WO2021230134A1 - Procédé de formation d'image - Google Patents

Procédé de formation d'image Download PDF

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Publication number
WO2021230134A1
WO2021230134A1 PCT/JP2021/017385 JP2021017385W WO2021230134A1 WO 2021230134 A1 WO2021230134 A1 WO 2021230134A1 JP 2021017385 W JP2021017385 W JP 2021017385W WO 2021230134 A1 WO2021230134 A1 WO 2021230134A1
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Prior art keywords
fluorescent
substance
fluorescence
tissue
image
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PCT/JP2021/017385
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English (en)
Japanese (ja)
Inventor
武寿 磯田
愛奈 安藤
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コニカミノルタ株式会社
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Priority to JP2022521860A priority Critical patent/JPWO2021230134A1/ja
Publication of WO2021230134A1 publication Critical patent/WO2021230134A1/fr

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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

Definitions

  • the present invention relates to an image forming method, and more particularly to an image forming method capable of observing a fluorescent color derived from a target substance conspicuously, reducing noise fluorescence such as autofluorescence, and accurately measuring a fluorescence signal.
  • a specific target substance for example, a substance overexpressed in a specific cell or a substance specifically expressed in a specific cell
  • a fluorescent signal is extracted from a fluorescent image obtained by imaging a tissue specimen labeled with a specific target substance using fluorescent nanoparticles containing a large number of fluorescent dyes (fluorescent luminescent substances), and the specific target substance in the fluorescent image is used.
  • the fluorescence brightness derived from the fluorescent nanoparticles that label the target substance is signaled (S), and the brightness of the noise fluorescence derived from a substance other than the fluorescent nanoparticles is measured. If the S / N ratio in the case of noise (N) is low, there is a high possibility that even noise fluorescence will be included in the fluorescence derived from fluorescent nanoparticles, which may cause an error, and thus quantitative analysis is also difficult. there were.
  • Patent Document 1 a technique for improving the fluorescence intensity of a target fluorescent label by labeling with an aminocoumarin compound having a specific structure as a green dye is disclosed (for example).
  • noise fluorescence such as autofluorescence is often detected in green, and the fluorescence signal cannot be measured correctly, so that the red dye is preferable.
  • the present invention has been made in view of the above problems and situations, and the solution thereof is that the fluorescence color of the target substance can be observed conspicuously, noise fluorescence such as autofluorescence is reduced, and the fluorescence signal is accurately measured. It is to provide an image forming method which can be done.
  • the present inventor identifies the excitation light in the tissue sample by utilizing the fact that the timing of the fading of the autofluorescence and the fading of the fluorescent luminescent substance are different in the process of examining the cause of the above problem.
  • An image forming method using a fluorescent substance which is an image forming method.
  • a tissue sample in which the target substance contained in the tissue section is stained with the fluorescent substance is irradiated with excitation light for at least 5 seconds, and at least a part of the fluorescence emitted from the fluorescent substance is forcibly faded.
  • An image forming method comprising a step of obtaining a forced fading fluorescent image obtained by forcibly fading at least a part of noise fluorescence emitted from a substance different from the fluorescent light emitting substance.
  • the image forming method according to item 1 further comprising a step of adding an autofluorescence inhibitor that suppresses autofluorescence generated from the tissue section when preparing the tissue specimen before the step of acquiring the forced fading fluorescence image. ..
  • the present invention it is possible to provide an image forming method capable of observing the fluorescence color of a target substance conspicuously, reducing noise fluorescence such as autofluorescence, and accurately measuring a fluorescence signal.
  • the mechanism of expression or the mechanism of action of the effect of the present invention has not been clarified, it is inferred as follows.
  • a tissue specimen in which the target substance is stained with a fluorescent substance is irradiated with excitation light for at least 5 seconds to forcibly fade at least a part of the fluorescence emitted from the fluorescent substance, or the fluorescent substance.
  • Noise emitted from a substance different from that of The fluorescent color stands out, and the fluorescence intensity of the fluorescent substance is relatively improved. As a result, the fluorescence signal can be accurately measured.
  • the image forming method of the present invention is an image forming method using a fluorescent luminescent substance, and an excitation light is applied to a tissue sample in which the target substance contained in the tissue section is stained with the fluorescent luminescent substance for at least 5 seconds.
  • the forced fading obtained by forcibly fading at least a part of the fluorescence emitted from the fluorescent substance or forcibly fading at least a part of the noise fluorescence emitted from a substance different from the fluorescent substance by the above irradiation. It has a step of acquiring a faded fluorescence image.
  • This feature is a technical feature common to or corresponding to each of the following embodiments.
  • the present invention there is a step of adding an autofluorescence inhibitor that suppresses autofluorescence generated from the tissue section when preparing the tissue specimen before the step of acquiring the forced fading fluorescence image.
  • an autofluorescence inhibitor that suppresses autofluorescence generated from the tissue section when preparing the tissue specimen before the step of acquiring the forced fading fluorescence image.
  • the fluorescence signal can be measured more accurately.
  • a sulfonated compound as the autofluorescence inhibitor, and it is particularly preferable to use a sulfonated perylene compound as the sulfonated compound in that the blocking effect on the site that emits autofluorescence is high.
  • the fluorescent substance is fluorescent particles because it is excellent in maintaining labeling performance.
  • the image forming method of the present invention is an image forming method using a fluorescent luminescent substance, and an excitation light is applied to a tissue sample in which the target substance contained in the tissue section is stained with the fluorescent luminescent substance for at least 5 seconds.
  • the forced fading obtained by forcibly fading at least a part of the fluorescence emitted from the fluorescent substance or forcibly fading at least a part of the noise fluorescence emitted from a substance different from the fluorescent substance by the above irradiation. It has a step of acquiring a faded fluorescence image. Further, it is preferable to have a step of adding an autofluorescence inhibitor that suppresses autofluorescence generated from the tissue section when preparing the tissue specimen before the step of acquiring the forced fading fluorescence image.
  • noise fluorescence refers to fluorescence derived from a substance other than a fluorescent luminescent substance among the fluorescence emitted from a tissue specimen after immunostaining.
  • noise fluorescence there is typically fluorescence (so-called autofluorescence) that is naturally emitted by a fluorescent substance (coenzyme, amino acid, protein, intracellular pigment substance, etc.) that originally exists in a tissue sample.
  • autofluorescence fluorescence
  • a substance other than the fluorescent luminescent substance contained in the tissue specimen and fluorescence emitted by an instrument such as a slide glass can be mentioned.
  • a sulfonated compound or the like can be used, and in particular, the use of the sulfonated compound has a high blocking effect on the site that emits autofluorescence. preferable.
  • the sulfonated compound it is preferable to use a sulfonated perylene-based compound in terms of blocking effect.
  • the sulfonated perylene-based compound for example, the following compound 1-7 (molecular weight 1399) is preferably used.
  • the step of adding the autofluorescence inhibitor is an activation treatment or blocking treatment of a specimen preparation step, a primary antibody reaction or a secondary antibody reaction or fluorescent labeling treatment of an immunostaining step, and morphological observation staining in a tissue section staining method described later. It is preferable to carry out at least one of the steps. Further, it is more preferable to carry out after the activation treatment or after the morphological observation staining step, and in particular, it is preferable to carry out after the activation treatment step in that the blocking effect on the site emitting autofluorescence is high.
  • the tissue sample is irradiated with excitation light for at least 5 seconds after the self-fluorescence inhibitor addition step to forcibly fade at least a part of the fluorescence emitted from the fluorescent luminescent material.
  • a forced fading fluorescence image obtained by forcibly fading at least a part of noise fluorescence emitted from a substance different from the fluorescent light emitting substance is acquired.
  • the excitation light it is preferable to change the wavelength of the excitation light according to the spectral characteristics of the tissue sample, but in general, it is preferable to use the excitation light in the wavelength range of 575 to 600 nm through, for example, an optical filter. ..
  • the excitation light source for irradiating such excitation light for example, it is preferable to use the fluorescence observation illumination "U-HGLGPS" attached to the fluorescence microscope "BX-53" (manufactured by Olympus Corporation).
  • the time for irradiating the excitation light is at least 5 seconds or longer, preferably 60 seconds or longer.
  • the upper limit is preferably 300 seconds or less.
  • the microscopic image acquisition device irradiates the tissue sample with excitation light for at least 5 seconds after the step of adding the autofluorescent inhibitor to forcibly fade at least a part of the fluorescence emitted from the fluorescent luminescent material, or the above-mentioned.
  • a forced fading fluorescence image obtained by forcibly fading at least a part of noise fluorescence emitted from a substance different from the fluorescent light emitting substance is acquired.
  • the acquired forced fading fluorescent image is subjected to image processing and analysis steps in, for example, an image processing apparatus.
  • the microscope image acquisition device and the image processing device are connected so as to be able to transmit and receive data via an interface such as a cable.
  • 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 by a LAN (Local Area Network) or may be wirelessly connected.
  • the microscope image acquisition device is a known fluorescence microscope with a camera, which acquires a microscope image of a tissue section on a slide placed on a slide fixing stage and transmits it to an image processing device.
  • the microscope image acquisition device is configured to include an irradiation means, an image forming means, an imaging means, a communication I / F, and the like.
  • the irradiation means is composed of an excitation light source, an optical filter, etc., and irradiates the tissue section on the slide placed on the slide fixing stage with excitation light for at least 5 seconds or more, and at least a part of the fluorescence emitted from the fluorescent luminescent material. Is forcibly faded, or at least a part of the noise fluorescence emitted from a substance different from the fluorescent luminescent substance is forcibly faded.
  • the excitation light source examples include the above-mentioned light sources, and it is preferable that the excitation light has a wavelength in the range of 575 to 600 nm by passing through, for example, an optical filter. Further, it is preferable that the wavelength range of the fluorescence to be observed is also set within the wavelength range of 612 to 692 nm by passing through an optical filter.
  • the imaging means is composed of an eyepiece, an objective lens, or the like, and forms a transmitted light, a reflected light, or a fluorescence emitted from a tissue section on a slide by the irradiated light.
  • the image pickup means is a microscope-installed camera equipped with a CCD (Charge Coupled Device) sensor or the like, and images an image formed on an image forming surface by the image forming means to generate digital image data of a forced fading fluorescence image.
  • the communication I / F transmits the image data of the generated forced fading fluorescent image to the image processing device.
  • the microscope image acquisition device is provided with a bright-field unit that combines irradiation means and imaging means suitable for bright-field observation, and a fluorescence unit that combines irradiation means and imaging means suitable for fluorescence observation, and the units are switched. This makes it possible to switch between bright field and fluorescence.
  • the microscope image acquisition device is a known fluorescence microscope with a camera, and is not particularly limited, and a microscope in which a camera is installed in any known microscope (for example, a phase contrast microscope, a differential interference microscope, an electron microscope, etc.) is used. It can be used as a microscope image acquisition device. For example, it is preferable to use a fluorescence microscope (“BX-53” manufactured by Olympus Corporation), a digital camera for a microscope (“DP73” manufactured by Olympus Corporation), or the like.
  • the microscope image acquisition device is not limited to a microscope with a camera.
  • a virtual microscope slide creation device for example, a special table
  • a virtual microscope slide creating device it is possible to acquire image data that allows the entire image of the tissue section on the slide to be viewed at once on the display unit.
  • the image processing device identifies the in-focus position of each cell in the tissue section to be observed by analyzing the forced fading fluorescent image transmitted from the microscope image acquisition device.
  • the tissue specimen is a tissue section containing a target substance, which is a specimen stained with a fluorescent luminescent substance contained as an immunostaining agent and to which an autofluorescent inhibitor is added.
  • the tissue specimen after such staining is placed on the stage of the microscope image acquisition device.
  • Target substance A target substance is a substance that is the target of immunostaining using a fluorescent luminescent substance, mainly for detection or quantification from the viewpoint of pathological diagnosis.
  • the target substance is not particularly limited, but in pathological diagnosis, a biomarker such as an antigen according to the purpose is generally selected.
  • a biological substance such as a protein (antigen) expressed in a tissue section can be used as a target substance.
  • a unit smaller than a protein such as a peptide or a biomarker such as RNA may be used as a target substance.
  • the target substance does not have to be unique to the living body as long as it is present in the sample.
  • the target substance may be a drug introduced into the living body from outside the body.
  • Typical target substances include biological substances that are expressed on the cell membranes of various cancer tissues and can be used as biomarkers.
  • the fluorescent luminescent substance according to the present invention is preferably fluorescent luminescent particles because it is excellent in labeling performance.
  • the fluorescent substance is preferably used as a conjugate (immunostainer) bound to an antibody. That is, as an immunostaining agent, in order to improve the efficiency of fluorescent labeling and suppress the passage of time leading to deterioration of fluorescence as much as possible, the primary antibody and fluorescent nanoparticles are shared indirectly, that is, by utilizing an antigen-antibody reaction or the like. It is preferable to use a complex that is linked by a bond other than the bond. In order to simplify the staining operation, a complex in which fluorescent nanoparticles are directly linked to the primary antibody or the secondary antibody can also be used as the immunostaining agent.
  • immunostaining agent examples include [primary antibody against the target substance] ... [antibody against the primary antibody (secondary antibody)] to [fluorescent nanoparticles].
  • "" indicates that they are bound by an antigen-antibody reaction, and the mode of binding indicated by "" is not particularly limited.
  • covalent binding, ion binding, hydrogen binding, coordination binding, antigen-antibody binding examples thereof include biotin avidin reaction, physical adsorption, chemical adsorption, etc., and may be mediated by a linker molecule if necessary.
  • Antibody 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.
  • an anti-HER2 antibody can be used
  • HER3 is the target substance
  • an anti-HER3 antibody can be used.
  • an antibody (IgG) that specifically recognizes and binds to the primary antibody as an antigen can be used.
  • Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferable.
  • the type of animal (immune animal) that produces an antibody is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like as in the past.
  • Fluorescent nanoparticles are nano-sized particles that emit fluorescence when irradiated with excitation light, and have sufficient intensity of fluorescence to represent the target substance as a signal one molecule at a time. It is a particle that can emit light.
  • fluorescent nanoparticles quantum dots (semiconductor nanoparticles) and fluorescence-emitting substance-integrated nanoparticles are preferably used.
  • the brightness of the fluorescent nanoparticles is preferably 5 times or more the brightness of noise fluorescence.
  • Quantum Dots As the quantum dots, semiconductor nanoparticles containing a II-VI group compound, a III-V group compound or an IV group element are used. For example, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge and the like can be mentioned.
  • the Fluorescent Luminescent Substance Accumulated Nanoparticles are based on particles made of an organic substance or an inorganic substance, and a plurality of fluorescent luminescent substances (for example, the quantum dots, fluorescent dyes, etc.). Is a nano-sized particle having a structure contained therein and / or adsorbed on its surface.
  • the fluorescent substance-accumulated nanoparticles it is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges and have an electrostatic interaction with each other.
  • quantum dot-accumulated nanoparticles, fluorescent dye-accumulated nanoparticles and the like are used as the fluorescent substance-accumulated nanoparticles.
  • the emission wavelength of the fluorescent substance integrated particles is arbitrary as long as it is within the sensitivity range of the image pickup device of the fluorescence microscope. Specifically, it is preferable that the emission wavelength is in the range of 400 to 700 nm.
  • the average particle size of the fluorescent substance-accumulated particles is not particularly limited, but those with a large particle size have difficulty in accessing the antigen, and those with a small particle size and low brightness value emit fluorescence as background noise (camera noise and cells). Since it is buried in the noise fluorescence of the above, it is preferable to use a particle size of about 20 to 200 nm. Further, it is preferable that the coefficient of variation of the particle size is 15% or less.
  • the average particle size is the diameter of the circle when an electron micrograph is taken using a scanning electron microscope (SEM), the cross-sectional area is measured for a sufficient number of particles, and each measured value is the area of the circle. Obtained as the diameter.
  • SEM scanning electron microscope
  • the arithmetic mean of the particle sizes of 1000 particles is taken as the average particle size.
  • the fluctuation coefficient was also a value calculated from the particle size distribution of 1000 particles.
  • organic substances generally include melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, furan resin and the like.
  • Resins classified as thermosetting resins generally heat such as styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), ASA resin (acrylonitrile-styrene-methyl acrylate copolymer), etc.
  • Resins classified as plastic resins; other resins such as polylactic acid; polysaccharides can be exemplified.
  • examples of the inorganic substance include silica and glass.
  • Quantum dot integrated nanoparticles are structures in which the quantum dots are contained in and / or adsorbed on the surface thereof. Have. When the quantum dots are contained in the mother body, the quantum dots may or may not be chemically bonded to the mother body itself as long as they are dispersed inside the mother body.
  • Fluorescent dye-accumulated nanoparticles have a structure in which a fluorescent dye is contained in the mother body and / or is adsorbed on the surface thereof. ..
  • the fluorescent dye include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyrromesen-based dye molecules.
  • fluorescent dye examples include Alexa Fluor (registered trademark, manufactured by Invigen) dye molecule, BODIPY (registered trademark, manufactured by Invigen) dye molecule, Cy (registered trademark, manufactured by GE Healthcare) dye molecule, and HiLyte (registered).
  • the method for preparing a tissue section (also referred to simply as a “section” and including a section such as a pathological section) to which this staining method can be applied is not particularly limited, and a tissue section prepared by a known procedure can be used.
  • Specimen preparation step (5.1) Specimen preparation step (5.1.1) Deparaffin treatment
  • the section is immersed in a container containing xylene to remove paraffin.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably in the range of 3 to 30 minutes. If necessary, xylene may be replaced during immersion.
  • the section is immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably in the range of 3 to 30 minutes. If necessary, ethanol may be replaced during immersion.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably in the range of 3 to 30 minutes. If necessary, the water may be replaced during the immersion.
  • the activation treatment of the target substance is carried out according to a known method.
  • the activation conditions are not particularly specified, but the activating solution includes 0.01 M citric acid buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, and 0.1 M Tris-hydrochloric acid buffer.
  • a liquid or the like can be used.
  • the pH condition is such that a signal is emitted from the pH range of 2.0 to 13.0 at 25 ° C. and the tissue roughness is such that the signal can be evaluated, depending on the tissue section to be used.
  • the pH is in the range of 6.0 to 8.0, but for special tissue sections, for example, pH 3.0 is also used.
  • an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used as the heating device.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • 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 (phosphate buffered saline) and washed.
  • PBS phosphate buffered saline
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the immersion time is preferably in the range of 3 to 30 minutes. Further, if necessary, the PBS may be replaced during the immersion.
  • an autofluorescence inhibitor in the activation treatment.
  • the section after the activation treatment is washed with PBS, and then the above-mentioned autofluorescence inhibitor is added.
  • the amount of the autofluorescence inhibitor added may be as long as it can cover the section, and is preferably in the range of 20 uL to 100 uL per slide. Even after the addition of the autofluorescence inhibitor, it is preferable to further wash with PBS.
  • (5.2) Immunostaining step In the immunostaining step, fluorescent nanoparticles having a site that can directly or indirectly bind to the target substance in order to stain the target substance (fluorescent emission property) based on the immunostaining method.
  • a solution of the immunostaining agent containing the substance) is placed on a section and reacted with the target substance.
  • the solution of the immunostaining agent used in the immunostaining step may be prepared in advance before this step.
  • the immunostaining method is not particularly limited, but the following are typical examples.
  • a method of preparing a fluorescently labeled primary antibody in which a fluorescent substance and a primary antibody are linked, and directly fluorescently labeling and staining the target protein with the fluorescently labeled primary antibody (primary antibody method); Prepare a primary antibody and a fluorescently labeled secondary antibody in which a fluorescent luminescent substance and a secondary antibody are linked, react the primary antibody with the target protein, and then react the primary antibody with the fluorescently labeled secondary antibody. Therefore, a method of indirectly fluorescently labeling and staining the target protein (secondary antibody method).
  • a method in which a phosphor is reacted and the target protein is indirectly fluorescently labeled and stained using the avidin-biotin reaction (primary antibody method in combination with avidin-biotin); Prepare a biotin-modified secondary antibody in which the primary antibody and the secondary antibody are linked to biotin, and an avidin-modified phosphor in which the phosphor is linked to avidin or streptavidin, react the target protein with the primary antibody, and then biotin-modify.
  • a method in which an avidin-modified phosphor is further reacted after the secondary antibody is reacted, and the target protein is indirectly fluorescently labeled and stained using the avidin-biotin reaction (secondary antibody method in combination with avidin-biotin).
  • a hapten which has no immunogenicity but exhibits antigenicity and can react with an antibody
  • a hapten which has no immunogenicity but exhibits antigenicity and can react with an antibody
  • Combinations of low molecular weight substances and anti-hapten antibodies such as dicoxygenin and anti-dicoxygenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, as well as other substances with similar specific reactivity can also be utilized. can.
  • the immunostaining method may be performed according to the standard procedure and treatment conditions for each of the various methods as described above.
  • the sample slide on which the sample is placed may be immersed in one or more kinds of reagents according to the immunostaining method under appropriate temperature and time conditions.
  • the temperature is not particularly limited, but it can be carried out at room temperature.
  • the reaction time is preferably 30 minutes or more and 24 hours or less.
  • reagents required for immunostaining that is, fluorescently labeled primary / secondary antibody, biotin-modified primary / secondary antibody, avidin-modified secondary antibody / phosphor, etc. are dissolved, and BSA (bovine serum albumin) is required.
  • a solution such as a buffer solution to which a blocking agent such as (bovine serum albumin)) is added can be prepared according to a known method and can also be obtained as a commercially available product.
  • the sample slide is preferably washed by immersing it in a washing solution such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the temperature of the washing treatment using PBS performed after the treatment with this labeling solution is room temperature, and the time is 3 to 30 minutes. If necessary, the PBS may be replaced during the immersion. It is preferable to drop a known blocking agent such as PBS containing BSA or a surfactant such as Tween 20 before performing the treatment as described above.
  • the autofluorescence inhibitor at least in any of the primary antibody reaction, the secondary antibody reaction and the fluorescent labeling in the various methods described above.
  • the avidin-modified phosphor is further reacted.
  • an autofluorescence inhibitor it is preferable to add an autofluorescence inhibitor.
  • the amount of the autofluorescence inhibitor added may be as long as it can cover the section, and is preferably in the range of 20 uL to 100 uL per slide. It is preferable to wash with PBS after adding the autofluorescence inhibitor.
  • Morphological observation staining step Apart from the immunostaining step, morphological observation staining may be 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 with eosin, in which cytoplasm, stroma, various fibers, erythrocytes, and keratinized cells are stained in red to deep red, is standardly used.
  • hematoxylin which stains cell nuclei, lime, cartilage tissue, bacteria, and mucus in blue-blue to pale blue
  • the method of performing these two stainings at the same time is hematoxylin / eosin staining).
  • HE staining HE staining
  • the morphological observation staining step it may be performed after the immunostaining step or before the immunostaining step.
  • an autofluorescence inhibitor in the morphological observation staining step.
  • the autofluorescence inhibitor is added after hematoxylin staining or, in the case of eosin staining, eosin staining.
  • the amount of the autofluorescence inhibitor added may be as long as it can cover the section, and is preferably in the range of 20 uL to 100 uL per slide. It is preferable to wash with PBS after adding the autofluorescence inhibitor.
  • tissue specimen after the morphological observation staining step is subjected to treatments such as immobilization / dehydration, permeation, and encapsulation so as to be suitable for observation.
  • the tissue specimen may be immersed in an immobilization treatment solution (crosslinking agent such as formalin, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol).
  • the permeation treatment may be performed by immersing the tissue specimen after the immobilization / dehydration treatment in a permeation liquid (xylene or the like).
  • the encapsulation treatment may be performed by immersing the tissue specimen that has been subjected to the permeation treatment in the encapsulation liquid.
  • the conditions for performing these treatments for example, the temperature and soaking time when immersing the tissue specimen in a predetermined treatment solution, may be appropriately adjusted so as to obtain an appropriate signal according to the conventional immunostaining method. can.
  • the tissue sample obtained by staining the tissue section as described above is placed on the stage of the above-mentioned microscopic image acquisition device, and the tissue sample is irradiated with excitation light to expose it for at least 5 seconds or more, and the fluorescence emission is performed.
  • a forced fading fluorescence image is obtained in which at least a part of the fluorescence of the sex substance is forcibly faded, or at least a part of the noise fluorescence emitted from a substance different from the fluorescence emitting substance is forcibly faded.
  • the acquired forced fading fluorescent image is subjected to image processing and analysis steps in, for example, an image processing apparatus.
  • the Rf value of the target product was 0.73 with respect to the Rf value of 0.88 of the raw material, and the convergence of the reaction and the formation of the target product were confirmed from the TLC data.
  • Ice was placed in a 50 mL vial until the 8th minute (30 mL), and the reaction solution was added little by little. A suspension in which the resulting dye was suspended was obtained. The suspension was centrifuged, the supernatant was removed, and the dye was recovered as a precipitate. Disperse the precipitate with 10 mL of pure water, centrifuge this dispersion to remove the supernatant, collect the precipitate, disperse again with 10 mL of pure water, centrifuge this dispersion to obtain the supernatant. It was removed and the precipitate was collected.
  • the recovered precipitate was dispersed with ethanol, the dispersion was centrifuged, and the supernatant was removed to obtain an aminocoumarin compound II represented by the following formula (II) as a precipitate.
  • the yield of aminocoumarin compound II was 80%.
  • the obtained precipitate was dried, the obtained powder was added to pure water, and then neutralized with an aqueous NaOH solution to dissolve the precipitate, and the pH of the solution was adjusted to 7 to 8. This solution was dried in a freeze-dryer to obtain a Na salt of aminocoumarin compound II. It was confirmed that the aminocoumarin compound II has poor solubility in water as a sulfonic acid form, whereas it is rapidly dissolved in water by using a Na salt.
  • 0.3 mL of the obtained organoalkoxysilane compound solution was mixed with 24 mL of 99% ethanol, 0.3 mL of tetraethoxysilane (TEOS), 0.75 mL of ultrapure water, and 0.75 mL of 28% by mass of ammonia water at 25 ° C. 3 Time mixed.
  • the mixed solution prepared in the above step was centrifuged at 10000 G for 20 minutes to remove the supernatant. Ethanol was added to this precipitate to disperse the precipitate, and the precipitate was rinsed again for centrifugation. Further, the same rinse was repeated twice to obtain aminocoumarin compound-encapsulating particles II. SEM observation was performed on 1000 of the obtained particles, and the average particle size was measured. As a result, the average particle size was 60 nm.
  • This streptavidin solution was desalted with a gel filtration column (Zaba Spin Deserting Colors: Funakoshi) to obtain streptavidin capable of binding to the silica-based particles.
  • the total amount of streptavidin (containing 0.04 mg) and 740 ⁇ L of silica-based particles adjusted to 0.67 nM above were mixed with PBS containing 2 mM of EDTA, and reacted at room temperature for 1 hour. 10 mM mercaptoethanol was added to terminate the reaction.
  • the linker reagent "(+)-Biotin-PEG6-NH-Mal" (PurePEG, product number 246106-250) having a spacer length of 30 angstroms becomes 0.4 mM using DMSO. Adjusted to. 8.5 ⁇ L of this solution was added to the antibody solution, mixed and reacted at 37 ° C. for 30 minutes.
  • This reaction solution was purified by subjecting it to a desalting column "Zeba Desert Spin Columns" (Cat. # 89882, manufactured by Thermo Scientific Co., Ltd.). The absorption of the desalted reaction solution at a wavelength of 300 nm was measured with a spectroscopic altimeter (“F-7000” manufactured by Hitachi) 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 the solution was used as a solution of the biotinylated secondary antibody.
  • Specimen treatment step (1-1) Deparaffinization treatment step HER2 (3+) and HER2 (-) tissue array slides ("CB-A712 series” manufactured by Cosmo Bio Co., Ltd.) are used as tissue sections for staining. board. This tissue array slide was deparaffinized.
  • (1-2) Activation treatment step The deparaffinized tissue array slide was washed by replacing it with water. The washed tissue array slides were autoclaved in 10 mM citrate buffer (pH 6.0) at 121 ° C. for 15 minutes to activate the antigen. The tissue array slides after the activation treatment were washed with PBS, and the washed tissue array slides were blocked with PBS containing 1% BSA for 1 hour.
  • tissue sections that had completed the immunostaining step and the morphological observation staining step were washed and dehydrated by immersing them in pure ethanol for 5 minutes four times. Subsequently, the operation of immersing in xylene for 5 minutes was performed four times to perform transparency. Finally, a tissue section was encapsulated using an encapsulant (“Enteran New” manufactured by Merck & Co., Inc.) to prepare a tissue array slide of a sample for observation.
  • an encapsulant (“Enteran New” manufactured by Merck & Co., Inc.
  • the tissue section after the immobilization treatment step was irradiated with a predetermined excitation light to emit fluorescence.
  • the tissue section in that state was observed and imaged with a fluorescence microscope (“BX-53” manufactured by Olympus Corporation) and a digital camera for a microscope (“DP73” manufactured by Olympus Corporation).
  • the excitation light was set to 575 to 600 nm by passing it through an optical filter.
  • the range of the wavelength (nm) of the fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter.
  • the conditions of the excitation wavelength at the time of microscopic observation and image acquisition were such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • the excitation light irradiation time at the time of image acquisition was set to 0.5 seconds for imaging.
  • the number of signals in the tissue of HER2 (3+) was taken as the average value of 1000 cells measured by the ImageJ FindMaxims method based on the image taken at 400 times.
  • the number of signals S on the cell membrane in the visual field and the number N of extracellular signals in the visual field were measured, and the S / N ratio was calculated.
  • the number of signals and the S / N ratio are shown in Table I below.
  • Comparative Example 2 In Comparative Example 1, the excitation light irradiation time was set to 0.05 seconds before image acquisition in "(5) Observation / measurement step", and then the excitation light irradiation time at the time of image acquisition was set to 0. The same procedure was performed except that the image was taken with the setting set to 5 seconds.
  • the excitation wavelength condition before image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • Comparative Example 3 In Comparative Example 1, the excitation light irradiation time was set to 0.5 seconds before image acquisition in "(5) Observation / measurement step", and then the excitation light irradiation time at the time of image acquisition was set to 0. The same procedure was performed except that the image was taken with the setting set to 5 seconds.
  • the excitation wavelength condition before image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • Example 1 In Comparative Example 1, before image acquisition in "(5) Observation / Measurement Step", the excitation light irradiation time was set to 5 seconds and light was irradiated to perform forced fading, and then the excitation light irradiation time at the time of image acquisition was performed. The same procedure was performed except that the image was taken with the setting set to 0.5 seconds.
  • the excitation wavelength condition before image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • Example 2 In Comparative Example 1, before image acquisition in "(5) Observation / measurement step", the excitation light irradiation time is set to 60 seconds and light is irradiated to perform forced fading, and then the excitation light irradiation time at the time of image acquisition is performed. was set to 0.5 seconds and the image was taken in the same manner. The conditions for the excitation wavelength before image acquisition were such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • Example 3 In Comparative Example 1, before image acquisition in "(5) Observation / Measurement Step", the excitation light irradiation time was set to 300 seconds and light was irradiated to perform forced fading, and then the excitation light irradiation time at the time of image acquisition was performed. The same procedure was performed except that the image was taken with the setting set to 0.5 seconds.
  • the excitation wavelength condition before image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • the excitation light irradiation time was set to 60 seconds before image acquisition, light irradiation was performed to perform forced fading, and then the excitation light irradiation time at the time of image acquisition was 0.5. I set it to seconds and took an image.
  • the excitation wavelength condition before image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.
  • Example 5 PBS containing 1% of the following compound 1-7 (sulfonated perylene-based imide) in TrueVIEW (manufactured by Vector) solution (autofluorescence inhibitor) in "(1-2) Activation treatment step". The procedure was the same except that the solution was changed. The addition amount is the same.
  • Example 6 In Comparative Example 1, "(3) Morphological observation and dyeing step” and “(5) Observation / measurement step” were changed as follows, but the same procedure was performed. (3) Morphological observation staining step After immunostaining, hematoxylin staining (H staining) was performed. The immunostained sections were stained with Meyer hematoxylline solution for 1 minute for hematoxylin staining. Then, the tissue section was washed with running water at 45 ° C. for 3 minutes. For the stained tissue array slide, 50 uL of TrueVIEW (manufactured by Vector) solution as an autofluorescence inhibitor was placed on the slide and treated.
  • TrueVIEW manufactured by Vector
  • Example 7 In Example 6, the TrueVIEW (manufactured by Vector) solution (autofluorescent inhibitor) in "(3) Morphological observation staining step" was added to a PBS solution containing 1% of the compound 1-7 (sulfonated perylene-based imide). I did the same except for the change. The addition amount is the same.
  • the present invention can be used as an image forming method capable of observing the fluorescence color derived from the target substance conspicuously, reducing noise fluorescence such as autofluorescence, and accurately measuring the fluorescence signal.

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Abstract

Le procédé de formation d'image de l'invention fait appel à une substance fluorescente émettrice de lumière, le procédé de formation d'image comportant une étape dans laquelle : un échantillon de tissu, dans lequel une substance cible contenue dans une tranche de tissu est colorée avec la substance émettrice de lumière fluorescente, est irradié avec une lumière d'excitation pendant au moins 5 secondes pour atténuer de manière forcée au moins une partie de la fluorescence émise par la substance émettrice de lumière fluorescente, ou pour atténuer de manière forcée au moins une partie de la fluorescence du bruit émise par une substance différente de la substance émettrice de lumière fluorescente, et en conséquence, une image fluorescente atténuée de manière forcée est obtenue.
PCT/JP2021/017385 2020-05-13 2021-05-06 Procédé de formation d'image WO2021230134A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006342135A (ja) * 2005-06-10 2006-12-21 Kyushu Univ ペリレン誘導体、過酸化脂質計測用試薬及び過酸化脂質の計測方法
WO2012029269A1 (fr) * 2010-09-02 2012-03-08 国立大学法人東北大学 Méthode de détermination de l'apparition d'un cancer ou du risque d'apparition d'un cancer
US20180080874A1 (en) * 2016-09-21 2018-03-22 University Of Houston System Blue emitting persistent phosphor compositions as diagnostic reporters
JP2018169469A (ja) * 2017-03-29 2018-11-01 Jsr株式会社 表示パネルの製造方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006342135A (ja) * 2005-06-10 2006-12-21 Kyushu Univ ペリレン誘導体、過酸化脂質計測用試薬及び過酸化脂質の計測方法
WO2012029269A1 (fr) * 2010-09-02 2012-03-08 国立大学法人東北大学 Méthode de détermination de l'apparition d'un cancer ou du risque d'apparition d'un cancer
US20180080874A1 (en) * 2016-09-21 2018-03-22 University Of Houston System Blue emitting persistent phosphor compositions as diagnostic reporters
JP2018169469A (ja) * 2017-03-29 2018-11-01 Jsr株式会社 表示パネルの製造方法

Non-Patent Citations (1)

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Title
HIKAGE ET AL. ET AL.: "Nano-imaging of the lymph network structure with quantum dots", NANOTECHNOLOGY, vol. 21, 2010, pages 185103, XP020174849 *

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