JP2021113724A - Tissue morphology detection method and image acquisition device - Google Patents

Abstract

To allow for detecting tissue morphology while maintaining living tissue morphology.SOLUTION: A tissue morphology detection method is provided, comprising a section sample preparation step of attaching a tissue section to a membrane having a protein binding ability of 50 μg/cm2 or greater to prepare a section sample, and a detection step of detecting tissue morphology of the section sample.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to a method for detecting tissue morphology and an image acquisition device.

Conventionally, a technique for observing a tissue section by immunohistochemical staining is known (Non-Patent Document 1).

Comparative Endocrinology vol.43 No.162 (September 2017) p145-149.

However, in the prior art, the tissue structure at the time of survival could not be sufficiently maintained in a tissue having a low cell density, and the morphology of intercellular spaces and intercellular spaces in the tissue could not be sufficiently detected.

Therefore, an object of the present disclosure is to maintain the tissue structure at the time of survival and detect the tissue morphology.

The method for detecting the tissue morphology according to some embodiments includes ^{a section sample preparation step of attaching a tissue section to a membrane having a protein binding ability of 50 μg / cm 2} or more to prepare a section sample, and a tissue morphology of the section sample. It has a detection step for detecting. In this way, by attaching the tissue section to ^{a membrane having a protein binding ability of 50 μg / cm 2} or more to prepare a section sample, the tissue morphology can be detected while maintaining the tissue structure at the time of survival. In addition, in this specification, intercellular space is included in the tissue and between different tissues.

In the method for detecting the tissue morphology according to the embodiment, the extracellular protein of the section sample may be detected in the detection step. As described above, according to the method for detecting tissue morphology according to one embodiment, extracellular proteins that could not be detected by conventional immunohistochemical staining or the like can be detected.

In the method for detecting a tissue morphology according to an embodiment, the tissue morphology may be detected by dye staining. By detecting the tissue morphology by dye staining in this way, the tissue morphology can be detected with high detection sensitivity without being affected by the autofluorescence of the section sample.

The method for detecting a tissue form according to an embodiment may include a fixing step of immersing the tissue in a fixing solution before the section sample preparation step. In this way, by immersing the tissue in the fixation solution before the section sample preparation step, the tissue morphology of the embryo or the like is fixed, so that the tissue morphology can be detected in a state where the structure at the time of survival is more maintained. can.

The method for detecting a tissue form according to an embodiment may include a fixing step of immersing the tissue in a fixing solution without perfusing the tissue before the section sample preparation step. In this way, by immersing the tissue in the fixation solution without perfusion before the section sample preparation step, the tissue morphology such as embryos is fixed without being moved by perfusion, so that the tissue morphology is fixed at the time of survival. Can be detected while maintaining the structure of.

The method for detecting a tissue form according to an embodiment includes an embedding step of embedding the tissue with an embedding agent to form an embedding structure after the fixing step and before the section sample preparation step. The membrane is a porous membrane, and in the section sample preparation step, the embedded tissue section cut out from the embedded tissue is brought into contact with one side of the membrane to dissolve the embedding agent. The liquid may be passed from one side of the membrane to the other side to attach the tissue section from which the embedding agent has been removed to the membrane. In this way, after embedding the embryo to form an embedding tissue, an embedding tissue section cut out from the embedding tissue is brought into contact with one side of the membrane, and an embedding agent lysing solution for dissolving the embedding agent is applied to the membrane. By passing the tissue section from one side to the other side and attaching the tissue section from which the embedding agent has been removed to the membrane, the tissue section is attached to the membrane without disturbing the tissue morphology by the flow of the embedding agent solution. Can be made to.

In the method for detecting a tissue morphology according to an embodiment, the tissue section is a tissue section of an embryo, and before the fixation step, the amount of body fluid outflow from the embryo is suppressed to 1% by mass or less of the embryo. May have an embryo preparation step to prepare. In this way, by suppressing the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo, the outflow of substances existing in the body fluid is prevented, and the tissue morphology is maintained in a state in which the structure at the time of survival is more maintained. Can be detected.

In the method for detecting a tissue morphology according to an embodiment, a cut may be made in at least one place of the embryo in the embryo preparation step. In this way, by making a cut in at least one place of the embryo in the embryo preparation step, the fixative or the like can be easily immersed in the embryo in the subsequent fixing step, and the time required for embryo fixation can be shortened. can.

In the method for detecting the tissue morphology according to one embodiment, it is preferable to irradiate the section sample with a plurality of light sources using an upright microscope. By irradiating the section sample with a light source from the side that does not adhere to the film of the section sample using an upright microscope, the section sample is not subjected to any additional treatment and regardless of the light transmission of the film. , Tissue morphology can be detected.

The tissue morphology image acquisition apparatus according to some embodiments has a section sample portion for preparing a section sample in which a tissue section is attached to a membrane ^{having a protein binding ability of 50 μg / cm 2 or more, and a tissue morphology of the section sample.} It has a detection unit to detect and detects tissue morphology. According to such an image acquisition device, the tissue morphology can be detected while maintaining the tissue structure at the time of survival.

The tissue morphology image acquisition device according to one embodiment may have a protein detection unit that detects extracellular proteins of the tissue section. As described above, according to the image acquisition device having a protein detection unit, it is possible to detect extracellular proteins that could not be detected by conventional immunohistochemical staining.

The image acquisition device for the tissue form according to one embodiment refers to an image acquisition unit that acquires a different image by imaging the section sample subjected to different labeling treatments on the same section sample, and a different image. It may have a position information acquisition unit that acquires the position information of the tissue section in each image, and a trace unit that superimposes different images with reference to the position information. In this way, the image acquisition unit captures images of the same section sample subjected to different labeling treatments to acquire different images, and the trace unit superimposes the images to detect the detection results by the different labeling treatments. Can be evaluated in correspondence with each other.

According to the present disclosure, tissue morphology can be detected while maintaining the tissue structure at the time of survival.

It is a schematic diagram which shows an example of the outline of the tissue form detection method. It is a flowchart of an example of the organization form detection method. In one embodiment, it is a figure for demonstrating the direction in which the embedding agent solution flows at the time of embedding agent dissolution. It is a figure for demonstrating the laparotomy position of an embryo. It is a functional block diagram which shows an example of the basic structure of an image acquisition apparatus. It is a figure which shows an example of the schematic structure of the detection part. It is a figure which shows the result of having examined the amount of protein binding on the surface of the membrane, the slide glass, and the slide glass for the conventional tissue section of which the protein binding ability is 50 μg / cm ^{2 or more before and after washing.} It is a figure which shows the detection result of the tissue morphology of a mouse embryo by HE staining observed using the tissue morphology detection method and an image acquisition apparatus. It is a figure which shows the detection result of the tissue morphology of a mouse embryo by HE staining observed using the tissue morphology detection method and an image acquisition apparatus. It is a figure which shows the detection result of the tissue morphology of a mouse embryo by the immunohistochemical staining observed using the tissue morphology detection method and an image acquisition apparatus. It is a figure which shows the detection result of the tissue morphology of a mouse embryo by the immunohistochemical staining observed using the tissue morphology detection method and an image acquisition apparatus. It is a figure for demonstrating the method of fixing the position of a section sample on a microscope using a positioning jig. It is a figure for demonstrating the method of fixing the position of a section sample on a positioning jig. It is a figure which shows the detection result of the tissue morphology of a mouse embryo by immunohistochemical staining observed by using the image acquisition apparatus which has the position information acquisition means and the trace means using the tissue morphology detection method.

The method for detecting the tissue morphology of the present disclosure includes ^{a section sample preparation step of attaching a tissue section to a membrane having a protein binding ability of 50 μg / cm 2} or more to prepare a section sample, and a detection step of detecting the tissue morphology of the section sample. And have.

FIG. 1 shows an outline of an outline of a method for detecting tissue morphology. FIG. 2 is a flowchart showing a flow of a method for detecting a tissue morphology. In the illustrated example, first, the tissue 1 is prepared (tissue preparation step S2). When the tissue 1 is an embryo, a cut A is appropriately made in the embryo as shown in FIG. Next, the tissue 1 is fixed (fixing step S4). Next, the tissue 1 is optionally embedded to form an embedded tissue (embedding step S6). Next, the tissue 1 or the embedded tissue is cut out to obtain a tissue section 10 or an embedded tissue section, and then the embedded tissue section is ^{attached to a membrane 2 having a protein binding ability of 50 μg / cm 2} or more to form a section sample 100. (Section sample preparation step S8). Next, the tissue morphology of the section sample 100 is detected using an antibody or the like (detection step S10). The details of each step will be described below.

[Organizational preparation process]
First, in the tissue preparation step, it is preferable to prepare the tissue 1 in a state that reflects the state of the living body as much as possible. Here, as a method of preparing the tissue 1 in a state that reflects the state of the living body as much as possible, for example, the tissue 1 is ligated and excised without perfusion, and the amount of body fluid outflow from the target tissue 1 is 5 of the tissue 1. For example, it should be suppressed to mass% or less. The preparation of the tissue 1 includes both the removal of the tissue 1 from the living body and the preparation of the artificially cultured tissue 1.

[Fixing process]
Next, the tissue 1 is optionally immersed in a fixing solution before the section sample preparation step described later. By immersing the tissue 1 in the fixing solution before the section sample preparation step, the tissue morphology is fixed, so that the tissue morphology can be detected in a state where the structure at the time of survival is more maintained. It is preferable that perfusion is not performed when preparing and fixing the tissue 1. Traditionally, tissue morphology such as brain, spinal cord, pituitary gland, ganglion, liver, kidney, spleen, pancreas, tongue, lymph node, fat, mammary gland, and salivary gland, especially for small experimental animals (including embryos). In the case of detection, in order to improve the stainability of the tissue 1 and to prevent the blood cells in the blood vessel from diverging to other sites when preparing the tissue section 10, the tissue 1 is detected from the blood vessel of the tissue 1. It is common to perform perfusion by injecting a fixed solution of. However, when perfusion is performed, in the process of replacing blood in tissue 1 with a fixed solution, free cells in blood vessels including blood cells and substances such as extracellular proteins flow out and move on a large scale, so that they are intravascular. The location of substances such as free cells and extracellular proteins does not reflect the state of the living body. In the fixation step, by not performing perfusion, etc., the outflow of blood from the tissue 1 is prevented, and the tissue morphology including the distribution of free cells and extracellular proteins in the blood vessels is reflected as much as possible in the state of the living body. It can be detected in the state of being. According to this fixation step, the tissue morphology can be suitably detected even when a liver having many blood vessels and a heart having many lumens are used as the tissue 1. In particular, in the liver, there is a Disse cavity containing plasma inside in the region between the hepatocytes and the sinusoids. In conventional immunohistochemical staining, perfusion causes extracellular protein inside the Perisinusoidal space to flow out, making detailed analysis impossible. On the other hand, according to this fixing step, even the tissue morphology inside the Disse cavity can be detected.

The type of fixative is not particularly limited, but alcohol, acetone, formaldehyde, paraformaldehyde, glutaraldehyde, dimethyl subliminoate, picric acid, or a mixture thereof can be used. It is desirable that the tissue 1 is immersed in these fixing solutions within 1 minute after the tissue 1 is removed from the living body.

[Embedding process]
After the fixing step and before the section sample preparation step described later, the tissue 1 is optionally embedded with an embedding agent to obtain an embedding structure. Prior to embedding, the tissue 1 may be appropriately dehydrated and replaced by a known method. The type of embedding agent is not particularly limited, but for example, paraffin, carboxymethyl cellulose and the like can be used. The embedding method is not particularly limited, and a conventionally known method may be used.

Instead of the fixing step and the embedding step described above, the tissue 1 may be immobilized and embedded by a freezing method in which the tissue 1 is frozen. The freezing method may be carried out according to a conventionally known method. In one example, in order to prevent the formation of ice crystals in the tissue 1, the tissue 1 is first immersed in a sucrose solution. Next, the tissue 1 is immersed in formaldehyde, paraformaldehyde, or the like to dehydrate it. The dehydrated tissue 1 is then frozen with dry ice. Alternatively, the dehydrated tissue 1 is immersed in a refrigerant such as cooled acetone, hexane, isopentane, and a mixed solution thereof to freeze. When the freezing method is used, unlike the case where the tissue 1 is embedded with an embedding agent, it is not necessary to use an embedding agent solution in the section sample preparation step described later. Therefore, when the freezing method is used, a membrane 2 having low durability against the embedding agent solution can be used as the membrane 2 to which the tissue section 10 is attached.

[Section sample preparation process]
Next, an arbitrarily fixed tissue 1 is cut out and used as a tissue section 10. The method for cutting out the tissue 1 is not particularly limited, and the tissue 1 can be sliced by a known microtome or the like. Further, the thickness of the tissue section 10 is not particularly limited, and can be the same as the known technique. Next, the tissue section 10 is attached to a membrane 2 having a protein binding ability of 50 μg / cm ² or more to obtain a section sample 100.

As the membrane 2 to which the tissue section 10 is attached, a membrane 2 having a protein binding ability of ^{50 μg / cm 2 or more is used.} In conventional immunohistochemical staining, the tissue section 10 was attached on a slide glass for the tissue section. Various coatings are applied to the surface of the slide glass for the tissue section in order to enhance the affinity with the tissue section 10. However, the coating had a low binding ability to a single protein. Therefore, in the conventional slide glass for tissue sections, most of the proteins flow out of the tissue sections during the washing process of the tissue sections 10, and the tissue structure at the time of survival cannot be sufficiently maintained. On the other hand, according to the present disclosure, ^{by attaching the tissue section 10 to the membrane 2 having a protein binding ability of 50 μg / cm 2} or more, the protein contained in the tissue section 10 is retained on the membrane 2, and the tissue section 10 is retained. Since the protein can be prevented from flowing out of the tissue section 10 during the washing process, the tissue morphology can be detected while maintaining the tissue structure at the time of survival. Further, by adhering the tissue section 10 to the membrane 2, the tissue section 10 is hard to be peeled off from the membrane 2 even if the labeling treatment and the washing accompanying the labeling treatment are repeated. In conventional immunohistochemical staining, repeated labeling and subsequent washing may cause tissue sections on glass slides to come off. On the other hand, in the present disclosure, by attaching the tissue section 10 to ^{the membrane 2 having a protein binding ability of 50 μg / cm 2} or more, the same tissue section 10 can be repeatedly labeled.

The protein binding ability of the membrane 2 is preferably 70 μg / cm ² or more, more preferably 100 μg / cm ² or more, from the viewpoint of preferably retaining the tissue morphology. The upper limit of the protein binding ability of the membrane 2 is not particularly limited, but the protein binding ability of the membrane 2 is preferably 1000 μg / cm ² or less, more preferably 800 μg, in order to prevent the background from increasing during various labeling treatments. / Cm ² or less. The protein binding ability of the membrane 2 is measured as follows. 2 μL of a bovine serum albumin solution having a protein concentration of 0.2 to 30 mg / mL is added dropwise onto the membrane 2 and dried. The membrane is then washed with phosphate buffered saline for 1 minute x 3 times and then measured by detecting the amount of protein retained on the membrane 2.

The membrane 2 is not particularly limited as long as it has the above-mentioned protein-binding ability, and is, for example, a nitrocellulose (NC) membrane, a polyvinylidene fluoride (PVDF) membrane, a polymethylmethacrylate (PMMA) membrane, and a polysulfonic acid (PS). A film, a nylon film, or the like can be used. Among these, it is particularly preferable to use a PVDF membrane having a high protein binding ability. When the tissue 1 is embedded with an embedding agent, it is preferable to use an NC film or a PVDF film having high durability against the embedding agent solution.

The membrane 2 is preferably a porous membrane in order to allow the embedding agent solution or the like described later to pass through. When the membrane 2 is a porous membrane, the pore size is preferably 0.1 μm or more and 0.8 μm or less so that proteins and the like do not permeate. The thickness of the film 2 is also not particularly limited.

The tissue section 10 is placed on one side of the above-mentioned membrane 2 and attached to the membrane 2. When the tissue 1 is a frozen tissue immobilized and embedded by a freezing method, the frozen tissue is cut out to obtain a tissue section 10, and then the tissue section 10 is placed on one side of the membrane 2 and placed on the membrane 2. Attach. The tissue section 10 is preferably placed on the membrane 2 in a frozen state. By placing the tissue section 10 on the membrane 2 in a frozen state, the tissue section 10 can be attached to the membrane 2 before the tissue morphology is impaired by the thawing of the tissue section 10. The section sample 100 can be prepared in a state in which the morphology of 10 is more faithfully reflected.

When the tissue 1 is an embedded tissue embedded with an embedding agent, the embedding tissue is cut out to form an embedding tissue section, and then the embedding agent is dissolved with an embedding agent solution before the detection step described later. do. The embedding agent solution may be selected according to the type of embedding agent. The embedding agent solution is, for example, xylene, limonene, or other xylene substitute solution.

The timing of dissolving the embedding agent is not particularly limited as long as it is after preparing the embedding tissue section. Preferably, the embedding agent is dissolved after adhering the embedding tissue section to the membrane 2. By dissolving the embedding agent after adhering the embedding tissue section to the membrane 2, the embedding agent can be dissolved while retaining the morphology of the embedded tissue 1 on the membrane 2. It is possible to prevent the tissue morphology from being impaired in the process of dissolving the embedding agent.

In one embodiment, the membrane 2 is a porous membrane, and in the section sample preparation step, the embedded tissue section cut out from the embedded tissue is brought into contact with the two side surfaces of the membrane to dissolve the embedding agent. The embedding agent solution is passed from one side to the other side of the membrane 2 to attach the tissue section from which the embedding agent has been removed to the membrane 2. FIG. 3 is a diagram for explaining the direction in which the embedding agent dissolution liquid flows when the embedding agent is dissolved in the present embodiment. In the figure, arrow B indicates the flow direction of the embedding agent solution. In the present embodiment, since the membrane 2 is a porous membrane, the embedding agent solution supplied from the one-sided side in contact with the embedding tissue section 10a of the membrane 2 passes through the pores of the membrane. Pass to the other side. In conventional immunohistochemical staining, the embedded tissue section 10a is placed on a slide glass, and then the embedded tissue section 10a together with the slide glass is immersed in an embedding agent solution to dissolve the embedding agent. There is. In the conventional immunohistochemical staining, most of the proteins flowed out of the tissue section 10 in the process of dissolving this embedding agent, and the tissue morphology including the morphology between tissues could not be sufficiently maintained. On the other hand, by attaching the tissue section 10 to the porous membrane 2 instead of the slide glass, the tissue section 10 is embedded while the embedding agent solution is passed through the pores of the membrane 2. The embedding agent can be dissolved and removed. Therefore, the membrane 2 is a porous membrane, and the embedded tissue section 10a cut out from the embedded tissue is brought into contact with one side of the membrane 2, and the embedding agent solution that dissolves the embedding agent is applied to the membrane 2. By passing the tissue section 10 from one side to the other side and adhering the tissue section 10 from which the embedding agent has been removed to the membrane 2, the tissue section can be attached to the membrane without disturbing the tissue morphology by the flow of the embedding agent solution. Can be attached to. It is preferable that the other surface side of the membrane 2 (the side where the embedded tissue section 10a is not in contact) is brought into contact with a water-absorbent material 3 such as filter paper or sponge. By bringing the water-absorbent material 3 into contact with the other surface side of the membrane 2, the embedding agent solution that has passed from one side to the other side of the membrane 2 is quickly absorbed by the water-absorbent material 3. Will be done. As a result, the embedding agent solution supplied to one side of the membrane 2 does not spread on the membrane 2 but moves toward the other side of the membrane 2 in a direction substantially vertical to the membrane 2. Therefore, the embedding agent embedding the tissue section 10 can be dissolved while more preferably preventing the tissue morphology from being disturbed by the flow of the embedding agent dissolving liquid. When the embedding agent is dissolved after the embedding tissue section 10a is attached to the membrane 2 as in the present embodiment, the membrane 2 having durability against the embedding agent dissolving liquid is used as the membrane 2. ^{The flow rate of the embedding agent solution in the present embodiment is 0.5 mL / (cm 2} · min) or more in the region where the tissue section 10 is present on the membrane 2 from the viewpoint of more preferably dissolving the embedding agent. It is preferably 5, mL / (cm ² · min) or less. The time for flowing the embedding agent solution is preferably 1 min or more and 10 min or less from the viewpoint of more preferably dissolving the embedding agent.

After removing the embedding agent as described above, it is preferable to infiltrate the section sample 100 with an embedding agent dissolving solution washing solution such as alcohol in order to remove the embedding agent dissolving solution.

Prior to the detection step described later, the section sample 100 after washing the embedding agent and the embedding agent solution is placed on a slide glass, sealed with an encapsulant, and the cover glass is placed on the section sample 100. You may place it.

The unnecessary film 2 of the section sample 100 may be appropriately trimmed. The size of the section sample 100 after trimming is preferably smaller than that of the slide glass. More preferably, the size of the section sample 100 after trimming is smaller than that of a commercially available cover glass (for example, 20 mm × 30 mm or less). The trimming may be performed before the tissue section 10 is attached to the membrane 2, and may be performed at an arbitrary timing after the section sample 100 is prepared until the tissue morphology is detected.

[Detection process]
Next, the tissue morphology of the section sample 100 is detected. The tissue morphology may be detected without labeling the section sample 100, or after some labeling treatment. The type of labeling treatment is not particularly limited and may be selected according to the detection target. The labeling treatment can be immunohistochemical staining, hematoxylin / eosin (HE) staining, hematoxylin staining, in situ hybridization and the like. The immunohistochemical staining may be either fluorescent staining or dye staining. Preferably, tissue morphology is detected by dye staining. By detecting the tissue morphology by dye staining, the tissue morphology can be detected with high detection sensitivity without being affected by the autofluorescence of the section sample 100. In addition, immunohistochemical staining may be performed by a direct method or an indirect method. In the detection step, molecules such as proteins, sugar chains, nucleic acids, and fatty acids can be detected in addition to the tissue morphology. In particular, when proteins are detected by immunohistochemical staining, a calibration curve is prepared by dropping a fluorescent substance or a coloring substance having a known concentration onto the membrane 2, and the protein concentration on the section sample 100 is estimated based on the calibration curve. can do. In the detection step, the tissue morphology can be detected by using an optical microscope, a fluorescence microscope, a radioisotope detector, or the like, depending on the type of labeling treatment appropriately applied to the section sample 100.

Further, in the detection step, in addition to the method of staining the section sample 100 as described above, the tissue is stained with a radioisotope or a fluorescent dye in advance to detect the radioisotope or the fluorescent dye. The tissue containing a radioisotope, a fluorescent dye, or the like may be detected. For example, a molecule labeled with a radioisotope or a fluorescent dye is supplied to a living body from which the tissue is to be removed in advance, and the tissue containing the radioisotope or the fluorescent dye is detected by detecting the radioisotope or the fluorescent dye. be able to. In addition, the tissue containing the fluorescent protein may be detected by removing the tissue from the living body expressing the fluorescent protein by genetic recombination.

In the detection step, not only the molecules in the tissue but also the molecules between the tissues can be detected. For example, in the detection step, the extracellular protein of the section sample 100 may be detected. As described above, in the conventional immunohistochemical staining, most of the proteins have flowed out of the tissue section 10 during the washing process of the tissue section 10. On the other hand, in the present disclosure, ^{by retaining the protein contained in the tissue section 10 on the membrane 2 having a protein binding ability of 50 μg / cm 2} or more, an extracellular protein that could not be detected by conventional immunohistochemical staining. Can be detected. In recent years, as it becomes clear that the homeostasis of the living body is maintained by the organic network between tissues in the living body, between tissues by information transmission between cells, and between cells, information transmission between tissues and cells There is a need for technology to visualize substances. In the method for detecting tissue morphology of the present disclosure, extracellular proteins containing signal transmitters can be detected. Therefore, according to the method for detecting this tissue morphology, it is possible to clarify the organic network between tissues in a living body, between tissues by information transmission between cells, and between cells.

Hereinafter, an example of a procedure for performing immunohistochemical staining as a labeling treatment for the section sample 100 will be described. The section sample 100 prepared as described above is appropriately solvent-substituted with a buffer solution such as phosphate buffered saline (PBS) or water, and then the tissue section 100 on a slide glass is subjected to general immune tissue staining. Similarly, the section sample 100 is subjected to a blocking treatment. For the blocking treatment, bovine serum albumin (BSA), skim milk, and other commercially available blocking agents can be used. The antibody reaction is then performed on the section sample in the same manner that is performed on the tissue section 10 on the slide glass in general immunohistochemical staining. After the antibody reaction, the section sample 10 is placed on a slide glass, and the tissue morphology of the section sample 100 is detected with a microscope or the like.

Both a transmission type microscope and an epi-illumination type microscope can be used for detecting the tissue morphology, but it is preferable to use an epi-illumination type microscope. In the present disclosure, unlike the case where the tissue section 10 is placed on a slide glass, the tissue section 10 is attached to the membrane 2. Since the film 2 has a lower light transmittance than the slide glass, there is a possibility that the brightness required for observation may be insufficient depending on the light transmittance of the film 2 in a transmission type microscope. Therefore, in order to suitably detect the tissue morphology regardless of the light transmittance of the film, a side that does not adhere to the film 2 of the section sample 100 (the side where the film 2 does not exist) using an epi-illumination microscope. It is preferable to irradiate the section sample 100 from the above. In the epi-illumination type microscope referred to here, the light emitted to the section sample 100 is not only visible light for detecting the tissue morphology when the epi-illumination type microscope is an optical microscope, but also epi-illumination type. It also includes excitation light emitted to detect tissue morphology when the microscope is a fluorescence microscope.

The number of light sources is not particularly limited in detecting the tissue morphology, but when an epi-illumination type microscope is used, the number of light sources is preferably a plurality. The number of light sources with respect to the thickness of the film 2 is preferably 2 to 6.

Further, in detecting the tissue morphology, it is preferable to use an upright microscope instead of an inverted microscope. In the present section sample 100, since the film 2 is present on the slide glass side of the tissue section 10, in order to observe the section sample 100 using an inverted microscope, observe from the surface side of the section sample 100. Is preferable. In order to observe the section sample 100 using an inverted microscope, it is necessary to protect the surface of the section sample 100 in order to prevent the section sample 100 from being damaged by contact with the stage of the inverted microscope. be. Therefore, in order to suitably detect the tissue morphology without performing additional treatment on the section sample 100, an upright microscope is used, and the section sample 100 is sectioned from the side not attached to the film 2 by a light source. It is preferable to irradiate the sample 100.

Hereinafter, in the method for detecting the tissue morphology of the present disclosure, a case where an embryo is to be observed as the tissue 1 will be described. In the following, description of the same configuration as the above-described tissue morphology detection method will be omitted as appropriate.

[Embryo preparation process]
The tissue preparation step when the tissue 1 is an embryo is referred to as an embryo preparation step. First, in the embryo preparation step, before the fixation step described later, the embryo is prepared while suppressing the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo. Here, preparing an embryo includes both removing the embryo from the adult body and preparing an artificially generated embryo. By suppressing the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo, the outflow of substances such as proteins existing in the body fluid is prevented, and the tissue morphology is detected in a state where the structure at the time of survival is more maintained. can do. Conventionally, when immobilizing an animal embryo during immunohistochemistry, the embryo is perfused, or in the case of an immature embryo that cannot be perfused, the embryo is cut and immersed in a fixing solution. The method is adopted. In either method, it was unavoidable that intracellular cells and extracellular proteins flowed out with the outflow of blood, and the intertissue morphology could not be sufficiently detected. On the other hand, according to the embryo preparation step, by suppressing the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo, it is possible to prevent the outflow of cells and extracellular proteins in blood vessels. Intertissue morphology can be detected. The amount of body fluid outflow in the embryo preparation step is preferably 1% by mass or less, more preferably 0.2% by mass or less. More preferably, the amount of body fluid outflow is 0% by mass. The fact that the amount of body fluid outflow was 0% by mass means that when a cut was made in the embryo under a microscope, blood was used as an index of body fluid and no outflow of blood was visually observed. The amount of body fluid outflow is measured by using blood as an index of body fluid when making a cut in an embryo under a microscope. The amount of body fluid outflow is measured by sucking the blood that has flowed out from the tissue 1 and analyzing it by quantitative PCR (polymerase chain reaction). First, the blood flowing out of the tissue 1 is sucked up with a micropipette or a syringe. When the amount of blood flowing out is so small that it cannot be sucked up by a micropipette or a syringe, a cellulose membrane is applied to the cut portion of the tissue 1 to suck up the blood flowing out from the tissue 1. Next, the sucked blood is dissolved in physiological saline, or the cellulose membrane containing blood is immersed in physiological saline to dissolve or elute the blood in physiological saline to obtain a sample for blood measurement. .. On the other hand, 0.01 to 10 μL of blood of the same species as the sample is dissolved in physiological saline to obtain a control sample. Quantitative PCR is performed on this control sample to prepare a calibration curve showing the relationship between a specific gene amount and blood volume. The blood measurement sample is also subjected to quantitative PCR in the same manner, and the blood volume in the blood measurement sample is calculated using the above calibration curve. In addition to GAPDH, genes to be detected by quantitative PCR may be 18S rRNA, β-actin, peptidylprolyl isomerase A, GAPDH, β-2-microglobulin, β-glucuronidase, TATA box binding protein and the like. Quantitative PCR is performed using various primers for quantitative PCR that specifically bind to these gene sequences. In addition, quantitative PCR can be performed using universal primers that do not limit the target sequence to a specific gene.

In the embryo preparation step, as long as the amount of body fluid outflow from the embryo is suppressed to 1% by mass or less of the embryo, the embryo may be damaged. In order to suppress the outflow of body fluid to 1% by mass or less of the embryo, it is preferable not to damage the blood vessels and internal organs in the embryo preparation step. In order to suppress the outflow of body fluid to 1% by mass or less of the embryo, it is more preferable not to damage the artery in the embryo preparation step. In addition, in order to suppress the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo, it is preferable to immerse the embryo in physiological saline and perform the work under a microscope.

In the embryo preparation step, a cut may be made in at least one place of the embryo. At this time, the amount of body fluid outflow from the embryo is suppressed to 1% by mass or less of the embryo throughout the embryo preparation step. Preventing the outflow of cells and extracellular proteins in blood vessels by making cuts in at least one part of the embryo while keeping the amount of fluid outflow from the embryo to 1% by mass or less of the embryo throughout the embryo preparation process. In the subsequent fixation step, the fixation solution can be easily immersed in the embryo, and the time required for embryo fixation can be shortened. The number, length, depth, position, and range of the cuts are not particularly limited and may be determined according to the type of embryo to be used. For example, if the embryo is a mouse embryo after the 13th day of gestation, it is preferable to insert the cut so that the embryo is laparotomized. Laparotomy of an embryo means cutting the abdominal surface of the embryo from the head to the tail with a scalpel to expose the internal organs. In one example, a cut is made along the dotted line in FIG. 4 to open the embryo. By making a cut so as to open the embryo, the fixing solution can be suitably flowed into the abdomen or the chest, and the time required for embryo fixation can be further shortened.

[Fixing process] to [Detection process]
Examples of the fixing step, the embedding step, the section sample preparation step, and the detection step include the same steps as described above, and the preferred step described above is preferable.

The tissue morphology detection method of the present disclosure can also be applied when the observation target is not an embryo but a mature adult.

[Image acquisition device]
Next, an image acquisition device that can use the above-mentioned tissue morphology detection method will be described. FIG. 5 is a functional block diagram showing an example of the basic configuration of the image acquisition device. As shown in FIG. 5, the tissue morphology image acquisition device 200 includes a section sample portion 5 for preparing a section sample in which a ^{tissue section 10 is attached to a membrane 2 having a protein binding ability of 50 μg / cm 2 or more, and a section sample 100.} It has a detection unit 6 for detecting the tissue morphology of the above, and detects the tissue morphology. Further, the image acquisition device 200 optionally includes an image processing unit 11 including a position information acquisition unit 7, a trace unit 8, and a storage unit 9.

The outline of the operation of the image acquisition device 200 will be described. The section sample section 5 prepares the section sample 10 by adhering the tissue section 100 to the membrane 2 having a protein binding ability of 50 μg / cm ^{2 or more.} The detection unit 6 detects the tissue morphology of the section sample that has been appropriately labeled. The detection unit 6 acquires the detection result as an image and supplies the image to the storage unit 9.

Next, each component of the image acquisition device 200 described above will be specifically described.

The section sample section 5 includes a section sample preparation section and an observation table. The section sample section 5 prepares a section sample 100 by adhering the tissue section 10 to the membrane 2 having a protein binding ability of 50 μg / cm ^{2 or more.} The method for preparing the section sample 100 is as described in the section sample preparation step of the method for detecting the tissue morphology. In one example, the section sample preparation section includes a tissue preparation section provided with an extraction device for preparing the tissue 1, a fixing section provided with a fixing tank for fixing the tissue 1, and a packet for embedding the tissue 1. It includes an embedding part provided with a burial tank, a tissue section preparation part provided with a microtome for cutting out the embedding tissue, and a section sample placing part for attaching the tissue section 10 to the membrane. The image acquisition device 200 appropriately uses a tissue preparation section for preparing the tissue 1 using the above-mentioned tissue preparation step, a fixing section for fixing the tissue using the above-mentioned fixing step, and a structure using the above-mentioned embedding step. It may have an embedding portion for embedding. The observation table is a table on which the prepared section sample 100 is placed for observation. The observation table can be constructed in the same manner as a general microscope stage in one example.

The detection unit 6 detects the tissue morphology of the section sample 100 placed on the observation table. The method for detecting the tissue morphology of the section sample 100 is as described above. The detection unit 6 can be a protein detection unit that detects the protein of the tissue section 10. The protein detection unit may detect the extracellular protein of the tissue section 10. The method for detecting the protein in the tissue section 10 is as described above. FIG. 6 shows an example of the schematic configuration of the detection unit 6. As shown in FIG. 6, the detection unit 6 may include a light receiving optical system including a lens 12 and a light receiving unit (camera) 13. In the illustrated example, the section sample 100 is placed on the slide glass 16 and observed by the detection unit 6. Further, the detection unit 6 may optionally include various excitation light sources 14 for detecting fluorescence, a condenser lens 15, a bandpass filter (BF), and a dichroic mirror (DM). More specifically, the detection unit 6 may be a microscope provided with a camera. Since the preferable configuration of the microscope has been described above, the description thereof is omitted here.

The storage unit 9 may be, for example, a non-volatile memory or storage medium such as an SSD (Solid State Drive), an HDD (Hard Disk Drive), or an optical disk, which can be written and read at any time. The storage unit 9 may be provided in the image acquisition device 200, or may be provided on a computer connected to the image acquisition device 200 via a network.

As described above, the image processing unit 11 has a CPU (Central Processing Unit) and a program memory constituting the computer, and has the position information acquisition unit 7 and the position information acquisition unit 7 as parts for performing the control necessary for carrying out the present embodiment. A trace unit 8 is provided. All of these parts are realized by causing the CPU to execute the program stored in the program memory.

The image acquisition device 200 according to one embodiment refers to each image by referring to an image acquisition unit that acquires a different image by imaging the section sample subjected to different labeling treatment on the same section sample and the different image. It has a position information acquisition unit 7 for acquiring the position information of the tissue section 10 inside, and a trace unit 8 for superimposing different images with reference to the position information. Here, the term "labeled" includes the case where the section sample is not labeled at all.

An outline of the operation of the image acquisition device 200 according to the present embodiment will be described. First, the section sample section 5 prepares a section sample 100 in which the tissue section 10 is attached to the membrane 2 having a protein binding ability of 50 μg / cm ^{2 or more (step S200).} Next, the detection unit 6 detects the tissue morphology of the section sample 100 subjected to the first labeling treatment. The detection unit 6 acquires the detection result as a first image and supplies the first image to the storage unit 9 (step S202).

Next, the same section sample 100 is subjected to the second labeling treatment. The second labeling process may be performed by the detection unit 6, another device, or a manual operation. The detection unit 6 detects the tissue morphology of the section sample 100 subjected to the second labeling treatment. The detection unit 6 acquires the detection result as a second image and supplies the second image to the storage unit 9 (step S204). The storage unit 9 supplies the first image and the second image to the position information acquisition unit 7. The position information acquisition unit 7 refers to the first image and the second image, respectively, and acquires the position information of the tissue section 10 in each image in each of the first image and the second image. The position information acquisition unit 7 supplies the acquired position information to the trace unit 8 (step S206). The trace unit 8 superimposes the first image and the second image with reference to the position information of the tissue section 10 in the first image and the second image (step S208). The trace unit 8 appropriately supplies an image obtained by superimposing the first image and the second image to the storage unit 9. In this way, the image acquisition unit captures the same section sample 100 with different labeling treatments to obtain different images (first image and second image in this example). By superimposing the images on the trace unit 8, the detection results of different labeling processes can be evaluated in correspondence with each other. For example, by superimposing a plurality of images in which the proteins to be detected are different from each other, the distributions of the plurality of proteins in the tissue section 10 can be evaluated in correspondence with each other. In the above description, the example in which the image acquisition unit acquires the first image and the second image has been described, but the number of images acquired by the image acquisition unit is not particularly limited as long as it is plural.

The trace unit 8 may move and rotate the XY position of one image among the plurality of superposed different images, or enlarge or reduce one image to more accurately superimpose the plurality of images. ..

Further, the image processing unit 11 may have a difference generation unit that refers to a plurality of different images superimposed by the trace unit 8 and takes a difference between the plurality of different images. The difference generation unit supplies the generated difference to the storage unit as appropriate. Further, the image processing unit 11 provides an image correction unit that adjusts brightness, contrast, and gamma correction for each image acquired by the detection unit 6 and a plurality of different images superimposed by the trace unit 8. You may also have more. The image correction unit appropriately supplies the corrected image to the storage unit.

When the section sample 100 subjected to different labeling treatments is imaged to obtain different images, the position of the section sample 100 on the microscope may be fixed by using an auxiliary positioning jig. By fixing the position of the section sample 100 on the microscope using the positioning jig in this way, the section sample 100 can be fixed at the same position on the microscope before and after the different labeling treatments, so that they are the same. When observed at the same magnification and in the field of view, the positions of the section sample 100 are almost the same in the different images obtained by imaging the section sample 100 subjected to different labeling treatments. Therefore, the position information acquisition unit 7 can acquire the position information of the section sample 100 more accurately based on the different images, and can superimpose the different images more accurately. FIG. 12 shows an outline of a method for positioning the section sample 100 on a microscope using the positioning jig. First, the section sample 100 from which the film 2 has been trimmed is placed on a slide glass 20, and the slide glass 20 is fixed at a predetermined position A on the positioning jig 22 according to a method described later. Next, by inserting the positioning pin 17 through the positioning pin hole 18a provided in the positioning jig 22 and the positioning pin hole 18b provided in the stage 19 of the microscope, the positioning jig 22 is inserted into the stage 19 of the microscope. It can be fixed at the predetermined position B above, and thus the section sample 100 can be fixed at the predetermined position on the microscope.

A method of fixing the section sample 100 at a predetermined position on the positioning jig 22 will be described with reference to FIG. The positioning jig 22 has at least one positioning guide 21. The positioning guide 21 suppresses the movement of the slide glass 20 pressed from the two directions indicated by the arrows in the drawing in the X-axis direction and the Y-axis direction, and suppresses the movement of the slide glass 20 on the positioning jig 22 in the X-axis direction and the X-axis direction. The position in the Y-axis direction is fixed. In the figure, an example in which the positioning guide 21 is an L-shaped frame-shaped member is shown, but the form of the positioning guide 21 is particularly limited as long as the positions of the slide glass 20 in the X-axis direction and the Y-axis direction can be fixed. Not done. For example, the positioning guide 21 is separated from the first plate-shaped member that suppresses the movement of the slide glass 20 in the X-axis direction and the first plate-shaped member, and is perpendicular to the first plate-shaped member. A second plate-shaped member provided in the above may be included.

A film with a protein binding capacity of 50 μg / cm ² or more (PVDF; manufactured by Bio-Rad), a slide glass with no surface coating (white edge polishing; manufactured by Matsunami Glass), and a conventional structure coated on the surface. The amount of protein binding on the surface of the slide glass for section (MAS coated slide glass; manufactured by Matsunami Glass) was examined before and after washing. On a membrane or a slide glass, 2 μL of each of the colored protein solution (BSA / DAB / PBS solution) prepared with a dilution series of 8 points in the range of 0.2 to 30 mg / ml was added dropwise and dried. Next, the film and the slide glass were imaged, and a calibration curve was created to calculate the BSA concentration on the film or the slide glass from the shade of color of the dropped solution. Next, 2 μL of 1 to 30 mg / ml BSA / DAB / PBS was added dropwise onto the film or slide glass in the same manner as above, and the film and slide glass were imaged while being allowed to stand for 1 min under wet conditions. Then, it was washed with PBS for 1 minute × 3 times, and the film and the slide glass after washing were photographed again. Based on the shade of color of the dropped solution, the BSA concentration on the membrane or slide glass was calculated based on the prepared calibration curve, and used as the amount of protein after washing on the membrane or slide glass. The amount of protein before washing was taken as the initial amount of protein, and the ratio of the amount of protein on the membrane or slide glass after washing to the initial amount of protein was determined. The results are shown in FIG. As shown in FIG. 7, most of the protein was retained even after washing on the membrane having a protein binding ability of 50 μg / cm ^{2 or more.} On the other hand, it was found that most of the proteins were washed away after washing in the uncoated slide glass and the conventional slide glass for tissue sections coated on the surface.

[Example 1: Observation of mouse tissue by HE staining of liver tissue]
<Invention example>
The tissue morphology of the liver tissue of mouse embryos was detected by using the tissue morphology detection method and the image acquisition device according to the present disclosure. First, mouse 16.5 day embryos were removed from adults, and mouse embryos were opened so that the amount of body fluid outflow from the embryos was 0% by mass. After laparotomy, mouse embryos were immersed in formalin and fixed. Mouse embryos were removed from the fixation solution and embedded with paraffin to give an embedded tissue. Using a microtome, an 8 μm-thick embedded tissue section containing liver tissue was obtained. The embedded tissue section was placed on one side of a PVDF membrane having a pore size of 0.22 μm and a thickness of 0.5 mm. On the other side of the PVDF film, three 2 mm filter papers were laid on top of each other. ^{Remosol A was supplied at a flow rate of 0.5 mL / (cm 2} · min) from one side in contact with the embedded tissue section of the membrane to dissolve the paraffin embedding the tissue section. The section sample was then washed with running water. The section sample was solvent replaced with distilled water. Section samples were immersed in Meyer's hematoxylin solution for 4 minutes. The section samples were then washed with running water for 15 minutes. Section samples were immersed in 1.0% eosin solution for 2 minutes. Section samples were washed with 70% ethanol. Section samples were further washed with Remosol A. An unnecessary part of the PVDF membrane of the section sample was trimmed to obtain a trimmed sample, leaving the part to which the tissue section was attached. Next, the trimmed sample was placed on a slide glass (white edge polishing frost; manufactured by Matsunami Glass). The trimmed sample was sealed with a water-soluble mounting medium and covered with a cover glass. The tissue morphology was detected by an epi-illumination type upright microscope (M7000 manufactured by Thermo Fisher Scientific) with two light sources. The detection result is shown in FIG.

<Comparison example>
Rather than opening the abdomen and then dipping it in formalin to fix it, the mouse embryo was cut and then washed with PBS and then soaked in formalin and fixed; a glass slide without attaching tissue sections to the membrane. Placed on (MAS coated slide glass; made by Matsunami Glass); and when the paraffin is dissolved, a filter paper is laid on the other side of the PVDF film and instead of flowing Remosol A, it is wrapped in a container containing Remosol A. The tissue morphology of the liver tissue of the mouse embryo was detected in the same manner as in the invention except that the slide glass on which the buried tissue section was placed was immersed 3 times for 1 minute. The detection result is shown in FIG. In addition, in FIGS. 8 and 9, the stained part is shown in dark color.

As shown in FIG. 9, in the example of the invention, blood cell cells remaining in the blood vessel could be detected. On the other hand, as shown in FIG. 8, in the comparative example, most of the blood cell cells in the blood vessel flowed out of the section sample.

[Example 2: Observation of mouse embryo liver tissue by immunohistochemical staining]
Using the tissue morphology detection method and image acquisition device according to the present disclosure, the liver tissue of mouse embryos was observed with OSM antibody. First, mouse embryos on the 18th day of pregnancy were removed from adult BALB / c mice, and the mouse embryos were opened so that the amount of body fluid outflow from the embryos was 0% by mass. After laparotomy, mouse embryos were immersed in 30% paraformaldehyde / phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) for immobilization treatment. Mouse embryos were removed from the fixation solution and embedded with paraffin to give an embedded tissue. Using a microtome, an 8 μm-thick embedded tissue section containing liver tissue was obtained. The embedded tissue section was placed on one side of a PVDF membrane having a pore size of 0.22 μm and a thickness of 0.5 mm. On the other side of the PVDF film, three 2 mm filter papers were laid on top of each other. Xylene was supplied from one side in contact with the embedded tissue section of the membrane to dissolve the paraffin embedding the tissue section. The section sample was then washed with alcohol. After the section sample was solvent-substituted with PBS, a few drops of Peroxizard 1 (manufactured by Funakoshi Co., Ltd.) were added dropwise onto the sample, and the sample was treated at room temperature for 10 minutes to remove the endogenous peroxidase in the section sample. Next, 30 μL to 100 μL of 1% BSA / PBS was added dropwise, and blocking treatment was performed at room temperature for 60 minutes. An OSM antibody (manufactured by Abcam) was diluted 200-fold with 1% BSA / PBS to prepare an antibody diluent. The antibody diluent was added dropwise onto the section sample and reacted at 4 ° C. overnight. Section samples were washed with PBS at room temperature for 5 minutes x 3 times. As the secondary antibody, anti-rabbit POD conjugated (manufactured by Takara Bio Inc.) was used. This secondary antibody solution was added dropwise onto the section sample, reacted at room temperature for 1 hour, and washed with PBS at room temperature for 5 minutes × 3 times. Next, a DAB substrate (manufactured by Takara Bio Inc.) was added dropwise to develop color, and then the cells were washed with PBS at room temperature for 3 minutes × 3 times. After washing, the sample was trimmed to obtain a trimmed sample, leaving the portion of the membrane containing the section of the section sample. The trimmed sample was placed on a slide glass (white edge polishing frost; manufactured by Matsunami Glass), the trimmed sample was sealed with a water-soluble mounting medium, and a cover glass was covered. The tissue morphology was detected by an epi-illumination type upright microscope (M7000 manufactured by Thermo Fisher Scientific) with two light sources. The detection results are shown in FIGS. 10 and 11. FIG. 10 is a 4x observation image, and FIG. 11 is a 40x observation image. In addition, in FIG. 10 and FIG. 11, the stained portion is shown in dark color.

In FIG. 10, the liver is shown in the center. In FIG. 11, the liver portion of FIG. 10 is enlarged and shown, and the dotted line portion indicates a blood vessel. As is clear from FIG. 11, according to the method for detecting this tissue morphology, cells in blood vessels can also be detected.

[Example 3: Detection of tissue morphology of liver tissue using an image acquisition device having a position information acquisition means and a tracing means]
In the same manner as in Example 2 described above, a pre-stained section sample containing liver tissue was prepared from mouse embryos on the 18th day of pregnancy. After performing a primary antibody reaction and washing with an HGF antibody (manufactured by Abcam) diluted 200-fold with 1% BSA / PBS with respect to the section sample before staining, the portion containing the tissue section in the membrane. A section sample (trimmed sample) trimmed to a size of 20 mm × 40 mm or less was prepared, and image 1 before staining was acquired with an epi-illumination type upright microscope (M7000 manufactured by Thermo Fisher Scientific) using two light sources. Next, after performing color staining with the secondary antibody reaction and DAB in the same procedure as in Example 2, image 2 after staining was acquired at the same position and imaging conditions as before staining. In order to prevent the trimmed sample from drying during imaging of images 1 and 2, the trimmed sample after cleaning is placed on a general-purpose slide glass (white edge polishing frost; manufactured by Matsunami Glass) in a wet state, and the trimmed sample is placed on it. A 24 mm x 50 mm gaped cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) was placed so as to cover the section sample, and imaging was performed with the gap filled with 1% BSA / BPS. The background was removed or reduced by subtracting the image 1 (first image) before staining from the image 2 (second image) after staining. Specifically, a microscope and an imaging system (CQ-1 manufactured by Yokogawa Electric Co., Ltd.) that can set the Z direction of the stage in 1 μm units and can set the amount of light to irradiate the sample and the exposure time at the time of imaging. used. A positioning jig capable of fixing the XY position of the section sample was installed on the stage of the microscope described above as shown in FIG. 12, and the section sample was fixed at a specific position by the positioning guide shown in FIG. Image 1 of the section sample before staining was obtained under these conditions. Next, the section sample was subjected to a primary / secondary antibody reaction and color staining with DAB in the same manner as in Example 2 described above, and then a washing operation was performed. The section sample after color staining was fixed again at the same XY position and Z position as when the image 1 was acquired by using the positioning jig described above, and the same imaging conditions as the image 1 (same magnification, field of view, light intensity, Image 2 was obtained by imaging at (exposure time). Next, with respect to the acquired images 1 and 2, the image processing software Cell Path finder (manufactured by Yokogawa Electric Co., Ltd.) or Image J (manufactured by NIH) having a position information acquisition unit and a trace unit is used in the section sample. After accurately superimposing the XY positions of the tissue sections, the difference between the two images was taken and the black and white of the image was inverted to obtain image 3. The observation results of images 1 to 3 are shown in FIG.

In image 2 of FIG. 14, the stained area is shown as dark color, and in image 3, the stained area is shown as light color. Before taking the difference between the two images, as shown by the arrows in images 1 and 2 of FIG. 14, the background due to autofluorescence that develops color is observed both before and after staining. However, by taking the difference between images 1 and 2 using image processing software having a position information acquisition unit and a trace unit, extracellular HGF in the tissue section is significantly reduced while the background as shown by the arrow is significantly reduced. The protein could be detected (Image 3).

The tissue morphology detection method and image acquisition device according to the present disclosure can be used for analysis of the tissue morphology of a living body.

1 Tissue 10 Tissue section 10a Embedded tissue section 100 Section sample 2 Film 3 Water-absorbent material 5 Section sample part 6 Detection part 7 Position information acquisition part 8 Trace part 9 Storage part 11 Image processing part 12 Lens 13 Camera 14 Excitation light source 15 Condensing lens 16 Slide glass 17 Positioning pin 18a, b Positioning pin hole 19 Microscope stage 20 Slide glass 21 Positioning guide 22 Positioning jig 200 Image acquisition device A position B position

Claims (9)

A section sample preparation step of attaching a tissue section to a membrane having a protein binding ability of 50 μg / cm ² or more to prepare a section sample, and a step of preparing a section sample.
A detection step for detecting the tissue morphology of the section sample and
A method for detecting tissue morphology. The method for detecting a tissue morphology according to claim 1, wherein in the detection step, the extracellular protein of the section sample is detected. The method for detecting a tissue morphology according to claim 1 or 2, further comprising a fixing step of immersing the tissue in a fixing solution before the section sample preparation step. After the fixing step and before the section sample preparation step, there is an embedding step of embedding the tissue with an embedding agent to obtain an embedding structure.
The membrane is a porous membrane,
In the section sample preparation step, an embedding tissue section cut out from the embedding tissue is brought into contact with one side of the membrane, and an embedding agent solution for dissolving the embedding agent is applied from the one side to the other side of the membrane. The method for detecting a tissue morphology according to claim 3, wherein the tissue section from which the embedding agent has been removed is attached to the membrane by passing it to the side. The tissue section is the tissue section of the embryo.
The method for detecting a tissue morphology according to claim 3 or 4, further comprising an embryo preparation step of preparing the embryo while suppressing the amount of body fluid outflow from the embryo to 1% by mass or less of the embryo before the fixation step. The method for detecting a tissue morphology according to claim 5, wherein in the embryo preparation step, a cut is made in at least one place of the embryo. A section sample portion in which a section sample in which a tissue section is attached to a membrane having a protein binding ability of 50 μg / cm ^{2 or more is prepared, and a section sample portion.}
It has a detection unit that detects the tissue morphology of the section sample.
An image acquisition device for tissue morphology that detects tissue morphology. The image acquisition device for a tissue morphology according to claim 7, further comprising a protein detection unit for detecting extracellular proteins in the tissue section. An image acquisition unit that obtains different images by imaging the same section sample that has been subjected to different labeling treatments.
A position information acquisition unit that acquires the position information of the tissue section in each image by referring to the different images, and
With reference to the position information, a trace unit that superimposes different images and
The image acquisition device for the tissue form according to claim 7 or 8.

Images