prior art keywords
autofluorescence
tissue
image forming
forming method
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/005553
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English (en)
Japanese (ja)
Inventor
賢司 西川
愛奈 安藤
健 瀬戸川
洋 根岸
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Konica Minolta Inc
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Konica Minolta Inc
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Publication date
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Priority to JP2023506871A priority Critical patent/JPWO2022196203A1/ja
Publication of WO2022196203A1 publication Critical patent/WO2022196203A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to an image forming method, and more particularly to an image forming method that enables accurate measurement of fluorescence intensity derived from a target substance by reducing noise due to autofluorescence.
  • a specific target substance e.g., a substance overexpressed in a specific cell, a substance specifically expressed in a specific cell, etc.
  • PID particles fluorescent substance-integrated nanoparticles
  • PID particles fluorescent substance-integrated nanoparticles
  • the present invention has been made in view of the above problems and circumstances, and the problem to be solved is to provide an image forming method that enables accurate measurement of fluorescence intensity derived from a target substance by reducing noise due to autofluorescence. That is.
  • the present inventors used a combination of fluorescent substance-accumulating nanoparticles and an autofluorescence inhibitor to reduce noise due to autofluorescence.
  • the inventors have found that it is possible to provide an image forming method capable of accurately measuring the fluorescence intensity derived from a target substance, and have arrived at the present invention. That is, the above problems related to the present invention are solved by the following means.
  • An imaging method using a fluorescent material comprising: a step of adding an autofluorescence inhibitor to a biological tissue containing a target substance and labeling the target substance with fluorescent substance-accumulating nanoparticles to prepare a tissue specimen; and irradiating the tissue sample with excitation light to form a fluorescence image.
  • the autofluorescence suppressor comprises a fluorescence resonance energy transfer autofluorescence suppressor or an oxidative autofluorescence suppressor.
  • heteropolyacid or a salt thereof is a heteropolyacid represented by the following general formula (1) or a salt thereof.
  • General formula ( 1 ) HnAbDcOy.xH2O (Each symbol in the formula has the following meaning.
  • A represents phosphorus, boron, silicon, germanium, tin, arsenic, antimony, copper, nickel, cobalt, iron, cerium, thorium, chromium, or a composite element of two or more of these elements.
  • D represents molybdenum, tungsten, vanadium, or a composite element of two or more of these elements.
  • n represents the number of acidic hydrogens and is 1 or more.
  • b represents a number from 0.1 to 10;
  • c represents a number from 6 to 18;
  • x represents the number of moles of water of hydration.
  • y represents the number of oxygens in the heteropolyacid.
  • the present invention enables an image forming method that enables accurate measurement of fluorescence intensity derived from a target substance by reducing noise due to autofluorescence by using PID particles and an autofluorescence inhibitor in combination. is.
  • the image forming method of the present invention is an image forming method using a fluorescent substance, wherein an autofluorescence inhibitor is added to a biological tissue containing a target substance, and the target substance is labeled with fluorescent substance-accumulated nanoparticles. and forming a fluorescence image by irradiating the tissue sample with excitation light.
  • This feature is a technical feature common to or corresponding to each of the following embodiments.
  • the autofluorescence suppressing agent includes a fluorescence resonance energy transfer autofluorescence suppressing agent or an oxidized autofluorescence suppressing agent.
  • the autofluorescence suppressing agent may contain both the fluorescence resonance energy transfer autofluorescence suppressing agent and the oxidized autofluorescence suppressing agent. preferable.
  • the oxidized autofluorescence suppressing agent contains an oxidized autofluorescence suppressing compound, and the oxidized autofluorescence suppressing compound is an oxidizing autofluorescence suppressing compound.
  • a metal compound is preferred.
  • the oxidizable metal compound is preferably a heteropolyacid or a salt thereof, more preferably a heteropolyacid represented by the general formula (1) or a salt thereof.
  • the oxidized autofluorescence inhibitor further contains a reducing agent.
  • the reducing agent preferably contains a compound having a hydroxy group and a carbonyl group, or a compound having a carboxy group or a salt thereof, and is selected from ascorbic acid, tartaric acid, potassium antimonyl tartrate, and 2-furancarboxylic acid. It is more preferable to include at least one of
  • the biological tissue is preferably a fibrous tissue having interstitial cells, and the fibrous tissue is preferably lung tissue.
  • the image forming method of the present invention is an image forming method using a fluorescent material, and is characterized by comprising the following steps (1) and (2).
  • a step of adding an autofluorescence inhibitor to a biological tissue containing a target substance and labeling the target substance with fluorescent substance-accumulating nanoparticles to prepare a tissue sample (hereinafter referred to as “tissue sample preparation step”) Also called.)
  • tissue sample preparation step a tissue sample
  • fluorescence image forming step a step of irradiating the tissue sample with excitation light to form a fluorescence image
  • a fluorescence image obtained by the image forming method of the present invention is image-processed and used for analysis, etc., in an image processing apparatus, for example. Specifically, it is used to quantify a target substance in a biological tissue used as a specimen, to grasp the state of distribution, and the like.
  • the image forming method of the present invention may have steps other than the steps (1) and (2) depending on the purpose of use of the fluorescent image to be obtained. Each step of the image forming method of the present invention will be described below.
  • the tissue specimen preparing step according to the present invention is a step of preparing a biological tissue containing a target substance and preparing a tissue specimen by the following steps (1-1) and (1-2). is.
  • a tissue sample is, for example, a sample in which a target substance in a biological tissue prepared as a tissue section is stained with a fluorescent substance, and an autofluorescence inhibitor is added to the biological tissue (tissue section).
  • a step of adding an autofluorescence suppressor to a biological tissue containing a target substance (hereinafter also referred to as an "autofluorescence suppressor addition step”).
  • Step of labeling the target substance with fluorescent substance-accumulating nanoparticles (in this specification, labeling the target substance with PID particles and staining the target substance with PID particles are used interchangeably (1-2) is hereinafter also referred to as the “dyeing process”.)
  • steps (1-1) and (1-2) does not matter in the tissue preparation process.
  • various treatments described later in the dyeing process in (1-2) may reduce the ability of the autofluorescence inhibitor. Therefore, from the viewpoint of exhibiting the effect of the present invention, it is preferable to perform the step (1-1) after the step (1-2).
  • step (1-1) may be performed during step (1-2).
  • the step (1-1) may be performed after the morphological observation and staining step below. From the viewpoint of obtaining a more pronounced effect of the autofluorescence inhibitor, the step (1-1) is particularly preferably carried out after the morphological observation staining step and before the post-treatment step.
  • the tissue sample preparation step may have steps other than steps (1-1) and (1-2). For example, before the steps (1-1) and (1-2), a deparaffinization step and an activation treatment step may be provided in that order, and the steps (1-1) and (1-2) may be followed by a morphological observation staining step. Furthermore, a post-treatment step may be included as the final step in the tissue specimen preparation step.
  • the living tissue to which the image forming method of the present invention is applied is not particularly limited as long as it is a living tissue.
  • the effects of the present invention are more pronounced when the biological tissue is a tissue containing a large amount of autofluorescent substances, for example, a fibrous tissue having stromal cells.
  • Such fibrous tissue includes lung tissue.
  • a biological tissue used in the present invention contains a target substance.
  • a target substance is a substance that is targeted for immunostaining using a fluorescent substance, mainly for detection or quantification from the viewpoint of pathological diagnosis.
  • the target substance is not particularly limited, but in pathological diagnosis, biomarkers such as antigens are generally selected according to the purpose.
  • biomarkers such as antigens are generally selected according to the purpose.
  • biological substances such as proteins (antigens) expressed in biological tissues can be used as target substances.
  • units smaller than proteins such as peptides and biomarkers such as RNA may be used as target substances.
  • the target substance does not have to be unique to the living body as long as it exists in living tissue.
  • the target substance may be a drug or the like introduced from outside the body into the body.
  • Typical target substances include biological substances that are expressed in the cell membranes of various cancer tissues and can be used as biomarkers.
  • an autofluorescence suppressor is added to a living tissue containing a target substance (hereinafter, also simply referred to as “living tissue”).
  • the biological tissue to which the autofluorescence inhibitor is added is prepared, for example, as a tissue section having a size to be used as a tissue specimen.
  • the tissue section is preferably, for example, one that has undergone a deparaffinization step and an activation treatment step, which will be described later, in that order.
  • the autofluorescence suppressing agent used in the present invention is not particularly limited as long as it has a function of suppressing fluorescence emitted by a biologically derived fluorescent substance (hereinafter also referred to as "autofluorescence suppressing ability").
  • the ability to suppress autofluorescence is typically determined by the fact that a biologically derived fluorescent substance (coenzyme, amino acid, protein, intracellular pigment, etc., hereinafter also referred to as "autofluorescent substance”) originally present in a tissue specimen. This is the function of suppressing emitted fluorescence (so-called “autofluorescence”).
  • the autofluorescence inhibitor may also have a function of suppressing fluorescence emission not derived from the living body.
  • fluorescence includes, for example, fluorescence emitted from non-biologically-derived fluorescent substances contained in living tissue and instruments such as slide glasses.
  • the autofluorescence suppressing agent used in the present invention contains at least a compound having autofluorescence suppressing function (autofluorescence suppressing compound).
  • the autofluorescence suppressing agent may be an autofluorescence suppressing compound alone or a composition containing the compound.
  • the autofluorescence-suppressing agent is a composition
  • the composition contains at least one autofluorescence-suppressing compound, and as other components other than the autofluorescence-suppressing compound, a component that does not inhibit the autofluorescence-suppressing ability of the autofluorescence-suppressing compound. can be used as appropriate.
  • Other components include, for example, solvents, oxidizing agents, reducing agents, chelating agents, surfactants, and the like.
  • the autofluorescence inhibitor it is preferable to use a fluorescence resonance energy transfer type autofluorescence inhibitor or an oxidized autofluorescence inhibitor, and it is more preferable to use both of them.
  • a fluorescence resonance energy transfer (hereinafter also referred to as "FRET (Fluorescence resonance energy transfer) type”) autofluorescence inhibitor contains a FRET-type autofluorescence-suppressing compound.
  • a FRET-type autofluorescence-suppressing compound is a compound capable of receiving, through resonance, energy possessed by an autofluorescent substance excited by excitation light in a system coexisting with the autofluorescent substance. As a result, the autofluorescent substance in the excited state can return to the ground state without substantially emitting fluorescence.
  • the FRET-type autofluorescence-suppressing compound emits the received energy as, for example, light that does not generate heat or noise.
  • FRET-type autofluorescence-suppressing compounds include porphyrin.
  • Porphyrin is a general term for macrocyclic compounds and derivatives thereof in which four pyrrole rings are alternately bonded to four methine groups at the ⁇ -positions.
  • Porphyrins also include complexes with a metal element in the center of the ring.
  • One of the FRET-type autofluorescence-suppressing compounds may be used alone, or two or more thereof may be used in combination.
  • a FRET-type autofluorescence suppressing compound for example, porphyrin
  • the solvent used in the composition is a solvent capable of dissolving the compound without inhibiting the ability of FRET-type autofluorescence-suppressing compounds, such as porphyrin, to suppress autofluorescence.
  • Specific examples of the solvent include water and the like.
  • the content of the FRET-type autofluorescence-suppressing compound, such as porphyrin, in the composition can be in the range of, for example, 1 ng/mL to 1 mg/mL as a total content relative to the total amount of the composition.
  • the above composition may further contain a pH adjuster, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), PBS (phosphate buffered saline), Tris (Trishydroxymethylaminomethane) and the like may be contained.
  • the pH of the composition is preferably in the range of 6.0-8.0.
  • the oxidized autofluorescence suppressing agent contains an oxidized autofluorescence suppressing compound.
  • the oxidized autofluorescence-suppressing compound has a function of suppressing autofluorescence by binding to an autofluorescent substance. Specifically, the autofluorescence is suppressed by converting the wavelength of the fluorescence emission of the autofluorescent substance or by reducing the fluorescence emission amount itself.
  • One of the oxidized autofluorescence-suppressing compounds may be used alone, or two or more thereof may be used in combination.
  • oxidized autofluorescence-suppressing compounds include oxidizing metal compounds, and heteropolyacids or salts thereof are preferable as oxidizing metal compounds.
  • Heteropolyacids are polyacids of metal oxyacids containing heteroatoms.
  • the heteropolyacid is generally in the form of containing water of hydration. Molybdenum, tungsten, vanadium, etc. are mentioned as a metal atom which the metal oxyacid in a heteropolyacid has. One or more metal atoms may be used.
  • Heteroatoms in heteropolyacids include phosphorus, boron, silicon, germanium, tin, arsenic, antimony, copper, nickel, cobalt, iron, cerium, thorium, chromium, and the like. One or more heteroatoms may be used.
  • heteropolyacid salts include salts with monovalent metal cations such as sodium salts, potassium salts and lithium salts, salts with divalent metal cations such as nickel salts, cobalt salts, copper salts and zinc salts, and iron salts. salts with trivalent metal cations, salts with quaternary ammonium cations (NR 4+ (R is, for example, a hydrogen atom, a methyl group, a butyl group)), salts with quaternary phosphonium cations, and the like. be done.
  • monovalent metal cations such as sodium salts, potassium salts and lithium salts
  • salts with divalent metal cations such as nickel salts, cobalt salts, copper salts and zinc salts
  • iron salts salts with trivalent metal cations
  • salts with quaternary ammonium cations NR 4+ (R is, for example, a hydrogen atom, a methyl group, a butyl group)
  • Heteropolyacids or salts thereof bind to autofluorescent substances selected from aldehyde-fixed tissues, erythrocytes, collagen and elastin, and have the effect of inhibiting their autoluminescence.
  • heteropolyacids include heteropolyacids represented by the following general formula (1) (hereinafter also referred to as heteropolyacid (1)).
  • n represents the number of acidic hydrogens and is 1 or more. n is preferably in the range of 2-10.
  • b represents a number from 0.1 to 10; b is preferably in the range of 0.5 to 5, more preferably 0.5 to 2.5. As for b, 1 is especially preferable in a predetermined aspect.
  • c represents a number from 6 to 18; c is preferably in the range of 9-15, more preferably 10-12. c is particularly preferably 12 in certain embodiments.
  • x represents the number of moles of water of hydration. x is typically in the range 0-40. y represents the number of oxygens in the heteropolyacid. y is typically in the range 10-70.
  • Examples of salts of heteropolyacid (1) include compounds in which the acidic hydrogen in general formula (1) is replaced with the cations exemplified above. The number of n is appropriately adjusted according to the valence of the replaced cation.
  • Phosphomolybdic acid is particularly preferred as the heteropolyacid (1).
  • an oxidized autofluorescence-suppressing compound such as a heteropolyacid or a salt thereof may itself be used as an autofluorescence-suppressing agent. You may use it as an agent.
  • the solvent used in the composition is a solvent capable of dissolving the compound without inhibiting the autofluorescence-suppressing ability of the oxidized autofluorescence-suppressing compound, for example, a heteropolyacid or a salt thereof.
  • Specific examples of the solvent include water, ethanol, and the like.
  • the content of the oxidized autofluorescence-suppressing compound in the composition can be in the range of, for example, 0.1 to 10 mg/mL as a total content relative to the total amount of the composition.
  • the composition may further contain reducing agents, pH adjusters, HEPES, PBS, Tris, and the like, if necessary. pH adjusters, HEPES, PBS, Tris, etc. are used as buffers.
  • the pH of the composition is preferably in the range of 6.0-8.0.
  • the oxidized autofluorescence suppressing agent as a composition preferably contains a reducing agent in addition to the oxidized autofluorescence suppressing compound.
  • reducing agent known reducing agents can be used without any particular restrictions.
  • Specific examples of reducing agents include organic reducing agents such as compounds having a hydroxy group and a carbonyl group, compounds having a carboxy group or a salt thereof, and inorganic reduction agents such as metal compounds, metal complexes, metal nanoparticles, and inorganic acids. and organic reducing agents are preferred.
  • inorganic reducing agents examples include tin (II) chloride, sodium cyanoborohydride, sulfuric acid, sodium borohydride, and hydrazine.
  • compounds having a hydroxy group and a carbonyl group include ascorbic acid, erythorbic acid, and the like.
  • Compounds having a carboxyl group or a salt thereof include tartaric acid, potassium antimonyl tartrate, 2-furancarboxylic acid, formic acid, oxalic acid, 3,4,5-trihydroxybenzoic acid and the like.
  • the reducing agent contains at least one selected from ascorbic acid, tartaric acid, potassium antimonyl tartrate, and 2-furancarboxylic acid, and three kinds of ascorbic acid, potassium antimonyl tartrate, and 2-furancarboxylic acid. It is more preferable to include In that case, the mixing ratio of ascorbic acid, potassium antimonyl tartrate, and 2-furancarboxylic acid is, for example, 100 to 300 parts by weight of potassium antimonyl tartrate and 10 parts by weight of 2-furancarboxylic acid per 100 parts by weight of ascorbic acid. up to about 50 parts by mass.
  • the type of reducing agent is appropriately selected according to the type of oxidized autofluorescence-suppressing compound to be combined.
  • a heteropolyacid or a salt thereof is used as an oxidative autofluorescence inhibitor, and a compound having a hydroxy group and a carbonyl group, or a compound having a carboxy group or a salt thereof is used as a reducing agent.
  • the reducing agent more preferably contains at least one selected from ascorbic acid, tartaric acid, antimonyl potassium tartrate, and 2-furancarboxylic acid, and ascorbic acid, antimonyl potassium tartrate, and 2-furancarboxylic acid. It is more preferable to include the three types of, and as the mixing ratio of the three types, the same mixing ratio as described above can be mentioned.
  • the reducing agent moderately reduces an oxidized autofluorescence-suppressing compound, such as a heteropolyacid or a salt thereof, to obtain an oxidized autofluorescence-suppressing agent.
  • an oxidized autofluorescence-suppressing compound such as a heteropolyacid or a salt thereof.
  • a function of sufficiently suppressing autofluorescence can be imparted.
  • the effect on PID particles is small, and the S/N ratio in image evaluation is very high.
  • a commercially available composition containing an oxidized autofluorescence inhibitor such as a heteropolyacid or a salt thereof, may be used.
  • Commercially available products include, for example, a kit consisting of a plurality of components, and a solution (composition) containing the heteropolyacid (1) or a salt thereof by mixing the components at the time of use.
  • a kit for phosphomolybdic acid solution the phosphomolybdic acid concentration in the phosphomolybdic acid solution at the time of use is about 1.5 to 2.4 mg/mL).
  • TrueVIEW consists of a phosphomolybdic acid undiluted solution in which phosphomolybdic acid is dissolved in a solvent at a predetermined ratio, a reducing agent solution in which a reducing agent is dissolved in a solvent at a predetermined ratio, and a buffer solution.
  • a stock solution (A) of a heteropolyacid or a salt thereof in which a heteropolyacid or a salt thereof is dissolved in a solvent, and a reducing agent solution (B) in which a reducing agent is dissolved in a solvent at a predetermined ratio. and buffer solution (C) are prepared separately and mixed at the time of use to use as an autofluorescence inhibitor (composition).
  • the content of the heteropolyacid or its salt in the stock solution (A) of the heteropolyacid or its salt and the content of the reducing agent in the reducing agent solution (B) are, for example, the stock solution of the heteropolyacid or its salt (A), the reducing agent
  • the autofluorescence inhibitor is prepared by mixing the liquid (B) and the buffer solution (C) at a predetermined mixing ratio, for example, 1:1:1 (volume), each component in the resulting autofluorescence inhibitor
  • the content may be adjusted so as to be a suitable content for use.
  • the content of the heteropolyacid or its salt in the autofluorescence inhibitor is preferably in the above range, for example, 0.1 to 10 mg/mL, and the content of the reducing agent is, for example, in the range of 1.6 to 17 mg/mL. can do.
  • a stock solution (A) of a heteropolyacid or a salt thereof, a reducing agent solution (B), and a buffer solution (C) are mixed at, for example, 1:1:1 (by volume) to prepare an autofluorescence inhibitor.
  • the content of the heteropolyacid or its salt in the undiluted solution (A) of the heteropolyacid or its salt is preferably 0.3 to 30 mg/mL.
  • the content of the reducing agent in the reducing agent liquid (B) is preferably 5 to 50 mg/mL.
  • the buffer (C) for example, a buffer adjusted to pH 7.0 to 8.0 by adding a pH adjuster to an aqueous solution containing 10 to 30% by mass of a buffer such as HEPES or Tris. Liquid is preferred.
  • the mixing ratio of the stock solution (A) of the heteropolyacid or its salt, the reducing agent solution (B), and the buffer solution (C) is, for example, 1:0.5 to 2:0. It may be adjusted in the range of 5 to 2 (volume). In that case, according to the selected mixing ratio, the content of the heteropolyacid or its salt in the autofluorescence inhibitor and the content of the reducing agent are within the above ranges. The content of the acid or salt thereof and the content of the reducing agent in the reducing agent liquid (B) may be appropriately adjusted.
  • Addition of the autofluorescence suppressing agent to the biological tissue is performed, for example, to a biological tissue prepared as a tissue section having a size to be a tissue specimen.
  • the fluorescent image forming step that is performed after the tissue sample preparation step is typically performed using a microscope image acquisition device. Therefore, the tissue section to which the autofluorescence inhibitor is added is prepared in a form adapted to the microscope image acquisition device, specifically on a slide glass.
  • the tissue section (also simply referred to as "section”, including sections such as pathological sections) may be prepared by any method without particular limitation, and a section prepared by a known procedure and placed on a slide glass can be used. .
  • an autofluorescence inhibitor prepared in a liquid form is added to the section placed on the slide glass in this way.
  • the section is preferably subjected to the following deparaffinization step and activation treatment step in that order before the autofluorescence inhibitor is added.
  • the section placed on the slide glass is immersed in a container containing xylene to remove paraffin.
  • the temperature is not particularly limited, it can be performed at room temperature.
  • the immersion time is preferably within the range of 3 to 30 minutes.
  • xylene may be exchanged during the immersion, if necessary.
  • the sections are immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, it can be performed at room temperature.
  • the immersion time is preferably within the range of 3 to 30 minutes. If necessary, ethanol may be exchanged during the immersion.
  • the sections are immersed in a container containing water to remove ethanol.
  • the temperature is not particularly limited, it can be performed at room temperature.
  • the immersion time is preferably within the range of 3 to 30 minutes.
  • the water may be exchanged during the immersion.
  • activation treatment step Following a known method, the target substance is activated. Activation conditions are not particularly defined, but activating solutions include 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, and 0.1 M Tris-HCl buffer. A liquid or the like can be used.
  • pH conditions are such that a signal is generated at pH 2.0 to 13.0 at 25°C, and the roughness of the tissue is such that the signal can be evaluated.
  • pH is within the range of 6.0 to 8.0, but for special tissue sections, pH 3.0, for example, can also be used.
  • Autoclaves, microwaves, pressure cookers, water baths, etc. can be used as heating equipment.
  • the temperature can be in the range of 50 to 130° C., and the time can be in the range of 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, it can be performed at room temperature.
  • the immersion time is preferably within the range of 3 to 30 minutes. Also, the PBS may be replaced during the immersion, if necessary.
  • the autofluorescence-suppressing compound itself is liquid, it can be added to the slice as it is.
  • the autofluorescence-suppressing compound is solid, for example, a liquid composition (solution) containing the autofluorescence-suppressing compound in the above content is prepared and added to the slice.
  • the amount of the autofluorescence suppressor to be added should be enough to cover the section, and is preferably within the range of 20 ⁇ L to 150 ⁇ L per slide glass.
  • the temperature is not particularly limited, it can be performed at room temperature.
  • the standing time is preferably within the range of 3 to 5 minutes.
  • the section may be immersed in the autofluorescence inhibitor together with the slide glass.
  • the ratio of the two to the FRET-type autofluorescence-suppressing compound and the oxidized autofluorescence-suppressing compound is A mass ratio of 1:1000 to 1000:1 is preferred.
  • the order of adding the FRET-type autofluorescence inhibitor and the oxidized autofluorescence inhibitor is not particularly limited. In addition, part or all of the following dyeing process may be performed between the steps of adding the FRET-type autofluorescence suppressor and adding the autofluorescence suppressor.
  • the addition amount of each self-fluorescence suppressing agent, the temperature, and the standing time can be the same as those described above. Note that the FRET-type autofluorescence inhibitor and the oxidized autofluorescence inhibitor may be mixed and then added at the same time.
  • the staining step is a step of staining a target substance contained in a biological tissue, for example, a tissue section, with PID particles.
  • PID particles fluorescent substance-integrated nanoparticles
  • PID particles are nano-sized particles obtained by accumulating fluorescent substances. By staining the target substance using such PID particles, the detection sensitivity for labeling the target substance can be increased as described above.
  • PID particles have a structure in which a particle composed of an organic or inorganic substance is used as a matrix, and a fluorescent substance is included therein and/or adsorbed on its surface.
  • a fluorescent substance is excited by irradiation with excitation light such as X-rays, ultraviolet rays, visible rays, or infrared rays from the outside, and emits fluorescence in the process from the excited state to the ground state.
  • excitation light such as X-rays, ultraviolet rays, visible rays, or infrared rays from the outside.
  • Fluorescent substances used for PID particles include light with a wavelength in the range of 400 to 900 nm (visible It preferably exhibits luminescence in the range of light to near-infrared light.
  • the matrix and the fluorescent light-emitting substance have substituents or moieties having mutually opposite charges so that electrostatic interaction works.
  • Fluorescent substances are roughly classified into, for example, organic fluorescent substances (fluorescent dyes) and inorganic fluorescent substances (quantum dots).
  • fluorescent dyes include fluorescein-based dyes, rhodamine-based dyes, squarylium-based dyes, coumarin-based dyes, pyrene-based dyes, cyanine-based dyes, perylene-based dyes, oxazine-based dyes, aromatic ring-based dyes, carbopyronine-based dyes, pyromecenes-based dyes. Fluorescent dyes such as dyes can be exemplified.
  • Alexa Fluor registered trademark, manufactured by Invitrogen
  • BODIPY registered trademark, manufactured by Invitrogen
  • Cy registered trademark, manufactured by GE Healthcare
  • HiLyte 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
  • CF registered trademark, Biotium
  • DY registered trademark, manufactured by DYOMICS
  • CAL registered trademark, manufactured by BioSearch Technologies
  • NBD registered trademark
  • Texas Red registered trademark
  • Quantum dots Semiconductor nanoparticles containing II-VI compounds, III-V compounds or IV elements are used as quantum dots. Examples include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.
  • a quantum dot in which the above quantum dot is used as a core and a shell is provided thereon can also be used.
  • the core is CdSe and the shell is ZnS
  • quantum dots with shells for example, CdSe/ZnS, CdS/ZnS, InP/ZnS, InGaP/ZnS, Si/SiO 2 , Si/ZnS, Ge/GeO 2 , Ge/ZnS, etc. can be used. , but not limited to.
  • Quantum dots may be surface-treated with an organic polymer or the like, if necessary.
  • an organic polymer or the like for example, CdSe/ZnS (manufactured by Invitrogen) having surface carboxyl groups, CdSe/ZnS (manufactured by Invitrogen) having surface amino groups, and the like can be mentioned.
  • the method for producing PID particles is not particularly limited, and they can be produced by known methods. Specifically, a manufacturing method can be used in which a fluorescent substance is encapsulated in particles whose base material is an organic substance or an inorganic substance and/or the fluorescent substance is immobilized on the surface.
  • Organic substances that make up the matrix include resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin; styrene resin, acrylic resin, acrylonitrile.
  • Resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer) resins generally classified as thermoplastic resins; other resins such as polylactic acid; polysaccharides can be exemplified.
  • Silica, glass, and the like can be exemplified as inorganic substances constituting the matrix.
  • the methods for producing PID particles will be described separately for the case where the fluorescent substance is a fluorescent dye and the case where the fluorescent substance is a quantum dot.
  • a method for producing PID particles using a fluorescent dye there is a method of forming resin particles having a diameter on the order of nanometers, in which the fluorescent dye is immobilized inside or on the surface of a matrix made of resin.
  • the method for preparing the PID particles is not particularly limited, but for example, (co)polymerizing a (co)monomer for synthesizing the resin (thermoplastic resin or thermosetting resin) forming the base of the PID particles
  • a method of adding a fluorescent dye and incorporating the fluorescent dye into the interior or surface of the resulting (co)polymer can be used.
  • Polystyrene nanoparticles encapsulating a fluorescent dye can be produced, for example, by a copolymerization method using an organic dye having a polymerizable functional group as described in US Pat. No. 4,326,008 (1982), or as described in US Pat. It can be produced using a method of impregnating a fluorescent dye into a polystyrene nanoparticle.
  • PID particles in which a fluorescent dye is immobilized inside or on the surface of a matrix made of silica.
  • Silica nanoparticles encapsulating a fluorescent dye can be synthesized, for example, with reference to the synthesis of FITC (fluorescein isothiocyanate) encapsulating silica particles described in Langmuir, Vol. 8, page 2921 (1992).
  • FITC fluorescein isothiocyanate
  • a variety of silica-based PID particles can be synthesized by substituting the desired fluorescent dye for FITC.
  • the fluorescent dye when the fluorescent dye is encapsulated in the matrix, the fluorescent dye may or may not be chemically bonded to the matrix itself as long as it is dispersed inside the matrix.
  • PID particles using quantum dots can be produced by a known method.
  • silica nanoparticles encapsulating quantum dots can be synthesized with reference to Synthesis of CdTe-encapsulating silica nanoparticles described in New Journal of Chemistry, Vol. 33, page 561 (2009).
  • Silica nanoparticles encapsulating quantum dots are, for example, silica nanoparticles in which CdSe/ZnS capped with 5-amino-1-pentanol and APS described in Chemical Communication, page 2670 (2009) are accumulated on the surface. Synthesis can be used as a reference.
  • Polymer nanoparticles encapsulating quantum dots can be produced, for example, using the method of impregnating quantum dots into polystyrene nanoparticles described in Nature Biotechnology, Vol. 19, p. 631 (2001).
  • silica nanoparticles are treated with a silane coupling agent to aminate the ends, and quantum dots having a carboxyl group end are accumulated by bonding to the surface of silica beads through amide bonds, and PID particles There is also a method of
  • a reverse micelle method and a sol-gel using a mixture of an organic alkoxysilane and an alkoxide having an organic functional group with good adsorption to quantum dots at the end of the molecule as a glass precursor.
  • glass-like particles in which quantum dots are dispersed and fixed are formed to form PID particles.
  • Yet another method for preparing PID particles is to mix amino-terminated and carboxy-terminated quantum dots in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Then, there is an example of manufacturing PID particles by accumulating quantum dots by bonding quantum dots via amide bonds.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • the quantum dots when included in the matrix, the quantum dots need only be dispersed inside the matrix, and may or may not be chemically bonded to the matrix itself.
  • the average particle size of the PID particles is not particularly limited, those of about 20 to 200 nm are suitable. Those having an average particle size larger than the above upper limit tend to be difficult to access the target substance. When the average particle size is smaller than the above lower limit, the amount of the fluorescent light-emitting substance contained in the PID particles tends to be small and the luminance value tends to be low. Therefore, the emitted fluorescence is buried in background noise (camera noise and cell noise fluorescence), making quantitative evaluation difficult.
  • the average particle size of the PID particles is more preferably in the range of 40-150 nm.
  • the coefficient of variation of the particle size of the PID particles is 15% or less. Since the variation in particle size of the PID particles is small, the luminance value of the fluorescence per particle is substantially constant, so that the accuracy of quantification is enhanced.
  • the average particle diameter of the PID particles is the diameter of the circle when the cross-sectional area is measured for a sufficient number of particles by taking an electron micrograph using a scanning electron microscope (SEM), and each measured value is the area of the circle. was obtained as the particle size.
  • SEM scanning electron microscope
  • the arithmetic mean of the particle diameters of 1000 particles was taken as the average particle diameter.
  • the coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
  • the PID particles described above are used to stain the target substance.
  • An immunostaining method is preferable as a method for staining a target substance with PID particles.
  • a conjugate immunostaining agent
  • the obtained immunostaining agent binds to the target substance through an antigen-antibody reaction to stain the target substance. do.
  • immunostaining there are various methods for immunostaining, and there is no particular limitation as long as the protein as the target substance can be stained.
  • Representative techniques include the following techniques, and immunostaining agents are used according to each technique as follows.
  • a fluorescence-labeled primary antibody is prepared by linking the PID particles and the primary antibody. Staining is performed by directly fluorescently labeling the target substance with the obtained immunostaining agent (fluorescently labeled primary antibody).
  • a fluorescence-labeled secondary antibody is prepared by linking the PID particles and a secondary antibody (an antibody against the primary antibody). Staining is performed by reacting a target substance with a primary antibody and then reacting the primary antibody with an immunostaining agent (fluorescently labeled secondary antibody) to indirectly fluorescently label the target substance.
  • a biotin-modified primary antibody in which a primary antibody and biotin are linked, and avidin-modified PID particles in which PID particles and avidin or streptavidin are linked as an immunostaining agent are prepared. Staining involves reacting a target substance with a biotin-modified primary antibody and then reacting it with an immunostaining agent (avidin-modified PID particles) to indirectly fluorescently label the target substance using the avidin-biotin reaction. is done in
  • Biotin-biotin combined secondary antibody method Prepare primary antibody.
  • a biotin-modified secondary antibody in which a secondary antibody is linked to biotin and an avidin-modified PID particle in which PID particles are linked to avidin or streptavidin as an immunostaining agent are prepared.
  • the target substance is reacted with a primary antibody, then with a biotin-modified secondary antibody, and then with an immunostaining agent (avidin-modified PID particles) to detect the target substance using the avidin-biotin reaction. is indirectly fluorescently labeled.
  • hapten non-immunogenic but antigenic antibody
  • anti-hapten antibodies such as dickoxigenin and anti-dicoxigenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antibodies, as well as other substance combinations with similar specific reactivity can also be used.
  • An antibody (IgG) that specifically recognizes and binds to a protein as a target substance as an antigen can be used as the primary antibody in the various immunostaining methods described above.
  • an anti-HER2 antibody can be used when HER2 is the target substance
  • an anti-HER3 antibody can be used when HER3 is the target substance.
  • An antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used as the secondary antibody in the various immunostaining methods described above.
  • Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of stability of quantification, monoclonal antibodies are preferred.
  • the type of animal that produces antibodies is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like, as in the past.
  • biotin-modified primary antibody or secondary antibody and the hapten-, dickoxigenin- or FITC-modified primary antibody or secondary antibody used in these modifications, are produced by conventionally known methods. can be done.
  • a linker molecule may optionally be introduced between the primary antibody or secondary antibody and biotin, hapten, dickoxigenin or FITC.
  • the immunostaining agent used is obtained by appropriately binding the primary antibody or secondary antibody and PID particles.
  • the binding between the primary antibody or secondary antibody and the PID particles includes, for example, covalent bond, ionic bond, hydrogen bond, coordinate bond, antigen-antibody bond, biotinavidin reaction, physical adsorption, chemical adsorption, etc.
  • a linker molecule may be interposed, if necessary.
  • the immunostaining agents used are avidin-modified PID particles in which PID particles and avidin or streptavidin are linked.
  • the immunostaining agents used are PID particles modified with anti-hapten antibodies, anti-dicoxigenin antibodies or anti-FITC antibodies. Modification of PID particles with avidin, anti-hapten antibody, anti-dicoxigenin antibody or anti-FITC antibody can be performed by known methods. For example, it can be produced by introducing functional groups capable of reacting with each other into both the avidin, anti-hapten antibody, anti-dicoxygenin antibody or anti-FITC antibody and the matrix of the PID particles and allowing them to react.
  • Immunostaining may be performed according to standard procedures and processing conditions for each of the various techniques described above.
  • a slide glass on which a tissue section containing a target substance is placed is coated with one or more reagents according to each immunostaining method in the order described above under appropriate temperature and time conditions. It should be immersed below.
  • the temperature is not particularly limited, it can be performed at room temperature.
  • the reaction time is preferably 30 minutes or more and 24 hours or less for each reagent.
  • each immunostaining method treatment with a reagent containing an immunostaining agent is performed last.
  • the slide glass on which the immunostained tissue section is placed is preferably immersed in a washing solution such as PBS and washed.
  • a washing solution such as PBS
  • the temperature of the washing treatment with PBS after the treatment with the reagent containing the immunostaining agent is room temperature, and the time is 3 to 30 minutes. If necessary, the PBS may be replaced during the immersion.
  • a known blocking agent such as BSA-containing PBS or a surfactant such as Tween 20 before performing the above-described treatment.
  • the dyeing step is preferably performed before the autofluorescence inhibitor addition step.
  • the biological tissue for example, the tissue section
  • the deparaffinization step and the activation treatment step in that order.
  • the avidin-biotin combination secondary antibody method after the primary antibody is reacted with the target substance, and then with the biotin-modified secondary antibody, the avidin-modified PID particles are further reacted.
  • a step of adding an autofluorescence inhibitor may be provided after each step.
  • morphological observation staining may be performed separately from the staining step (1-2) so that the morphology of cells, tissues, organs, etc. can be observed in a bright field. .
  • the morphological observation staining step can be performed according to a conventional method.
  • eosin which stains cytoplasm, stroma, various fibers, red blood cells, and keratinocytes in red to dark red
  • hematoxylin which stains cell nuclei, calcareous tissue, cartilage tissue, bacteria, and mucus in blue to pale blue
  • a method of performing these two stainings simultaneously is known as hematoxylin-eosin staining (HE staining).
  • the morphological observation and staining process may be performed after the staining process (1-2) or before the staining process.
  • (1-1) autofluorescence inhibitor addition process may be performed after the morphological observation and staining process.
  • an autofluorescence inhibitor is added.
  • the autofluorescence inhibitor addition step is preferably performed after the morphological observation and staining step and before the post-treatment step.
  • the tissue specimen may be subjected to post-treatments such as fixation/dehydration, clearing, and encapsulation so as to make it suitable for observation in the next (2) fluorescence imaging step. preferable.
  • the fixation/dehydration treatment is performed by immersing the tissue specimen in a fixation treatment solution (cross-linking agents such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, and methanol).
  • the clearing treatment is performed by immersing the tissue specimen that has been fixed and dehydrated in a clearing solution (xylene or the like).
  • the encapsulation treatment is performed by immersing the tissue specimen that has undergone the clearing treatment in an encapsulating fluid.
  • the conditions for performing these treatments for example, the temperature and immersion time when immersing the tissue specimen in a prescribed treatment solution, can be adjusted as appropriate to obtain an appropriate signal according to conventional immunostaining methods. can.
  • a tissue specimen to be subjected to the fluorescent imaging step (2) is prepared. That is, in the image forming method of the present invention, the tissue sample prepared in (1) is then subjected to the fluorescent image forming step (2).
  • the tissue specimen prepared in (1) is irradiated with excitation light to form a fluorescence image.
  • the acquired fluorescence image is image-processed and used for analysis or the like in an image processing device, for example.
  • a fluorescence image can be formed by a conventionally known method.
  • a fluorescence image is formed using, for example, the following microscope image acquisition device.
  • the microscope image acquisition device is a device that irradiates the tissue specimen prepared in (1) with excitation light and acquires a fluorescence image based on the fluorescence emitted from the fluorescent substance in the PID particles that fluorescently labeled the target substance. be.
  • the acquired fluorescence image is subjected to, for example, image processing and analysis steps in an image processing device.
  • the microscope image acquisition device and image processing device are connected via an interface such as a cable so that data can be sent and received.
  • the connection method between the microscope image acquisition device and the image processing device is not particularly limited.
  • the microscope image acquisition device and the image processing device may be connected via a LAN (Local Area Network), or may be connected wirelessly.
  • the microscope image acquisition device is typically a known camera-equipped fluorescence microscope, and the tissue specimen prepared in (1) above (the tissue section on the slide glass of (1-1) and (1-2) A tissue sample subjected to the process) is placed on a slide fixing stage, a fluorescence microscope image thereof is obtained, and the image is transmitted to an image processing device.
  • a microscope image acquisition device is configured with, for example, irradiation means, imaging means, imaging means, communication I/F, and the like.
  • the irradiation means is composed of an excitation light source, an optical filter, and the like, and irradiates the tissue sample on the slide glass mounted on the slide fixing stage with excitation light.
  • the excitation light source examples include light sources capable of emitting light of the wavelengths described above, and the excitation light is preferably excitation light having a wavelength in the range of 575 to 600 nm, for example, by passing through an optical filter. Also, it is preferable to set the wavelength range of fluorescence to be observed within the wavelength range of 612 to 692 nm by passing it through an optical filter.
  • the imaging means is composed of an eyepiece lens, an objective lens, etc., and forms an image of transmitted light, reflected light, or fluorescence emitted from the tissue specimen on the slide by the irradiated light.
  • the imaging means is a microscope-equipped camera that includes a CCD (Charge Coupled Device) sensor or the like, and captures an image formed on an imaging plane by the imaging means to generate digital image data of a fluorescence image.
  • CCD Charge Coupled Device
  • the communication I/F transmits image data of the generated fluorescence image to the image processing device.
  • the microscope image acquisition device is equipped with a bright field unit combining illumination means and imaging means suitable for bright field observation, and a fluorescence unit combining illumination means and imaging means suitable for fluorescence observation. By doing so, it is possible to switch between bright field and fluorescence.
  • a known camera-equipped fluorescence microscope used as a microscope image acquisition device is not particularly limited, and any known microscope (e.g., phase contrast microscope, differential interference contrast microscope, electron microscope, etc.) with a camera installed.
  • a fluorescence microscope with an attached light can be used.
  • the microscope image acquisition device is not limited to a microscope with a camera.
  • a virtual microscope slide creation device that scans a slide on a slide fixing stage of a microscope and acquires an entire microscope image of a tissue specimen (tissue section).
  • a tissue specimen tissue section
  • the virtual microscope slide creation device it is possible to acquire an entire image of a tissue sample (tissue section) on the slide as image data that can be viewed at once on the display unit.
  • the image processing device identifies the focal position for each cell in the tissue section to be observed by analyzing the fluorescence image transmitted from the microscope image acquisition device.
  • Example 1 Preparation of tissue specimen (biological tissue containing target substance) A human lung cancer tissue array slide (manufactured by US Biomax, model number LC241m; target substance is ATPase) was used as a biological tissue containing the target substance.
  • Step Staining of the target substance was performed by a combined secondary antibody method of avidin and biotin.
  • a solution containing a primary antibody, a biotin-modified secondary antibody, and an immunostaining agent (avidin-modified PID particles) as reagents was prepared as follows.
  • linker reagent "(+)-Biotin-PEG6-NH-Mal" manufactured by PurePEG, product number 2461006-250
  • DMSO dimethyl sulfoxide
  • This reaction solution was subjected to a desalting column "ZebaDesaltSpinColumns" (manufactured by Thermo Scientific, Cat. #89882) for purification.
  • the absorption of the desalted reaction solution at a wavelength of 300 nm was measured with a spectrophotometer (Hitachi "F-7000") to calculate the amount of protein contained in the reaction solution.
  • the reaction solution was adjusted to 250 ⁇ g/mL with a 50 mM Tris solution and further diluted to 6 ⁇ g/mL with PBS containing 1% BSA. This solution was used as a biotin-modified secondary antibody solution.
  • PBS containing 2 mM EDTA (ethylenediaminetetraacetic acid)
  • SM (PEG) 12 succinimidyl-[(N -maleimidopropionamido)-dodecaethylenegly
  • streptavidin (Wako Pure Chemical Industries, Ltd.) is subjected to a thiol group addition treatment, followed by filtration through a gel filtration column, and can be bound to maleimide-modified PID particles. , to obtain a streptavidin solution to which a thiol group was added.
  • SATA N-succinimidyl S-acetylthioacetate
  • streptavidin-modified PID particle 1 solution The streptavidin-modified PID particles 1 obtained above were subjected to ultrasonic dispersion treatment, and then diluted with PBS containing 1% BSA to adjust to 0.02 nM.
  • tissue array slide subjected to the primary reaction was washed with PBS, and then reacted with the biotin-modified secondary antibody solution obtained above at room temperature for 30 minutes.
  • hematoxylin staining was performed. Hematoxylin staining was performed by staining the immunostained sections with Mayer's hematoxylin solution for 5 minutes. The tissue sections were then washed with running water at 45°C for 3 minutes.
  • the three components of TrueVIEW consist of phosphomolybdic acid undiluted solution (A1), reducing agent solution (B1), and buffer solution (C1).
  • the solvent in the phosphomolybdic acid stock solution (A1) is water, and the content of phosphomolybdic acid is 7 mg/mL.
  • the reducing agent in the reducing agent liquid (B1) is a mixture of ascorbic acid, antimonyl potassium tartrate and 2-furancarboxylic acid in a ratio of 17:27:5 (mass ratio), and the reducing agent liquid (B1) is a solvent. It is a solution in which the above mixture is dissolved in water at a ratio of 49 mg/mL.
  • the buffer solution (C1) is a buffer solution prepared by adding sodium hydroxide to an aqueous solution of HEPES (23.8% by mass) to adjust the pH to 7.6.
  • the mixing ratio of the three components was 1:1:1 (by volume).
  • the concentration of the reducing agent in the autofluorescence inhibitor is 16.3 mg/mL.
  • tissue sections are immersed in pure ethanol for 5 minutes four times, and then washed. ⁇ I dehydrated. Subsequently, an operation of immersion in xylene for 5 minutes was performed four times to carry out penetration. Finally, using a mounting medium ("Enteran New" manufactured by Merck), the tissue sections were mounted to form tissue array slides for observation samples.
  • the tissue sections that have undergone the fixation treatment step were irradiated with predetermined excitation light to emit fluorescence.
  • the tissue section in that state was observed and photographed with a fluorescence microscope ("BX-53" manufactured by Olympus) and a digital camera for microscope ("DP73" manufactured by Olympus).
  • the excitation light was set to 575-600 nm by passing it through an optical filter.
  • the wavelength (nm) range of fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter.
  • the excitation wavelength conditions during microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 25 mW/cm 2 with excitation of 580 nm.
  • the excitation light irradiation time at the time of image acquisition was set to 0.5 seconds, and the image was captured.
  • Example 1 (Observation/measurement process) In Example 1, address C6 of the tissue array slide (LC241m) was observed. Image analysis was performed by ImageJ based on an image (300 ⁇ m ⁇ 200 ⁇ m) taken at 400 times.
  • Example 1 In Example 1 above, except for using AlexaFluor 647 (manufactured by Invitrogen) modified with streptavidin instead of streptavidin-modified PID particles 1 as an immunostaining agent, a human lung cancer tissue array slide ( A tissue sample was prepared from US Biomax, model number LC241m; target substance is ATPase), and a fluorescence image was formed in the same manner. The S/N ratio was calculated in the same manner as in Example 1 above based on the fluorescence image of address C6 of the tissue array slide (LC241m). Results are shown in Table I.
  • Example 2 to 9 (1) Preparation of Tissue Specimen A tissue array slide of a sample for observation was prepared in the same manner as in Example 1 above, except that the autofluorescence inhibitor was changed as follows.
  • the autofluorescence inhibitor consists of phosphomolybdic acid undiluted solution (A2), reducing agent solution (B2-B9), and buffer solution (C2).
  • phosphomolybdic acid stock solution (A2) phosphomolybdic acid is 12-molybdo (VI) phosphate n-hydrate (product name, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), the solvent is water, and phosphomolybdic acid content is 7.2 mg/mL.
  • the reducing agent liquids (B2 to B9) are solutions obtained by dissolving each of the reducing agents shown in Table II in water as a solvent at a ratio of 49 mg/mL.
  • the buffer solution (C2) is a buffer solution prepared by adding sodium hydroxide to an aqueous solution of HEPES (23.8% by mass) to adjust the pH to 7.6.
  • the mixing ratio of ascorbic acid, potassium antimonyl tartrate, and 2-furancarboxylic acid in the reducing agent liquid (B6) was 4:6:1 in mass ratio.
  • the total content of the above three reducing agents was 49 mg/mL.
  • the mixing ratio of the phosphomolybdic acid undiluted solution (A2), the reducing agent solutions (B2 to B9), and the buffer solution (C2) is 1:1:1 (by volume). Further, the concentration of phosphomolybdic acid in the autofluorescence inhibitor is about 2.4 mg/mL, and the concentration of the reducing agent is 16.3 mg/mL.
  • Comparative Example 2 A tissue array slide of a sample for observation was prepared in the same manner as in Comparative Example 1 except that the autofluorescence suppressor was changed to the same autofluorescence suppressor used in Example 2. Fluorescent images were formed as in 2-9. Based on the fluorescence image of address C6 of the tissue array slide (LC241m), the S/N ratio was calculated in the same manner as in Examples 2 to 9 above. Results are shown in Table II.
  • Example 11 A tissue specimen was prepared from a human lung cancer tissue array slide (manufactured by US Biomax, model number LC241m; target substance is ATPase) in the same manner as in Example 1 above, and a fluorescence image was formed in the same manner. Based on the obtained fluorescence images, in Example 11, address C6 (lung cancer cancer cells) of the tissue array slide (LC241m), and in Example 12, address C5 (lung cancer stroma) of the same tissue array slide, In Example 13, address B3 (blood vessel) of the same tissue array slide was observed in the same manner as in Example 1, and the S/N ratio was calculated. Results are shown in Table III.
  • Tissue specimens were prepared from human lung cancer tissue array slides (manufactured by US Biomax, model number LC241m; target substance is ATPase) in the same manner as in Examples 11 to 13, except that no autofluorescence inhibitor was added.
  • a fluorescence image was formed by Based on the obtained fluorescence images, in Comparative Example 11, address C6 (lung cancer cancer cells) of the tissue array slide (LC241m), and in Comparative Example 12, address C5 (lung cancer stroma) of the same tissue array slide, In Comparative Example 13, address B3 (blood vessel) of the same tissue array slide was observed in the same manner as in Example 1, and the S/N ratio was calculated. Results are shown in Table III.
  • the present invention it is possible to provide an image forming method that reduces noise due to autofluorescence and enables accurate measurement of fluorescence intensity derived from a target substance.

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé de formation d'image qui permet une mesure précise de la luminance fluorescente provenant d'une substance cible, par réduction du bruit dû à l'autofluorescence. Le présent procédé de formation d'image fait appel à une substance fluorescente et comprend : une étape d'ajout d'un agent de suppression d'autofluorescence à un tissu biologique qui comprend une substance cible, et de marquage de la substance cible à l'aide de nanoparticules intégrées à une substance fluorescente pour préparer un échantillon de tissu ; et une étape d'exposition de l'échantillon de tissu à une lumière d'excitation pour former une image de fluorescence.
PCT/JP2022/005553 2021-03-17 2022-02-14 Procédé de formation d'image Ceased WO2022196203A1 (fr)

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