Covering tool for observation sample, covering tool package, and covering method for observation sample

Abstract

The present invention provides a covering device (10) for observing a sample, characterized in that it comprises: an ultra-thin film (1) for covering the observed sample; and a main body (2) having a retaining portion (3) formed with an opening portion (4), wherein the ultra-thin film (1) is formed to be larger than the opening portion (4), and the ultra-thin film (1) is retained on the retaining portion (3) by being adhered to at least a portion of the upper surface and side surfaces of the retaining portion (3) by physical adsorption in a manner that seals the opening portion (4).

Description

Technical Field

[0001] The present invention relates to a covering tool for observation samples, a covering tool package, and a covering method for observation samples. Background Art

[0002] Microscope imaging technology is constantly improving, and it is an indispensable observation method for visualizing life phenomena on site and obtaining real-time information. Taking the development of two-photon excitation microscopes, total internal reflection fluorescence microscopes, and super-resolution microscopes as examples, there are amazing technologies in the development of hardware (microscope body, observation accuracy). In addition, recently, reagents that can make biological tissues such as internal organs transparent have been developed (non-patent literature 1, 2), making deep imaging of specific proteins in biological tissues possible, and the demand for imaging the entire biological tissue, which was previously impossible, has increased dramatically.

[0003] Here, for ease of explanation, the observation sample and the observation specimen are first defined. The observation sample is set to be a cell or biological tissue, or a cell or biological tissue placed on an observation substrate or a holder, and the sample covered with the observation sample by an ultra-thin film is defined as the observation specimen.

[0004] On the other hand, for the preparation of observation samples (software side), in the prior art, the cells or biological tissues to be observed are transparentized using a transparent reagent (non-patent literature 1, 2), and the biological tissues are placed on a cover glass (observation substrate). However, since the observation sample cannot prevent the drying of cells or biological tissues, the image is blurred due to inertial force, making it difficult to observe for a long time and obtain high-resolution images. For the drying problem of the prior art, it can be avoided by wrapping cells or biological tissues with hydrogels, but the transparent cells or biological tissues return to their original opaque state. Therefore, the following method is developed: the cells or biological tissues to be observed are placed on a cover glass (observation substrate), and the method of covering them with an ultra-thin film from above (observation specimen). By doing so, it is possible to observe for a long time while maintaining transparency. For the observation sample, a microscope is used to observe from the cover glass (observation substrate) side. In addition, the following method is also proposed: using an ultra-thin film as an observation substrate, covering cells or biological tissues with an ultra-thin film, and observing from the ultra-thin film side using a microscope. By this method, when observing with a microscope, there is no optical obstacle like a coverslip, so it is possible to observe the deeper part of cells or biological tissues. (Non-patent literature 3) In this way, when imaging with a microscope, a demand for covering with an ultra-thin film is generated. However, the ultra-thin film before covering is immersed in an aqueous solvent. Therefore, when the ultra-thin film is taken out from the aqueous solvent into the atmosphere with tweezers, etc., the ultra-thin film is distorted, and it is difficult to take it out in a flat state. As a result, there is the following problem: it is very difficult to cover the entire cell or biological tissue of the object with an ultra-thin film.

[0005] In order to solve this problem, the following method is proposed: a non-woven fabric is used as a substrate, an ultra-thin film is provided on it, the ultra-thin film side of the non-woven fabric is attached to a covering body, and the ultra-thin film is transferred to the covering body by rubbing the non-woven fabric with the fingertips. (Patent Document 1)

[0006] In addition, the following steps are performed: floating the ultra-thin film on an aqueous solvent, placing biological tissue on the ultra-thin film, and then placing a cover glass (observation substrate) from above it, and covering the biological tissue and the cover glass (observation substrate) with the ultra-thin film (non-patent document 4).

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: H. Hama, et al., Nat. Neurosci., 14, 1481 (2011)

[0010] Non-patent document 2: H. Hama, et al., Nat. Neurosci., 18, 1518 (2015)

[0011] Non-patent literature 3: Preparation of waterproof ultra-thin films and establishment of a cover glass-free deep biological imaging method, 8th CSJ Chemistry Festival 2018, student poster presentation, field: 2. Inorganic Chemistry / Analytical Chemistry, presentation number: P2-072, held on October 23, 2018

[0012] Non-patent document 4: Y. Okamura, et al., Adv. Mater., 29, 1703139 (2017)

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent Application Publication No. 2017-164930. Summary of the invention

[0015] Technical problem to be solved by the invention

[0016] However, the transfer method of the ultra-thin film described in Patent Document 1 can be transferred to the surface of the observation sample, but it is difficult to use the ultra-thin film to wrap the back of the observation sample to prepare the observation specimen. In addition, in order to transfer the ultra-thin film, it is necessary to rub the non-woven fabric side, so there is a possibility that cells or biological tissues are crushed, or cells or biological tissues are displaced from the specified position of the cover glass (observation substrate), and there is a possibility of pressure on the cells or biological tissues to be observed.

[0017] In addition, the method for preparing the observation specimen described in non-patent document 4 is troublesome and requires proficiency. In addition, there is a possibility that before the biological tissue and the cover glass (observation substrate) are covered with the ultra-thin film, the aqueous solvent exceeding the required amount enters between the cover glass (observation substrate) and the ultra-thin film, thereby affecting the cell or biological tissue environment to be observed. In addition, when the biological tissue is heavy, the deformation of the ultra-thin film downward sometimes becomes larger, and the biological tissue falls into the aqueous solvent, and the observation specimen cannot be prepared.

[0018] In addition, researchers observing cells or biological tissues need to obtain ultra-thin films in order to produce such observation specimens. However, in order to transport the ultra-thin films, in addition to providing them using non-woven fabrics with ultra-thin films attached as described above, they can only provide films with sacrificial layers and substrates used in the manufacture of ultra-thin films, or provide them for each solvent in a state where the ultra-thin film is floated in the solvent. This makes the transportation and distribution of the ultra-thin films very difficult.

[0019] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an observation sample covering tool, a covering tool package, and an observation sample covering method that are easy to work when preparing an observation sample.

[0020] Technical solutions for solving technical problems

[0021] In order to solve the above-mentioned problems, the covering device for observation sample involved in the present invention is characterized in that it comprises: an ultra-thin film for covering the observation sample; and a main body, having a retaining portion formed with an opening, the ultra-thin film is formed to be larger than the opening, and the ultra-thin film is retained on the retaining portion by physical adsorption and adhered to at least a part of the upper surface and side surfaces of the retaining portion in a manner that seals the opening.

[0022] According to such structure, the ultrathin film after sacrificial layer and substrate can be utilized immediately. In addition, the ultrathin film is adhered and held on the holding portion by physical adsorption, so when observing the preparation of the specimen, the observation sample can be easily covered without distortion etc. in the ultrathin film.

[0023] In the observation sample covering tool according to the present invention, the main body preferably includes a plate portion constituting the holding portion and having a through hole formed in the plate portion as the opening portion, and a wall portion extending downward from an outer periphery of the plate portion.

[0024] According to such a configuration, when the ultra-thin film floating on the solvent is scooped up and taken out by the main body, the wall portion is used to support the ultra-thin film, thereby facilitating the scooping operation of the ultra-thin film.

[0025] In the sample observation covering tool according to the present invention, preferably, the main body includes: a frame portion in which the holding portion is constituted by a wire member and the wire member is arranged in a frame shape as the opening; and a handle portion connected to the frame portion.

[0026] According to such a configuration, when the ultra-thin film floating on the solvent is scooped up by the main body and taken out, the handle is held, thereby facilitating the scooping operation of the ultra-thin film.

[0027] In the covering tool for observing samples according to the present invention, preferably, the holding portion has a step portion forming a recessed portion on the upper surface side where the ultra-thin film is held, the step portion is formed on the outer peripheral side of the holding portion closer to the outside than the opening portion, and the opening portion is opened on the bottom surface of the recessed portion.

[0028] According to such a structure, since the holding part has the step part, when preparing the observation specimen, even if the observation sample covering tool is placed on the workbench, it can stand on its own, and the ultra-thin film in the area covered with the observation sample will not stick to the workbench.

[0029] In the sample observation covering tool according to the present invention, it is preferred that the main body is made of metal, glass or resin, and the main body is insoluble in a solvent selected from the group consisting of aqueous solvents and organic solvents.

[0030] According to such a structure, when the ultra-thin film floating in the solvent is scooped up by the main body, the main body will not be dissolved in the solvent and deformed or disappear.

[0031] The observation sample covering tool according to the present invention preferably further comprises at least one of a lid portion covering the upper surface side of the holding portion and a receiving portion covering the lower surface side of the holding portion.

[0032] According to such a structure, at least one of the cover portion and the receiving portion covers at least one of the upper surface and the lower surface of the ultra-thin film, so dust or dirt does not adhere to the ultra-thin film.

[0033] In the observation sample covering tool according to the present invention, it is preferable that the opening is formed larger than the observation sample.

[0034] According to such a configuration, when preparing the observation sample, the observation sample covering tool can be easily moved to the lower side of the observation sample, so that the lower surface of the observation sample can be covered with the ultra-thin film.

[0035] The covering tool package according to the present invention preferably comprises: the observation sample covering tool; and a packaging bag that wraps the observation sample covering tool from the outside.

[0036] According to such a structure, since the packaging bag covers the observation sample covering tool, it is possible to prevent the ultra-thin film from being broken during transportation. In addition, if a disinfection bag is used for the packaging bag and the observation sample covering tool that has been sterilized is wrapped, a sterilized observation sample covering tool can be provided. Furthermore, if at least one surface of the disinfection bag is a material that transmits ultraviolet rays, after packaging with the disinfection bag, the observation sample covering tool can be irradiated with ultraviolet rays to sterilize it.

[0037] The covering method of the observation sample involved in the present invention preferably includes the following steps: a step of preparing the covering tool for the observation sample and the observation sample; and a covering step of arranging the covering tool for the observation sample on the observation sample and covering the observation sample with the ultra-thin film.

[0038] According to such a method, the step of removing the sacrificial layer and the base material from the ultra-thin film with a solvent can be omitted, and covering the observation sample becomes easy.

[0039] Effects of the Invention

[0040] According to the observation sample covering tool, the covering tool package, and the observation sample covering method according to the present invention, the work is facilitated when preparing the observation sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A It is a perspective view showing the structure of a covering tool for observing a sample.

[0042] Figure 1B yes Figure 1A A cross-sectional view of the IB-IB line.

[0043] Figure 2 This is a cross-sectional view of a covering tool for observation samples equipped with a cover material.

[0044] Figure 3 It is a cross-sectional view showing the structure of a covering tool package.

[0045] Figure 4 This is a flowchart showing the steps of a method for manufacturing a covering tool for an observation sample.

[0046] Figure 5A This is a perspective view schematically showing a substrate prepared in a substrate preparation step of a method for producing a covering tool for an observation sample.

[0047] Figure 5B This is a perspective view schematically showing a substrate after a sacrificial layer is formed in the sacrificial layer forming step of the method for producing the observation sample covering tool.

[0048] Figure 5C This is a perspective view schematically showing a substrate after an ultra-thin film is formed on a sacrificial layer in the ultra-thin film forming step of the method for manufacturing a cover tool for an observation sample.

[0049] Figure 5D This is a perspective view schematically showing an ultra-thin film immersed in a solvent in the ultra-thin film immersion step of the method for producing a cover for an observation sample.

[0050] Figure 5E This is a partially cutaway perspective view schematically showing a state in which an ultra-thin film immersed in a solvent is scooped up by a main body in an ultra-thin film taking-out step of a method for producing a covering tool for an observation sample.

[0051] Figure 6 This is a flowchart showing the process of the covering method of the observation sample.

[0052] Fig. 7A This is a cross-sectional view schematically showing a covering tool and an observation sample prepared in a preparation step of a covering method for an observation sample.

[0053] Figure 7BThis is a cross-sectional view schematically showing an observation sample after being covered with an ultra-thin film in a covering step of a covering method of an observation sample.

[0054] Fig. 8A It is a cross-sectional view schematically showing another form of a covering tool and an observation sample prepared in the preparation step of the observation sample covering method.

[0055] Figure 8B This is a cross-sectional view schematically showing another form of the observation sample after being covered with an ultra-thin film in the covering step of the observation sample covering method.

[0056] Fig. 9 This is a cross-sectional view schematically showing another form of a covering tool and an observation sample prepared in the preparation step of the observation sample covering method.

[0057] Fig.10 This is a cross-sectional view schematically showing another form of a covering tool and an observation sample prepared in the preparation step of the observation sample covering method.

[0058] Fig.11 This is a perspective view showing the structure of another form of the cover for observing the sample.

[0059] Fig. 12A This is a perspective view showing the structure of another form of the cover for observing the sample.

[0060] Fig. 12B yes Fig. 12A A cross-sectional view taken along line XIIB-XIIB.

[0061] Fig.13 It is a cross-sectional view showing the structure of another embodiment of the observation sample covering tool including the cover material.

[0062] Fig.14A This is a perspective view showing the structure of another form of the cover for observing the sample.

[0063] Fig. 14B yes Fig.14A Cross-sectional view of the XIVB-XIVB line.

[0064] Fig.15 This is a perspective view showing the structure of another form of the cover for observing the sample. DETAILED DESCRIPTION

[0065] A first embodiment of the present invention will be described with reference to the drawings.

[0066] <Observation sample cover>

[0067] First, the covering tool for observation samples according to the present invention (hereinafter referred to as “covering tool”) will be described.

[0068] like Figure 1A , Figure 1B As shown, the covering tool 10 according to the present invention comprises: an ultra-thin film 1; and a main body 2. Each structure will be described below.

[0069] (Ultra thin film)

[0070] The ultra-thin film 1 is used to cover the observation sample. Here, the observation sample refers to cells or biological tissues, or cells or biological tissues placed on the observation substrate or holder. In addition, according to the observation conditions, reagents or culture fluids required for observation together with the cells or biological tissues may also be included. In addition, the observation substrate here refers to a component located at a position on the side where light is seen, etc., under a microscope, etc.

[0071] Cells include eukaryotic cells such as animal cells and plant cells, and prokaryotic cells such as bacteria and archaea. As all cells extracted from organisms, for example, cells derived from skin, muscle, bone, adipose tissue, brain and nervous system, sensory system, heart and blood vessels and other circulatory systems, lungs, liver, spleen, pancreas, kidneys, digestive system, thymus, lymph, etc., and their cultures can be cited. Cells can also include body fluids such as blood (e.g., whole blood, serum, plasma), lymph, saliva, urine, ascites, and sputum.

[0072] As biological tissue, all tissues extracted from organisms can be cited, for example: skin, muscle, bone, adipose tissue, cranial nervous system, sensory system, circulatory system such as heart and blood vessels, lung, liver, spleen, pancreas, kidney, digestive system, thymus, lymph, etc. and their culture. Biological tissue can also include: body fluids such as blood (for example, whole blood, serum, plasma), lymph, saliva, urine, ascites, sputum. Among them, it is preferred that biological tissues such as cranial nervous system, sensory system, circulatory system, bone, muscle, etc. that use fluorescent imaging of fluorescent pigments are more utilized. As a substrate for observation, plastics, cover glass, slide glass, etc. are more utilized. In addition, if it is a material that can be used for observation, it is not limited to these. In addition, when the ultra-thin film 1 to be covered is set as a substrate for observation, and when cells or biological tissues are small, a holder is used. Plastics, cover glass, slide glass, etc. are more utilized by the holder. In addition, if it is a material that can hold cells or biological tissues, it is not limited to these.

[0073] In addition, the observation sample is not only a cell or a biological tissue, but also a fine particle / nanoparticle that is not derived from a biological body, or a fine particle / nanoparticle that is placed on an observation substrate or a holder. As fine particles / nanoparticles, for example, polymer particles, liposomes, polymer vesicles, droplets, metal colloids, etc. can be cited. In addition, if the particle size of the fine particles / nanoparticles is greater than 1 nm and less than 1 mm, the material is not particularly limited.

[0074] The ultra-thin film 1 is adhered to at least a portion of the upper surface 5a and the side surface 5b of the holding portion 3 (plate portion 5) by physical adsorption in a manner that seals the opening portion 4, and is held on the plate portion 5. In addition, when viewed from above, the shape of the ultra-thin film 1 can be, for example, a square shape, a rectangular shape, a circular shape, and an elliptical shape. In addition, if the ultra-thin film 1 is formed larger than the opening portion 4, the shape of the ultra-thin film 1 is not limited. With regard to the size of the ultra-thin film 1, it is preferred that the size of the ultra-thin film 1 when viewed from above is larger than the outer diameter of the plate portion 5, and preferably smaller than the size of the container that contains the solvent in which the ultra-thin film 1 is immersed and the substrate or sacrificial layer of the ultra-thin film 1 is dissolved.

[0075] The ultra-thin film 1 is a self-supporting film (no need to observe the support state of the substrate) with a thickness controlled to the nanometer level. It exhibits high adhesion unique to nanometer thickness. It does not use reactive functional groups or adhesives, but only utilizes physical adsorption such as van der Waals forces and electrostatic interactions to adhere to various interfaces (glass, plastic, biological tissues, etc.).

[0076] The film thickness of the ultra-thin film 1 is more than 20nm, preferably more than 30nm, more preferably more than 40nm, on the other hand, it is less than 200nm, preferably less than 180nm, more preferably less than 150nm. The film thickness is more than 20nm, thereby, it is easy to process the ultra-thin film 1, on the other hand, the film thickness is less than 200nm, thereby, the adhesion of the ultra-thin film 1 is good.

[0077] The resin included in the ultra-thin film 1 is preferably insoluble in the culture solution or buffer used when observing the biological tissue, and is insoluble in the solvent used to dissolve the sacrificial layer when manufacturing the ultra-thin film 1. In addition, preferably, the resin does not affect the biological tissue, for example, it is not a resin that brings biological stimulation or a resin that is toxic to the biological tissue.

[0078] When the ultra-thin film 1 is used as an observation substrate, the light used in the microscope is visible light (above 400nm), and the ultra-thin film with a film thickness of less than half of the wavelength, i.e. less than 200nm, has no effect on the optical system. The ultra-thin film 1 can be made of a resin with a refractive index that does not interfere with the optical system of the microscope, etc.

[0079] The resin, in order to prevent the drying of cells or biological tissues and inhibit their movement for the purpose, for example, can exemplify a waterproof resin. For water resistance, the water contact angle can be used to evaluate, for example, a contact angle meter can be used to measure. The water contact angle is preferably more than 90 degrees, more preferably more than 95 degrees, and more preferably more than 100 degrees. On the other hand, it is preferably less than 130 degrees, more preferably less than 125 degrees, and more preferably less than 120 degrees. As a waterproof resin, for example, a perfluorinated (1-butenyl vinyl ether) polymer, as a product, CYTOP (registered trademark) made by AGC Asahi Glass Co., Ltd. can be cited. However, if the resin is a material that can be formed into an ultra-thin film 1, it is not limited to a waterproof resin, and the material can be selected according to the purpose of the observation object or the data to be observed.

[0080] (Main body)

[0081] The main body 2 includes a holding portion 3 having an opening 4. Preferably, the main body 2 includes a plate 5, which is formed by a plate material to constitute the holding portion 3 and has a through hole formed in the plate material as the opening 4; and a wall 6, which is extended downward from the outer periphery of the plate 5. The ultra-thin film 1 is held on the upper surface 5a and the side surface 5b of the plate 5.

[0082] The shape of the plate portion 5 (holding portion 3) is not particularly limited as long as it can hold the ultra-thin film 1, but is preferably circular or elliptical in a plan view. The outer diameter and thickness of the plate constituting the plate portion 5 are also not particularly limited as long as it can hold the ultra-thin film 1, for example, the outer diameter is: 10 mm or more and 100 mm or less, and the thickness is: 0.1 mm or more and 5 mm or less.

[0083] The shape of the through hole (opening 4) formed by the plate portion 5 (holding portion 3) is not particularly limited, but is preferably circular or elliptical in plan view. The outer diameter of the through hole (opening 4) can be determined by moving the covering tool 10 downward to the observation sample S when the observation sample S is covered with the ultra-thin film 1, so that the ultra-thin film 1 can be cut to a size sufficient to cover the observation sample S (see Figure 7B ), is not particularly limited, and is, for example, greater than 10 mm and less than 100 mm.

[0084] Preferably, the wall portion 6 is arranged to extend downward from the outer periphery of the plate portion 5 as a whole, but it can also be arranged to extend downward from a part of the outer periphery. In addition, preferably, the wall portion 6 is arranged to extend downward from the plate portion 5 vertically, but it can also be inclined outward from the plate portion 5 at a specified angle, as long as the wall portion 6 can be used to support the ultra-thin film 1 floating on the solvent when the main body 2 is used to pick it up and take it out, the shape is not particularly limited. As for the length (height) and thickness of the wall portion 6, as long as the main body 2 can be used automatically, it is not particularly limited. For example, the length (height) is: more than 1mm and less than 20mm, and the thickness is: more than 0.3mm and less than 3mm.

[0085] Preferably, the plate 5 has a step portion 5d forming a recess 5c on the upper surface 5a side where the ultra-thin film 1 is held, and the step portion 5d is formed on the outer peripheral side of the plate 5, which is closer to the outside than the opening 4, and the opening 4 is opened on the bottom surface of the recess 5c. Preferably, the step portion 5d is formed along the entire periphery of the plate 5, but it can also be formed along a part of the periphery. As for the formation position of the step portion 5d, it is preferably formed at the outer end of the formation periphery (side 5b) of the plate 5, but it can also be formed at a predetermined interval on the side of the opening 4 than the side 5b. As for the height of the step portion 5d, when the covering tool 10 is placed on the workbench during the preparation of the observation specimen, a gap can be generated between the ultra-thin film 1 and the workbench to prevent the ultra-thin film 1 from sticking to the workbench, and it is greater than 0.1 mm and less than 5 mm.

[0086] The material of the main body 2 is not particularly limited as long as it does not change the properties of the ultra-thin film 1 held by it, but it is preferably insoluble in a solvent 53 selected from the group consisting of an aqueous solvent and an organic solvent used in the ultra-thin film impregnation step S14 described later (see Figure 5D ), but is made of metal, glass or resin. Examples of metal include aluminum, iron, copper, brass, stainless steel, etc., and examples of resin include PS, PC, PET, COP, PMMA, PEEK, PDMS, etc. As the material of the main body 2, resin is more preferably used.

[0087] like Figure 2 As shown, the covering member 10 preferably further includes at least one of a lid portion 9A covering the upper surface 5 a side of the holding portion 3 (plate portion 5 ) and a receiving portion 9B covering the lower surface 5 e side.

[0088] The shapes of the cover 9A and the support 9B are not particularly limited as long as they can cover the upper surface 5a side of the plate 5 or the lower surface 5e side of the plate 5, that is, the lower surface 6a of the wall 6, but are preferably circular or elliptical shapes similar to the plate 5 when viewed from above. The cover 9A and the support 9B cover the covering tool 10 by clamping it from above and below, and the cover 9A and the support 9B may have different heights of the side surfaces even if they have the same shape. The materials of the cover 9A and the support 9B are not particularly limited, but are preferably the same material as the plate 5 or the wall 6.

[0089] Since the ultra-thin film 1 is thin and nanometer-level, it has the property of being easily attached to anything. Therefore, once dust or dirt adheres to the ultra-thin film 1, it is difficult to remove it, and the ultra-thin film 1 becomes a film that is not suitable for observing biological tissues, etc. The covering tool 10 of the present invention is provided with a cover 9 and a support 9B, so that dust or dirt can be prevented from adhering to the ultra-thin film 1 held by the plate 5, and the ultra-thin film 1 suitable for observing biological tissues, etc. can be maintained.

[0090] Preferably, the cover 9 covering the upper surface 5a side of the plate 5 is in a form in which a gap 9a is formed between the cover 9 and the ultra-thin film 1 in such a manner that the ultra-thin film 1 is not attached to the inner surface of the cover 9. To form such a gap 9a, preferably, a step portion 5d is formed on the plate 5. In addition, a convex portion (not shown) may be formed on the inner surface of the cover 9 on the side of the ultra-thin film 1.

[0091] <Covering tool packaging>

[0092] Next, the covering tool package according to the present invention will be described.

[0093] like Figure 3 As shown in FIG. 1 , the covering tool package 30 includes: a covering tool 10; and a packaging bag 11 that wraps the covering tool 10 from the outside. Since the covering tool 10 has the same structure as described above, the description thereof will be omitted.

[0094] The packaging bag 11 can use, for example, a packaging bag or a disinfection bag that is previously known, such as a peeling package of a resin film. In addition, if the covering tool 10 can be packaged, even if it is not in a bag shape, the covering tool 10 can be wrapped by a sheet-shaped covering tool packaging body, and the shape is not particularly limited. In the covering tool packaging body 30, since the packaging bag 11 is provided to wrap the covering tool 10 from the outside, it is possible to prevent the cover 9 from falling off the ultra-thin film 1 during transportation and causing damage such as rupture. In addition, if a disinfection bag is used for the packaging bag, and the covering tool 10 that has been sterilized is wrapped, a sterilized covering tool 10 can be provided. In addition, if at least one surface of the disinfection bag is a material that transmits ultraviolet rays, after packaging using the disinfection bag, ultraviolet rays can be irradiated to sterilize the covering tool 10.

[0095] <Process of Manufacturing Method of Covering Tool for Observation Sample>

[0096] Next, the steps of the method for producing the observation sample covering tool according to the present invention will be described.

[0097] like Figure 4 As shown, the covering tool 10 can be manufactured by performing a substrate preparation step S11, a sacrificial layer forming step S12, an ultra-thin film forming step S13, an ultra-thin film impregnation step S14, an ultra-thin film removal step S15, and preferably a drying step S16. Specifically, the substrate preparation step S11 to the ultra-thin film forming step S13 refer to the manufacturing process of the ultra-thin film 1, and the ultra-thin film impregnation step S14 to the ultra-thin film removal step S15, or the ultra-thin film impregnation step S14 to the drying step S16 refer to the manufacturing process of the covering tool 10 in which the ultra-thin film 1 is attached to the main body 2.

[0098] (Ultra-thin film manufacturing process)

[0099] Here, as an example of a method for manufacturing the ultra-thin film 1, a manufacturing process of the ultra-thin film 1 by spin coating is described, but the manufacturing method is not particularly limited as long as the ultra-thin film 1 can be manufactured. Figure 5A As shown, in the substrate preparation step S11, a substrate 51 having a smooth surface and the same shape as the ultra-thin film 1, for example, a circular shape when viewed from above, is prepared by a common method such as a spin coating method. The thickness of the substrate 51 is appropriately set according to the type of the ultra-thin film 1, for example, to be set to be greater than 10 μm and less than 10000 μm. In addition, the shape of the substrate 51 can be exemplified by: a square shape, a rectangular shape, a circular shape, and an elliptical shape, but as long as it is a plane, the shape is not particularly limited.

[0100] Examples of the material of the substrate 51 include carbon materials such as silicon, silicone rubber, silicon dioxide, glass, mica, and graphite, polymer materials such as polyethylene, polypropylene, cellophane, and elastomers, and calcium compounds such as apatite. A preferred material is silicon, and a preferred substrate is a silicon wafer.

[0101] like Figure 5B As shown, in the sacrificial layer forming step S12, a sacrificial layer 52 is prepared on the substrate 51 by a common method such as spin coating. The thickness of the sacrificial layer 52 is appropriately set according to the type of the ultra-thin film 1, for example, to be 0.01 μm or more and 10 μm or less.

[0102] As the material of the sacrificial layer 52, in the following process, after the ultra-thin film 1 is prepared on the sacrificial layer 52, the sacrificial layer 52 is immersed in a solvent to dissolve it. Therefore, as long as it is soluble in the solvent at this time, it is not particularly limited. For example, when the solvent is an aqueous solvent, examples include: polymer electrolytes such as polyacrylic acid, polymethacrylic acid, and polystyrene sulfonic acid; polyethylene glycol, polyacrylamide, polyvinyl alcohol; and non-ionic water-soluble polymers such as polysaccharides such as starch and cellulose acetate.

[0103] like Figure 5C As shown, in the ultra-thin film forming step S13, the ultra-thin film 1 is prepared on the sacrificial layer 52 by a common method such as spin coating. The thickness and material of the ultra-thin film 1 are as described above.

[0104] (Manufacturing process of covering tool)

[0105] like Figure 5D As shown, in the ultra-thin film immersion step S14, the substrate 51 prepared with the sacrificial layer 52 and the ultra-thin film 1 is immersed in a solvent 53 contained in a container 54 and in which only the sacrificial layer 52 is dissolved, thereby dissolving the sacrificial layer 52 and leaving only the ultra-thin film 1 floating in the solvent 53. In addition, the substrate 51 sinks in the solvent 53 (not shown).

[0106] As the solvent, an aqueous solvent or an organic solvent can be used. Examples of the aqueous solvent include water, distilled water, water in which a salt is dissolved, water in which a surfactant is dissolved, and a buffer solution. When the sacrificial layer 52 is polyvinyl alcohol, water or distilled water is preferred.

[0107] like Figure 5EAs shown, in the ultra-thin film taking-out step S15, the ultra-thin film 1 floating (immersed) in the solvent 53 is picked up from the lower side of the ultra-thin film 1 by the main body 2, and the ultra-thin film 1 is held by the upper surface of the main body 2, specifically, the upper surface of the holding part 3 (plate part 5). In addition, the main body 2 can also be sunk in the solvent 53 in advance during the ultra-thin film immersion step S14 and then the ultra-thin film 1 is picked up, or it can be placed in the solvent 53 later during the ultra-thin film taking-out step S15 to pick up the ultra-thin film 1.

[0108] In the drying step S16, the ultra-thin film 1 held by the covering tool 10 is dried by a common method such as natural drying, freeze drying, vacuum drying, etc. Alternatively, the ultra-thin film 1 held by the main body 2 may be dried in a desiccator.

[0109] <Observation Sample Covering Method>

[0110] Next, a method of covering an observation sample using the covering tool according to the present invention will be described.

[0111] like Figure 6 As shown, the covering method includes: a preparation step S1; and a covering step S2.

[0112] [First covering method]

[0113] (Preparation process)

[0114] like Fig. 7A As shown, in the preparation step S1, the cover tool 10 is prepared with the ultra-thin film 1 arranged on the upper side, and the observation sample S is prepared on the workbench 110. In addition, for the observation sample S, the biological tissue 101 is placed on the observation substrate 102 together with the reagent or culture solution required for observation. Preferably, the workbench 110 is smaller than the observation substrate 102.

[0115] (Covering process)

[0116] Here, the step of covering the observation sample when the observation substrate is not an ultra-thin film is described. Fig. 7A , Figure 7BAs shown, in the covering step S2, the covering tool 10 is arranged on the observation sample S, and then the covering tool 10 is moved to the lower side of the observation sample S. As a result, the ultra-thin film 1 held by the main body 2 is cut by the peripheral portion of the opening 4, and the lower surface 1b of the ultra-thin film 1 after cutting exhibits high adhesion unique to nanometer thickness, and can be pasted without using reactive functional groups or adhesives, and the observation sample S can be covered only by physical adsorption such as van der Waals force and electrostatic interaction, so that the observation specimen can be easily prepared. In the case where the observation sample S is an observation substrate 102 carrying a biological tissue 101, the outer peripheral surface of the biological tissue 101 is covered by the lower surface 1b of the ultra-thin film 1, and not only the upper surface and side surface of the observation substrate 102, but also a part of the lower surface is covered by the lower surface 1b of the ultra-thin film 1.

[0117] [Second covering method]

[0118] (Preparation process)

[0119] like Fig. 8A As shown, in the preparation step S1, the process is the same as the first covering method except that the covering tool 10 is prepared in a posture where the ultra-thin film 1 is arranged at the bottom.

[0120] (Covering process)

[0121] like Figure 8B As shown, similar to the first covering method, in the covering step S2, the covering tool 10 is arranged on the observation sample S, and then the covering tool 10 is moved to the lower side of the observation sample S. As a result, the ultra-thin film 1 held by the main body 2 is cut by the peripheral portion of the opening 4, and the upper surface 1a of the ultra-thin film 1 after cutting exhibits high adhesion unique to nanometer thickness, and can be pasted without using reactive functional groups or adhesives, but only through physical adsorption such as van der Waals force and electrostatic interaction, and the outer peripheral surface of the biological tissue 101 can be covered, and not only the upper surface and side surface of the observation substrate 102, but also a part of the lower surface is covered by the upper surface 1a of the ultra-thin film 1, so that the observation specimen can be easily prepared.

[0122] [Third covering method]

[0123] (Preparation process)

[0124] like Fig. 9 As shown, in the preparation step S1, in addition to wetting the upper surface 1a of the ultra-thin film 1 with water W or a culture solution, the first covering method (see Fig. 7A )same.

[0125] (Covering process)

[0126] In the covering step S2, the covering tool 10 is turned upside down so that the upper surface 1a of the ultra-thin film 1 wetted with water W or a culture solution is in contact with the observation sample S. Thereafter, the covering tool 10 is moved to the lower side of the observation sample S. Thus, when the biological tissue 101 and the observation substrate 102 are covered with the ultra-thin film 1, the adhesion of the covering is improved. In addition, since the drying of the observation sample S can be prevented, the observation time of the biological tissue 101 can be extended.

[0127] [Fourth covering method]

[0128] (Preparation process)

[0129] like Fig.10 As shown, in the preparation step S1, in addition to wetting the lower surface 1b of the ultra-thin film 1 with water W or a culture solution, the second covering method (see Fig. 8A )same.

[0130] (Covering process)

[0131] In the covering step S2, the covering tool 10 is turned upside down so that the lower surface 1b of the ultra-thin film 1 wetted with water W or a culture solution is in contact with the observation sample S. Thereafter, the covering tool 10 is moved to the lower side of the observation sample S. Thus, when the biological tissue 101 and the observation substrate 102 are covered with the ultra-thin film 1, the adhesion of the covering is improved. In addition, since the drying of the observation sample S can be prevented, the observation time of the biological tissue 101 can be extended.

[0132] In addition, if Fig.11 As shown, in the covering tool 10A according to the first embodiment of the present invention, the plate portion 5 (holding portion 3) and the opening portion 4 formed by the plate portion 5 may be set to be rectangular in plan view, preferably square. Figure 1A The covering means 10 shown are identical.

[0133] A second embodiment of the present invention will be described with reference to the drawings.

[0134] <Observation sample cover>

[0135] First, the covering tool for observation samples according to the present invention (hereinafter referred to as “covering tool”) will be described.

[0136] like Fig. 12A , Fig. 12B As shown in FIG. 1 , the covering tool 20 according to the present invention includes an ultra-thin film 1 and a main body 2. Since the ultra-thin film 1 is the same as that of the first embodiment, the description thereof will be omitted.

[0137] (Main body)

[0138] The main body 2 includes a holding portion 3 having an opening 4. Preferably, the main body 2 includes a frame 7, the holding portion 3 is formed by a wire having a circular or elliptical cross-sectional shape, and the wire is arranged in a frame shape as the opening 4; and a handle 8 connected to the frame 7. The frame 7 holds the ultra-thin film 1.

[0139] The shape of the frame 7 (holding portion 3) is not particularly limited as long as it can hold the ultra-thin film 1, but preferably, it is circular or elliptical when viewed from above. The outer diameter of the wire constituting the frame 7 is not particularly limited as long as it can hold the ultra-thin film 1, for example, it is more than 10 mm and less than 100 mm. In addition, the wire may be a wire obtained by twisting a plurality of wires into one.

[0140] The shape of the opening 4 formed in the frame 7 (holding portion 3) by configuring the wire into a frame shape is not particularly limited, and preferably, it is a circular shape or an elliptical shape when viewed from above. For the outer diameter of the opening 4, the ultra-thin film 1 can be cut into a size larger than the observation sample S and fully covering the observation sample S, for example, 10 mm or more and 100 mm or less. In addition, the ultra-thin film 1 held by the frame 7 forms a repeating portion 1c on the inner peripheral side of the frame 7 by bonding the upper surface 1a and the lower surface 1b of the ultra-thin film 1.

[0141] The handle 8 is a rod-shaped component connected to the frame 7 and extending upward from the frame 7 at a predetermined angle. In the ultra-thin film removal step S15, the handle 8 has the function of serving as a gripping handle of the frame 7 when the ultra-thin film 1 floating (immersed) in the solvent is fished out (taken out) from the solvent by the frame 7. In addition, the handle 8 may also have a curved portion (not shown) that is curved in the middle of the length direction. In addition, the outer diameter and length of the handle 8 are appropriately set in consideration of the operability of taking out the ultra-thin film 1.

[0142] The material of the main body 2 is preferably a material that does not change the properties of the ultra-thin film 1 and is insoluble in the solvent 53 used in the ultra-thin film immersion step S14 (see Figure 5D ) is made of metal or resin, and more preferably is made of metal such as aluminum, iron, copper, brass, stainless steel, etc.

[0143] like Fig.13 As shown, the covering member 20 preferably further includes a cover 9 that covers the upper and lower surfaces of the holding portion 3 (frame 7). The cover 9 can prevent dust or dirt from adhering to the ultra-thin film 1.

[0144] The cover 9 accommodates the frame 7 holding the ultra-thin film 1 inside, and is configured as a box having an open end surface 9c at one end. In addition, the cover 9 prevents the ultra-thin film 1 accommodated therein from sticking, and therefore, preferably has a convex portion 9b forming a gap 9a between the cover 9 and the ultra-thin film 1. Furthermore, preferably, the cover 9 has a notch portion 9d for accommodating the handle 8 on the side of the open end surface 9c. In addition, preferably, the material of the cover 9 is the same material as that of the frame 7.

[0145] <Covering tool packaging>

[0146] The covering tool package includes: a covering tool 20; and a packaging bag made of a resin film that wraps the covering tool 20 from the outside. The covering tool 20 is configured as described above, and the packaging bag can be used in the same manner as the packaging bag 11 of the first embodiment (see Figure 3 )Same packaging bag.

[0147] <Observation Sample Covering Method>

[0148] The covering method of the observation sample is the same as that of the first embodiment except that the covering tool 20 is used.

[0149] In addition, if Fig.14A , Fig. 14B As shown, in the covering tool 20A according to the present invention, the holding portion 3 (frame portion 7) may be formed by using a plate, and the plate may be arranged in a frame shape as the opening 4. In addition, preferably, the frame portion 7 and the plate portion 5 (see Figure 1B ) Similarly, the outer peripheral side has a step portion 7d. Here, the width of the plate is set to be larger than the outer diameter of the wire, for example, 11 mm or more and 150 mm or less. In addition to the above-mentioned structure, Fig. 12A The covering means 20 shown are identical.

[0150] By using a plate material to form the frame 7, when the ultra-thin film 1 is held in the frame 7, the ultra-thin film 1 surrounding the lower surface 7b of the frame 7 is reduced, so that the overlapping portion 1c (see FIG. 1 ) where the upper surface 1a and the lower surface 1b of the ultra-thin film 1 are adhered and overlapped is not formed on the inner circumference of the frame 7. Fig. 12B As a result, when the observation sample S is covered with the ultra-thin film 1, the strength of the outer peripheral side of the ultra-thin film 1 does not increase due to the repeated portion 1c, and the ultra-thin film 1 can be easily cut by the end of the opening 4, making the covering operation easier.

[0151] In addition, if Fig.15 As shown, the covering tool 20B according to the second embodiment of the present invention may also be configured such that the frame 7 (holding portion 3) and the opening 4 formed by the frame 7 are rectangular in plan view, preferably square in shape. Fig. 12A The covering means 20 shown are identical.

[0152] <Other overwriting methods>

[0153] In the above-mentioned covering method, the method of making an observation specimen by covering an observation substrate that is not an ultra-thin film is described. However, when the observation substrate is set to an ultra-thin film, and the biological tissue (observation specimen) is sufficiently large, the biological tissue (observation specimen) can be placed on the workbench 110 instead of being placed on the holder, and the biological tissue (observation specimen) can be covered with an ultra-thin film by a covering tool through the same process to make an observation specimen. In addition, when the observation substrate is set to an ultra-thin film, and the cells or biological tissue are small, the cells or biological tissue can be placed on the holder to make the observation specimen, and the observation specimen can be covered with an ultra-thin film by a covering tool through the same process to make the observation specimen. By doing so, it is easy to observe cells or biological tissue from the ultra-thin film side.

[0154] Description of Reference Numerals

[0155] 1 ultra-thin film

[0156] 1a Upper surface

[0157] 1b Lower surface

[0158] 1c Repeating part

[0159] 2 Main body

[0160] 3. Holding part

[0161] 4 Opening

[0162] 5. Board

[0163] 5a Upper surface

[0164] 5b Side

[0165] 5c recess

[0166] 5d Step

[0167] 6 Wall

[0168] 6a Lower surface

[0169] 7 Frame

[0170] 7d Step

[0171] 8. Handle

[0172] 9A Cover

[0173] 9B Support

[0174] 9a Gap

[0175] 9b convex part

[0176] 9c Open end face

[0177] 9d Notch

[0178] 10 Covering tools

[0179] 10A Covering equipment

[0180] 11 Packaging bags

[0181] 20 Covering utensils

[0182] 20A Covering equipment

[0183] 20B Covering tools

[0184] 30 Covering equipment packaging

[0185] 51 Substrate

[0186] 52 Sacrificial layer

[0187] 53 Solvent

[0188] 54 Container

[0189] 101 Organisms

[0190] 102 Observation substrate

[0191] 110 Workbench

[0192] S Observe the sample.

Claims

1. A covering tool for observing a sample, It is characterized in that It comprises: an ultra-thin film for covering an observation sample placed on an observation substrate; and a main body having a holding portion formed with an opening, wherein the ultra-thin film covers the top surface and the peripheral side surface of the observation sample; The ultra-thin film is formed to be larger than the opening, The ultra-thin film is held by the holding portion by being adhered to at least a portion of the upper surface and the side surface of the holding portion by physical adsorption in a manner that seals the opening; The ultra-thin film has a thickness ranging from 20 nm to 200 nm, and is made of a hydrophobic resin, wherein: The main body portion includes a plate portion that forms the holding portion with a plate material and in which a through hole is formed as the opening portion; and a wall portion that extends downward from an outer peripheral portion of the plate portion.

2. The covering tool for observation sample according to claim 1, It is characterized in that The main body includes: a frame portion, in which the holding portion is formed of a wire material and the wire material is arranged in a frame shape as the opening portion; and a handle portion connected to the frame portion.

3. The covering tool for observation sample according to claim 1 or 2, It is characterized in that The holding portion has a step portion forming a recessed portion on the upper surface side where the ultra-thin film is held, the step portion is formed on the outer peripheral side of the holding portion, which is outside the opening, and the opening is opened on the bottom surface of the recessed portion.

4. The covering tool for observation sample according to any one of claims 1 to 3, It is characterized in that The main body is made of metal, glass or resin. The main body is insoluble in a solvent selected from the group consisting of an aqueous solvent and an organic solvent.

5. The covering tool for observation sample according to any one of claims 1 to 4, It is characterized in that The device further includes at least one of a cover portion that covers the upper surface side of the holding portion and a receiving portion that covers the lower surface side of the holding portion.

6. The covering tool for observation sample according to any one of claims 1 to 5, It is characterized in that The opening is formed larger than the observation sample.

7. A covering tool package, It is characterized in that have: The observation sample covering tool according to any one of claims 1 to 6; and a packaging bag that wraps the observation sample covering tool from the outside.

8. A covering method for observing a sample, It is characterized in that Including the following processes: A step of preparing the observation sample covering tool and the observation sample according to any one of claims 1 to 6; and The covering step includes placing the observation sample covering tool on the observation sample and covering the observation sample with the ultra-thin film.

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