WO2023223521A1 - Embedding and fixation composition for improving visibility in observation using electron microscope or the like, and observation method using same - Google Patents

Abstract

Provided are: an embedding and fixation composition that enables clear visualization of a microstructure of a solid material formed of a light element such as graphite and boron nitride and thereby improving the visibility thereof, in observation using an electron microscope or the like; and an observation method with an electron microscope or the like using the embedding and fixation composition. According to the present invention, a water-soluble polymer such as polyvinyl alcohol and a water-soluble heavy metal salt such as phosphotungstic acid are dissolved in water to obtain a liquid composition. A solid material is impregnated with the liquid composition, which is then solidified, to have a microstructure embedded and fixed. Then, the microstructure is observed using an electron microscope or the like.

Description

An embedding and fixing composition that improves visibility during observation using an electron microscope, etc., and an observation method using the same

The present invention provides an embedding and fixing composition that can clearly visualize the fine structure of a solid material containing light elements as a main component and improve visibility under observation with an electron microscope or an X-ray microscope. The present invention relates to a kit provided with the same, a method for manufacturing the same, and an observation method using the same using an electron microscope or an X-ray microscope.

When observing the morphology of a solid material using an electron microscope or the like, if the sample is a porous material, a powder material, a fibrous material, a material with low strength and easily deformed, etc., embedding in resin is performed. Embedding the solid material in resin facilitates subsequent handling. By exposing the internal structure by polishing or the like after embedding the material in a resin, the internal morphology of the material can also be observed.

This method has a long history in the field of metallographic observation, and is commonly used in material analysis using various microscopes such as optical microscopy, electron microscopy, and scanning probe microscopy.

In observation using an electron microscope or an X-ray microscope, the contrast obtained strongly depends on the atomic number of the sample. When a sample composed of light elements, a polymer material, a biological material, etc. is embedded in resin, even if the handling of the sample is improved and internal observation becomes possible, there is a clear contrast between the embedding resin and the sample. There is a problem in that the visibility of the observed image is poor because the image cannot be obtained.

Furthermore, since resin-embedded samples have low conductivity, charging and drifting of the sample may become a problem in measurement methods that use charged particles as probes, such as in electron microscopes. Therefore, conventionally, these problems have been avoided by using the following four methods.

One of the conventional methods is to add heavy metals such as ruthenium tetroxide, osmium tetroxide, and phosphotungstic acid to specific parts of the molecular structure when the sample is a polymeric material, and then embed it in a resin to make it conductive. Examples include electronic staining, which involves processing and observation. With this method, it is possible to observe the fine structure of a polymeric material whose main component is a light element with improved contrast.

The second method is to coat the surface of the sample with a noble metal such as Au, Pt, Pd, Os, etc., embed it in resin, then polish it and observe the cross section. In this method, the cross-sectional structure is "resin/noble metal/sample", so even fine uneven structures such as polymer resist on a silicon wafer can be observed with improved contrast.

A third method is negative staining, in which an aqueous solution of uranium acetate, gadolinium acetate, phosphotungstic acid, etc. is dropped around the sample, followed by conductive treatment and observation. With this method, it is possible to observe particle/fibrous samples such as carbon black, graphene, and cellulose nanofibers, as well as the microstructures of biological samples, with improved contrast.

A fourth method is to impregnate the microstructure of the sample with a contrast agent such as barium sulfate or organic iodine and then perform X-ray observation. With this method, defects such as minute cracks in concrete can be detected.

Further, the following Patent Document 1 discloses a protective agent for electron microscopy, which is characterized by containing a survival environment-imparting component, saccharides, and an electrolyte, and a method for observing a sample using an electron microscope using the same. There is. By using this protective agent, a biological sample in a water-containing state can be protected and observed in its living state without being deformed even under vacuum.

International Publication No. 2015/115502

When observing a resin-embedded sample using an electron microscope or an X-ray microscope, there is a problem that ``with a sample composed of light elements, it is impossible to distinguish between the embedding resin and the sample.'' The fundamental cause of this problem is that the main components of the embedding resin are light elements such as C, H, and O. Since the contrast obtained with an electron microscope or an X-ray microscope strongly depends on the atomic number of the sample, it is theoretically impossible to obtain clear contrast in the case of a sample whose constituent elements are similar to those of the embedding resin.

Therefore, various methods described above have been attempted to improve the contrast of observed images, but these methods are not always sufficient. With electronic staining, which is one of the conventional methods, there are many materials that cannot be electronically dyed, such as inorganic compounds, fluororesins, and engineering plastics, so the materials that can be observed are limited. In addition, it takes time to prepare the sample, and the reagents are highly toxic.

In the second method of coating metals, if the sample is porous or has minute irregularities, the coating tends to be uneven, and the forms that can be clearly observed are limited. Furthermore, in the case of a sample containing metal, depending on the constituent element species, the contrast may be similar to that of a metal film, which may impede visibility.

The third type of negative staining is not suitable for cross-sectioning because the sample cannot be embedded, and the internal morphology cannot be observed. Furthermore, since it cannot fill a large space, it is not suitable for porous bodies, etc., and the materials that can be observed are limited.

In the fourth method of impregnating a contrast agent and performing X-ray observation, the liquid contrast agent is only deposited and cannot be solidified, so the sample cannot be embedded and fixed and the internal morphology cannot be observed. Furthermore, it is difficult to handle the sample after it has been impregnated with a contrast medium.

Furthermore, in the method described in Patent Document 1, it is necessary to form a film by crosslinking a gel-like substance applied to the sample surface with an electron beam or plasma. Since the curing reaction does not proceed inside the sample where the electron beam or plasma does not reach, the inside of the sample cannot be embedded and fixed, making it impossible to observe the internal morphology. In addition, preparing the sample requires irradiation with an electron beam or plasma, which is laborious and costly.

Depending on the form and characteristics of the material to be observed, conventional techniques may be able to handle the problem, but as mentioned above, conventional techniques have technical issues in terms of versatility, operability, and ease of interpretation of the obtained images. The current situation is that there are multiple.

The present invention was made in view of the above-mentioned background art and its problems, and it is an object of the present invention to clearly visualize the microstructure of a solid material mainly composed of light elements under observation with an electron microscope or an X-ray microscope. An object of the present invention is to provide an embedding and fixing composition that can improve visibility. Another object of the present invention is to provide an observation method using an electron microscope or an X-ray microscope that allows the microstructure of a solid material to be observed simply and clearly.

As a result of intensive research to solve the above problems, the present inventors have discovered that by creating an aqueous solution in which a heavy metal salt with high electron density and a water-soluble polymer are dissolved in water, it is suitable for impregnating various forms of solid materials. It is possible to realize a composition for embedding and fixation that can be solidified and embedded and fixed by physical or chemical methods, and that the composition has no structure at the nano level and that allows the microstructure of the solid material to be clearly visualized. I found it. As a result of further research into its composition and observation method, the present invention was completed.

That is, the present invention is an aqueous solution containing a water-soluble heavy metal salt and a water-soluble polymer and/or a derivative thereof, in which these water-soluble components are dissolved in water, and a solid material mainly composed of light elements. In observation using an electron microscope or an X-ray microscope, the visibility of the observed image can be improved due to the contrast between the heavy metals constituting the water-soluble heavy metal salt and the light elements that are the main components of the solid material. It is a composition for use.

By forming an aqueous solution in which a water-soluble heavy metal salt and a water-soluble polymer and/or its derivative are dissolved in water, it can be easily impregnated into various forms of solid materials, and solid materials can be easily impregnated into solid materials by physical or chemical methods. It can be embedded and fixed. In addition, the solidified composition has no structure at the nano-level because the heavy metal salt with high electron density is uniformly dispersed in the polymer, and under observation with an electron microscope and The microstructure of solid materials can be clearly visualized with good contrast.

Further, the water-soluble heavy metal salt is one or more selected from the group consisting of phosphotungstic acid (PTA), gadolinium acetate, ammonium molybdate, phosphomolybdic acid, potassium ferrocyanate, lead nitrate, and lead acetate, and is highly water-soluble. The above-mentioned embedding/fixing molecule is one or more selected from the group consisting of polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), polyacrylamide (PAM), polyacrylic acid (PAA), and polyethylene oxide (PEO). It is a composition.

Furthermore, in the embedding and fixing composition, the water-soluble heavy metal salt is phosphotungstic acid (PTA), and the water-soluble polymer is polyvinyl alcohol (PVA).

Further, the composition for embedding and fixing contains 0.25 to 40% by mass of a water-soluble heavy metal salt and 0.5 to 15% by mass of a water-soluble polymer and/or a derivative thereof, based on the total amount of the composition. It is a thing.

By making the present invention have each of the above compositions, it is possible to further improve the stability and handleability as a reagent, the ability to impregnate the microstructure of the solid material, and the visibility of the observed image after solidification, and to reduce the manufacturing cost. can also be suppressed.

Further, the present invention provides two or more types of the embedding and fixing compositions having different contents and/or types of water-soluble heavy metal salts, and by changing the type of the embedding and fixing composition, the contrast of the observed image is improved. This is a kit for observing the embedding and fixing composition, which allows adjusting the embedding and fixing composition.

The embedding and fixing composition of the present invention allows the brightness of the background of an observed image to be adjusted by changing the amount and type of the water-soluble heavy metal salt, similar to adjusting the monotone background with black and white paint. Can be done. This makes it possible to change the type of composition used for embedding and optimize the contrast (shading) in accordance with the constituent elements of the solid material to be observed, thereby improving the visibility of the observed image.

Furthermore, the present invention comprises the composition for embedding and fixation and a dispersion in which a conductive polymer is dispersed in water, and the dispersion is added to the composition for embedding and fixation to impart conductivity. This is a kit for observing the embedding and fixing composition.

The present invention is also a kit for observing the embedding and fixing composition, in which the conductive polymer is a composite of polyethylene dioxythiophene and polystyrene sulfonic acid (PEDOT/PSS).

By adding conductive polymers to impart conductivity, charging phenomena (charge-up) can be prevented, and depending on the sample preparation conditions, conductive treatment after embedding and fixation can be omitted or simplified. can do. Furthermore, like conventional carbon pastes and carbon tapes, it can be used in applications where it is made into a sheet and particles or fiber samples are attached or temporarily fixed to the surface of the sheet for easy observation without conductive treatment.

Further, the present invention comprises a water-soluble heavy metal salt in the form of a solid or an aqueous solution, and a water-soluble polymer in the form of a solid or an aqueous solution, and the water-soluble heavy metal salt and the water-soluble polymer are added to water. This is a kit for producing an embedding/fixing composition, which allows the embedding/fixing composition to be produced by mixing or mixing with each other and dissolving or mixing.

By making the embedding fixation composition of the present invention into a two-component mixing kit that can be prepared immediately before use, it is necessary to weigh and mix the two component reagents and dissolve and mix them. Since the fixative composition can be prepared freely, the number of reagents can be reduced and costs can be reduced. Furthermore, by storing the two components separately, stable storage for a longer period of time becomes possible.

Furthermore, the present invention provides a method for producing the composition for embedding and fixing, which includes the steps of adding and dissolving a water-soluble polymer in water, and adding and dissolving a water-soluble heavy metal salt to the resulting aqueous solution. be.

The embedding and fixing composition of the present invention can be produced by simply dissolving two water-soluble components in water, resulting in a simple process and low cost. If necessary, it can also be prepared in-house from powdered raw materials.

The present invention also provides a step of impregnating a solid material with the embedding and fixing composition, and evaporating the water in the embedding and fixing composition or crosslinking the water-soluble polymer and/or its derivative. This is an observation method using an electron microscope or an X-ray microscope, which includes a step of solidifying the solid material by solidifying the material, and a step of processing the embedded and fixed solid material to expose the observation surface.

Further, the observation using the electron microscope or the X-ray microscope includes the step of optimizing the contrast of the observed image by using two or more types of the embedding and fixing compositions having different contents and/or types of water-soluble heavy metal salts. It's a method.

The observation method using an electron microscope or an X-ray microscope of the present invention is easy to operate and can be applied to various forms and types of solid materials. It can be clearly observed under a line microscope.

Furthermore, since the brightness of the background can be adjusted by changing the amount and type of water-soluble heavy metal salt in the embedding and fixing composition used, the contrast of the observed image can be controlled by pre-treating the sample.

The embedding and fixing composition of the present invention and the observation method using the same clearly visualize the fine structure of a solid material mainly composed of light elements under observation using an electron microscope or an X-ray microscope, thereby improving visibility. can be improved. Since the composition for embedding and fixing of the present invention contains a heavy metal salt, the smaller the atomic number of the elements constituting the solid material, the larger the contrast difference becomes, allowing sharp and clear observation.

A is an image obtained by observing the sample of Example 1 with a scanning electron microscope (photographing magnification: 5,000 times), and B is a partially enlarged image thereof (photographing magnification: 50,000 times). A is an image obtained by observing the sample of Comparative Example 1 with a scanning electron microscope (photographing magnification: 5,000 times), and B is a partially enlarged image thereof (photographing magnification: 50,000 times). A is an image obtained by observing the sample of Comparative Example 2 with a scanning electron microscope (photographing magnification: 5,000 times), and B is a partially enlarged image thereof (photographing magnification: 50,000 times). A and B are images obtained by observing the sample of Example 2 using a scanning electron microscope (magnifications of 2,500x and 5,000x), and C and D are partially enlarged images thereof (magnifications of 50,000x and 150,000x). times). A is an image taken from directly above the created vertical section. B, C, and D are cross-sectional images. A is an image of the sample of Example 3 observed with a transmission electron microscope (field size 91.51 nm x 91.51 nm), B is a partially enlarged image (field size 10.01 nm x 10.01 nm), and C is an electron beam This is an image showing a diffraction pattern. This is a spectrum measured by X-ray photoelectron spectroscopy (XPS) of samples obtained by solidifying the embedding and fixing compositions of Examples 1 and 7, polyvinyl alcohol (PVA), phosphotungstic acid (PTA), and vinylon. A is the C1s spectrum, and B is the W4f spectrum. A is an image of the sample of Example 4 observed using a low vacuum scanning electron microscope (250x magnification), and B is a partially enlarged image thereof (1,000x magnification). A is an image obtained by observing the sample of Example 5 using a low vacuum scanning electron microscope (field size: 600 μm x 90 μm), and B is an image obtained by observing the sample of Comparative Example 3 under the same conditions.

A is an image obtained by observing the sample of Example 6 using an X-ray microscope, and B is an image obtained by observing the sample of Comparative Example 4 under the same conditions. A is an image of the sample of Example 7 observed with a scanning electron microscope (100x magnification), B is a partially enlarged image thereof (10,000x magnification), and C is an external image of the sample of Example 7. This is a spectrum measured by X-ray photoelectron spectroscopy (XPS) of a sample obtained by solidifying the embedding and fixing composition of Example 7, phosphotungstic acid (PTA), and vinylon. A is the C1s spectrum, and B is the W4f spectrum. These are images obtained by observing the sample of Comparative Example 5 using a field emission scanning electron microscope, where A is a backscattered electron image and B is a secondary electron image (field size: 6.8 μm×6.6 μm). A to F are images obtained by observing samples of Example 8 in which the PVA:PTA mixing ratio was varied in the range of 20:1 to 1:10 using a field emission scanning electron microscope (field of view size 6.8 μm x 7.0 μm). A to F are images obtained by observing samples of Example 9 in which the type and blending ratio of water-soluble heavy metal salts were changed using a field emission scanning electron microscope (field size: 6.8 μm×7.0 μm). A is an image obtained by observing the sample of Example 10 using a scanning electron microscope (magnification: 20,000 times), and B is an image obtained by observing the sample of Comparative Example 6 under the same conditions.

Hereinafter, the embedding and fixing composition of the present invention, a kit including the same, a manufacturing method thereof, and an observation method using the same using an electron microscope and an X-ray microscope will be explained in detail. Note that components, manufacturing methods, observation methods, etc. whose explanations are omitted may be the same or substantially the same as those known to those skilled in the art.

The embedding and fixing composition of the present invention is an aqueous solution in which a water-soluble heavy metal salt, a water-soluble polymer and/or a water-soluble polymer derivative thereof are dissolved in water. When observing a solid material mainly composed of light elements using an electron microscope or an X-ray microscope, it is possible to detect The visibility of observed images can be improved.

In the present invention, "for embedding and fixation" refers to the use of embedding and fixing solid materials in observation with electron microscopes and X-ray microscopes, as well as the use of solid materials in the form of particles and fibers on the surface of This shall include use for attachment or temporary fixation.

In a scanning electron microscope (SEM) image, the higher the atomic number of the elements constituting the solid material, the more secondary electrons and reflected electrons increase, making the image brighter, and the lower the atomic number, the darker it becomes. On the other hand, in images observed using a transmission electron microscope (TEM) or X-ray microscope (XRM), the higher the atomic number of the elements constituting the sample, the lower the intensity of transmitted electrons and transmitted X-rays, making them darker; The brighter it becomes.

In the present invention, "heavy metal" means a metal whose atomic number is iron (Fe: atomic number 26) or higher. Moreover, the term "light element" means an element whose atomic number is fluorine (F: atomic number 9) or less. Furthermore, "main component" means that light elements account for 50% by mass or more of all elements constituting the solid material.

The light elements constituting the solid material to which the present invention is applicable are mainly carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and boron (B), with particular emphasis on carbon (C ) is assumed. The present invention becomes more effective as the proportion of these light elements in the entire solid material increases, such as 70% by mass or more, 90% by mass or more, and is particularly effective in solid materials composed entirely of light elements.

The water-soluble heavy metal salt contained in the present invention exhibits high solubility in water, has a sufficiently larger atomic number than the light elements constituting the solid material, and is a heavy metal salt that provides a large contrast when observed with an electron microscope and an X-ray microscope. and its hydrates are preferred. Specifically, phosphotungstic acid (H ₃ [P(W ₃ O ₁₀ ) ₄ ]·nH ₂ O) (PTA), gadolinium acetate ((CH ₃ COO) ₃ Gd·nH ₂ O), ammonium molybdate ( (NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O), phosphomolybdic acid (H ₃ (PMo ₁₂ O ₄₀ )·nH ₂ O), potassium ferrocyanate (K ₄ [Fe(CN) ₆ ]·nH ₂ O) ), lead nitrate (Pb(NO ₃ ) ₂ ), lead acetate (Pb(CH ₃ COO) ₂ ), and the like. Two or more types of these heavy metal salts may be contained.

Phosphotungstic acid (PTA), ammonium molybdate, phosphomolybdic acid, gadolinium acetate, and lead acetate are preferred because they have high solubility in water and are easy to handle. Phosphortungstic acid (PTA) is most preferred because it has a large amount of metal per molecule and can provide a large contrast with a small amount added.

The water-soluble polymer contained in the present invention is preferably a polymer that exhibits high solubility in water and has excellent stability and handleability. Specific examples include polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), polyacrylamide (PAM), polyacrylic acid (PAA), and polyethylene oxide (PEO). Two or more types of these polymers may be contained.

Polyvinyl alcohol (PVA) and carboxymethyl cellulose (CMC) are preferred because they have high solubility in water and are easy to handle. Polyvinyl alcohol (PVA) is most preferred because it contains few impurities such as Na and K that affect observation. The degree of polymerization and degree of saponification in the state dissolved in water before solidification are not particularly limited as long as they can exhibit the predetermined functions of the present invention. In the case of PVA, from the viewpoint of impregnating solid materials with appropriate viscosity and ease of handling, examples include a degree of polymerization in the range of 1500 to 1700 and a degree of saponification in the range of 90 to 100 mol%.

When a water-soluble polymer is dissolved in water and a water-soluble heavy metal salt is added, the functional groups of the water-soluble polymer react to form a derivative of the water-soluble polymer, depending on the type and blending ratio of the two water-soluble components. is formed. The embedding and fixing composition of the present invention also contains this water-soluble polymer derivative. Examples described later suggest hydrolysis of residual acetate groups in PVA by strong acidity and crosslinking between hydroxyl groups in PVA by PTA. The ratio of polymers in the composition that serves as a derivative and the ratio of functional groups in the polymer to be reacted vary depending on the types of water-soluble polymer and water-soluble heavy metal salt and their blending ratio, and the ratio of polymers in the composition to be a derivative and the ratio of functional groups in the polymer to be reacted vary depending on the types of water-soluble polymer and water-soluble heavy metal salt and their blending ratio. There is no particular limitation as long as it can exhibit the following. It may be part or all.

Although the water-soluble polymer derivative maintains its properties as a water-soluble polymer, the formation of the water-soluble polymer derivative can be expected to contribute to the effectiveness of the embedding and fixing composition of the present invention. For example, it is thought that by improving the dispersibility of the heavy metal salt and polymer in an aqueous solution, the dispersibility of the heavy metal salt and polymer also improves in the sample after embedding and fixation. In addition, the solubility of the solid in water after drying and solidification decreases, and the hardness of the solid after crosslinking and hardening increases, resulting in improved handling and processability of the sample after embedding and fixation. is possible.

The composition for embedding and fixing of the present invention comprises two components: the water-soluble heavy metal salt and the water-soluble polymer, the water-soluble heavy metal salt and the water-soluble polymer derivative, or the water-soluble heavy metal salt and the water-soluble polymer. It is an aqueous solution in which three components, a molecule and a water-soluble polymer derivative, are dissolved in water. It is preferable that the two or three water-soluble components are completely dissolved, but even if a small amount is not completely dissolved and is dispersed in the form of fine particles, it is possible to completely dissolve the two or three water-soluble components as long as the predetermined function of the present invention can be achieved. It can be equated with the state of being dissolved in. The water is preferably pure water, such as RO water, distilled water, ion-exchanged water, or purified water, which does not contain impurities such as Na or K.

It can be prepared according to a conventional method, and a water-soluble polymer and a water-soluble heavy metal salt are mixed in water and dissolved using a combination of stirring and heating. For example, in the case of PVA, resin powder is mixed with water at room temperature while stirring to disperse it well, and then heated to less than 80 to 100°C and stirred for 30 to 60 minutes to completely dissolve. Next, a water-soluble heavy metal salt is added, but if the aqueous solution is mixed at a high temperature, oxides will be generated and coloration will occur easily, so it is preferable to mix the aqueous solution with stirring after it has cooled down to completely dissolve it.

The amount of water-soluble polymer blended is sufficient to include the heavy metal salt in a dispersed state when solidified, and the resulting aqueous solution has a moderately low viscosity and is easy to impregnate into the microstructure of the solid material. It is set taking into account that no undissolved material will be left below the solubility (saturation concentration). For example, in the case of PVA, it is preferably 0.5 to 15% by mass, more preferably 1.0 to 10% by mass, even more preferably 2.0 to 7.5% by mass, based on the entire composition (aqueous solution). % range.

The amount of water-soluble heavy metal salt to be mixed must be such that it provides sufficient contrast to the solid material to be embedded, that no undissolved material is left below the solubility (saturation concentration), and that crystals do not precipitate when solidified. It is set taking into account the following. For example, in the case of PTA, it is preferably 0.25 to 40% by mass, more preferably 0.5 to 35% by mass, even more preferably 2.5 to 20% by mass, based on the total amount of the composition (aqueous solution). range.

The blending ratio of the water-soluble polymer and the water-soluble heavy metal salt is similarly set in consideration of obtaining sufficient contrast, not leaving any undissolved material, and not precipitating crystals. The range of water-soluble polymer: water-soluble heavy metal salt is preferably 5:1 to 1:20, more preferably 3:1 to 1:15, even more preferably 2:1 to 1:12.5. .

The embedding and fixing composition of the present invention allows the brightness of the background of an observed image to be adjusted by changing the amount and type of the water-soluble heavy metal salt, similar to adjusting the monotone background with black and white paint. Can be done. This makes it possible to optimize the contrast (shading) by changing the type of composition used for embedding in accordance with the constituent elements of the solid material to be observed, thereby improving the visibility of the observed image. Operability and convenience during observation are improved by preparing in advance two or more types of embedding and fixing compositions with varying contents and types of water-soluble heavy metal salts and filling them into a container to form a kit.

The embedding and fixing composition of the present invention may further contain a conductive polymer. By imparting conductivity, it is possible to prevent the charging phenomenon (charge-up) during electron microscopy observation, and depending on the sample preparation conditions, it is possible to omit or simplify the conductive treatment by sputtering or vapor deposition of precious metals. Can be done. Furthermore, like conventional carbon pastes and carbon tapes, it can be used in applications where it is made into a sheet and particles or fiber samples are attached or temporarily fixed to the surface of the sheet for easy observation without conductive treatment.

The conductive polymer is preferably a polymer that can impart suitable conductivity and has excellent dispersibility in an aqueous solution. Specific examples include a complex of polyethylene dioxythiophene and polystyrene sulfonic acid (PEDOT/PSS), polyaniline, P3HT, PCBM, and the like. PEDOT/PSS is most preferred from the viewpoint of utilizing gelation when mixed with PVA containing a metal salt. Adding a conductive polymer to the embedding and fixing composition may promote gelation of the water-soluble polymer, so instead of adding it to the embedding and fixing composition in advance, fill it in a separate container. Preferably, it is in the form of a kit, which is prepared in advance and mixed when necessary.

The amount of the conductive polymer to be added is determined taking into account that sufficient conductivity can be imparted after solidification, that it is uniformly dispersed in an aqueous solution, and that it does not interfere with the predetermined functions of the present invention. For example, in the case of PEDOT/PSS, it is preferably 0.1 to 2% by mass, more preferably 0.25 to 1.5% by mass, even more preferably 0.5 to 2% by mass, based on the total amount of the composition (aqueous solution). The range is 1.0% by mass.

A two-component mixture in which the water-soluble heavy metal salt and water-soluble polymer contained in the present invention are stored separately in a closed container in the form of a solid such as powder, granules, tablets, or an aqueous solution dissolved in water, and prepared at the time of use. It can also be used as a mold kit. As a result, it is possible to freely prepare an embedding and fixing composition of a desired concentration from the two-component reagents, thereby reducing the number of reagents for each concentration, simplifying the process, and reducing costs. Furthermore, by storing the two components separately, changes over time can be suppressed and stable storage can be achieved for a longer period of time.

Specifically, water-soluble heavy metal salts that are easily dissolved are stored in the form of powder or aqueous solution, and water-soluble polymers are stored in the form of aqueous solution, and the necessary amounts of water-soluble heavy metal salts and water-soluble polymers are collected during observation. For example, a composition for embedding and fixing having a desired concentration is prepared by weighing, dissolving and mixing a water-soluble heavy metal salt with a water-soluble polymer. The concentration of the two component aqueous solutions prepared in advance may be one type or two or more types. Examples of containers to be filled with solids such as powders include sealable plastic bags and containers, and examples of containers to be filled with aqueous solutions include glass vials.

The embedding and fixing composition of the present invention is an aqueous solution in which the two or three water-soluble components are dissolved in water, or an aqueous solution in which a conductive polymer is dispersed. In order to further improve the properties, other components such as surfactants may be added, but if Na or K is mixed in, it may have an adverse effect on observation using an electron microscope or an X-ray microscope, so the interface It is desirable that other components such as the activator do not contain Na, K, etc.

The present invention also provides an observation method using an electron microscope or an X-ray microscope, which includes the steps of impregnating an embedding and fixing composition, solidifying the composition, and exposing an observation surface. The microstructure of solid materials can be clearly observed with simple operations.

The embedding and fixing composition of the present invention is an aqueous solution of moderate viscosity and has excellent impregnation properties into the microstructure of solid materials. Therefore, the solid material may be immersed in the aqueous solution, the solid material and the aqueous solution may be mixed and stirred, or the aqueous solution may be applied to the surface of the solid material. Further, impregnation treatment such as reduced pressure may be used in combination.

The microstructure of the solid material can be solidified by impregnating the aqueous solution into the microstructure of the solid material and solidifying or curing the solution after the voids are filled, thereby embedding and fixing the microstructure of the solid material. Solidification by evaporating water through natural drying is the simplest method and has the least burden on the sample. Decompression and heating may be used together. The composition after being solidified or cured by impregnating, mixing, or coating a solid material may be in any form such as a hard solid, a soft solid, a gel, or a paste.

Alternatively, crosslinks may be chemically formed between water-soluble polymers and cured. This makes it possible to impart water resistance to the embedded and fixed sample. A conventional method can be used for chemical crosslinking. For example, in the case of PVA, crosslinking can be promoted by adding formaldehyde and hydrochloric acid while heating to 80° C. or higher.

By processing the embedded and fixed solid material to expose the observation surface, the internal structure of the solid material can be observed. Examples of processing methods include FIB (Focused Ion Beam) processing using a focused ion beam device, CP (Cross Section Polisher) processing using an ion milling device, and physical cutting using a blade.

Electron microscopes used in the observation method of the present invention include a scanning electron microscope (SEM), a low-vacuum pressure scanning electron microscope (LVP-SEM), and a focused ion beam device (Focused ion beam device). Ion Beam System (FIB) and Transmission Electron Microscope (TEM). Examples of the X-ray microscope (XRM) include a soft X-ray or hard X-ray scanning microscope, and a three-dimensional X-ray microscope (X-Ray Computed Tomography: X-ray CT).

Hereinafter, the embedding and fixing composition of the present invention, its manufacturing method, and observation method using the same will be specifically explained with reference to Examples, Comparative Examples, and Test Examples. Note that the present invention is not limited to these Examples, etc., and various changes can be made without departing from the technical idea of the present invention.

[Example 1]
5 g of polyvinyl alcohol (PVA) (manufactured by Tokyo Kasei Kogyo Co., Ltd., product code P0469) was gradually mixed with 90 g of pure water while stirring at room temperature to sufficiently disperse the mixture. Subsequently, the mixture was heated to 80° C. with stirring, and further heated stepwise to 90, 95, and 98° C., and stirred at each temperature for 30 minutes to completely dissolve. After this solution cooled to room temperature, 5 g of phosphotungstic acid (PTA) (manufactured by TAAB) was gradually mixed and dissolved with stirring to prepare an embedding and fixing composition (aqueous solution) of the present invention. Its pH was 1.40.

Add 0.01 to 0.1 g of dried carbon paste powder (C: atomic number 6) (manufactured by TED PELLA, product name: Aquaduck) to 1 g of this aqueous solution, mix, apply to the surface of a silicon wafer, and dry naturally. and solidified. Impregnation treatment such as reduced pressure was not performed.

After conducting conductive treatment by coating with C or Os by sputtering, FIB processing was performed using a focused ion beam device (manufactured by FEI, model: Scios) equipped with a scanning electron microscope (SEM), and the solidified sample and silicon wafer were A vertical cross section was formed on the surface of. The entire sample was tilted at 52 degrees from the horizontal position, and the exposed cross section was observed from above using an SEM. The observation results are shown in FIGS. 1A and B. FIG. 1B is an enlarged observation image of the sample portion in FIG. 1A.

[Comparative example 1]
A sample was prepared in the same manner as in Example 1, except that carbon powder was mixed in an aqueous solution in which only PVA was dissolved without adding PTA, and the sample was embedded and fixed, and its cross section was observed. The observation results are shown in FIGS. 2A and B.
[Comparative example 2]
A sample was prepared in the same manner as in Example 1, and its cross section was observed, except that carbon powder was mixed with a commercially available epoxy embedding resin (manufactured by GATAN, product name: G2 Epoxy) and embedded and fixed. The observation results are shown in FIGS. 3A and B.

From the results shown in Figures 1A and B, in Example 1, there was a large contrast between the dark color of the light element carbon powder and the light color of the solidified embedding composition containing the heavy metal tungsten, and the visibility of the observed image was improved. It can be seen that this has been improved and it is possible to identify fine structures down to the level of several tens of nanometers. On the other hand, from the results shown in FIGS. 2A and B and FIGS. 3A and B, in Comparative Examples 1 and 2, there was almost no contrast between the light element carbon powder and the solidified PVA and hardened epoxy embedding resin, and the observed images It can be seen that the visibility is poor and the microstructure cannot be sufficiently identified.

[Example 2]
Samples were prepared in the same manner as in Example 1, except that boron nitride powder aggregates (BN: atomic numbers 5 and 7) were used instead of carbon powder, and the cross sections were observed. The observation results are shown in FIGS. 4A to 4D.

From the results shown in FIGS. 4A to 4D, in Example 2, there was a large contrast between the dark color of the light element boron nitride powder and the light color of the solidified embedding composition, which was clearly visualized and the visibility of the observed image was improved. I can see that it is improving. A gap of approximately 10 nm between particles can also be identified from the observed image in FIG. 4D.

[Example 3]
Only the same embedding and fixing composition as in Example 1 was applied onto a silicon wafer and allowed to air dry to solidify. Next, a thin film with a thickness of about 100 nm was prepared using a focused ion beam device (FIB) (manufactured by FEI, model: Scios), and observed with a transmission electron microscope (TEM) (manufactured by Thermo Fisher Scientific, model: Talos). The observation results are shown in FIGS. 5A and B. Furthermore, the observation results of the electron beam diffraction image are shown in FIG. 5C.

From the results shown in Figures 5A and B, in the solidified composition of the present invention, what appears to be structures with a size of 1 nm or less can be seen here and there, such as in the arrow parts, but PTA is uniformly dispersed in PVA at the nano level. It can be seen that there is almost no structure. Further, from the results shown in FIG. 5C, it can be seen that the solidified composition is amorphous. It is thought that the nano-level dispersion of heavy metal salts in the polymer after solidification makes it possible to clearly visualize the microstructure.

[Test example 1]
The surface of the sample (PVA+PTA) obtained by drying and solidifying the embedding and fixing composition of Example 1, PVA and PTA, and vinylon synthesized according to a standard method was measured using an X-ray photoelectron spectroscopy (XPS) measuring device (manufactured by ULVAC-PHI). , Model Quantera SXM). The chemical bonding state of the constituent elements was analyzed using high-resolution analysis (narrow scan analysis). The acquired C1s and W4f spectra are shown in FIGS. 6A and B. The C1s and W4f spectra of a sample (vinylon+PTA) obtained by crosslinking and curing the embedding and fixing composition of Example 7, which will be described later, are also shown.

From the C1s spectrum in FIG. 6A, it can be seen that when PTA is added to PVA, the OC=O peak derived from the residual acetate group of PVA disappears. This suggests that the ester bond of the acetate group in PVA is hydrolyzed under strong acidity. It can also be seen that the ratio of C--O bonds in PVA is relatively reduced, and the ratio of C--C bonds is relatively increased. This suggests that the hydroxyl groups of PVA are chemically modified and crosslinked by PTA. These results indicate that PVA derivatives are formed in the aqueous solution.

As PVA derivatives are formed and residual acetate groups are greatly reduced, hydroxyl groups become relatively dominant in the functional groups of PVA. It is thought that this improves the dispersibility of PTA and PVA in the aqueous solution, and also improves the dispersibility of PTA and PVA in the sample after embedding and fixation. In addition, as the degree of saponification of PVA increases, the solubility of the solid in water after drying and solidification decreases, and the hardness of the solid after crosslinking and hardening increases, which increases the stability of the sample after embedding and fixation. It is conceivable that handling and processability will be improved.

On the other hand, from the W4f spectrum in FIG. 6B, it can be seen that even if PTA is added to PVA, the spectrum shape of tungsten (W) in PTA hardly changes. This shows that even if PTA is added to PVA, the structural change of PTA in an aqueous solution is extremely small.

[Example 4]
A cotton thread taken out from a commercially available garment was immersed in the same embedding and fixing composition as in Example 1, air-dried to solidify, and then embedded and fixed. Impregnation treatment such as reduced pressure was not performed. Next, it was cut with a razor, and its cross section was observed in the low vacuum mode of a scanning electron microscope (SEM) (manufactured by JEOL Ltd., Model JSM-7800F Prime). The observation results are shown in FIGS. 7A and B.

From the results shown in FIGS. 7A and B, it can be seen that by using the present invention, even fibers (organic substances) can be sufficiently embedded and fixed to the inside with simple processing, and the visibility of the microstructure is improved due to the high contrast.

[Example 5]
The surface of a commercially available polypropylene clear file was scratched with a cutter, and the same embedding and fixing composition as in Example 1 was applied thereto, followed by natural drying and solidification. Impregnation treatment such as reduced pressure was not performed. Next, it was cut with a razor, and its cross section was observed in the low vacuum mode of the SEM in the same manner as in Example 4. The observation results are shown in FIG. 8A.

[Comparative example 3]
A sample was prepared in the same manner as in Example 5, except that it was embedded using the same aqueous solution in which only PVA was dissolved as in Comparative Example 1, and its cross section was observed. The observation results are shown in FIG. 8B.

From the results in Figures 8A and B, although some peeling is observed, using the present invention, even with polymeric materials, even the details of defects can be sufficiently embedded with simple processing, and the high contrast makes it easy to see fine defects. I can see that it will improve.

[Example 6]
A commercially available polycarbonate washer (inner diameter 3 mm x outer diameter 8 mm x thickness 0.8 mm) was immersed in the same embedding and fixing composition as in Example 1, air-dried, solidified, and embedded. Next, it was observed from above using an X-ray microscope (XRM) (manufactured by Medi-Extech Co., Ltd., model MXT-160UU). The observation results are shown in FIG. 9A.

[Comparative example 4]
A sample was prepared and observed in the same manner as in Example 6, except that it was embedded using the same aqueous solution in which only PVA was dissolved as in Comparative Example 1. The observation results are shown in FIG. 9B.

From the results shown in FIGS. 9A and 9B, it can be seen that when the embedding and fixing composition of the present invention is used, polymeric materials can be observed with high contrast even when observed using an X-ray microscope. This indicates the possibility of embedding and fixing a porous polymer or a carbon filter using the present invention to visualize microstructures more clearly during observation using an X-ray microscope or X-ray CT.

[Example 7]
1 g of a mixture of the embedding and fixing composition and carbon powder was prepared under the same conditions as in Example 1, except that 10 g of PVA and 10 g of PTA were mixed and dissolved in 80 g of pure water. Subsequently, 0.08 mL of 13.4 mol/L formaldehyde was added and mixed well, and further 0.12 mL of 12.0 mol/L hydrochloric acid was added dropwise to chemically crosslink the PVA. The resulting gel composition was heated to 80° C. to evaporate water and harden it.

This cured composition was embedded and fixed in a commercially available epoxy embedding resin (manufactured by GATAN, product name: G2 Epoxy), and processed with an ion milling device (manufactured by JEOL Ltd., model IB-19520CCP) to cross-section it. was formed. Conductive treatment was performed by coating with C by sputtering, and the cross section was observed using a scanning electron microscope (SEM) (manufactured by JEOL Ltd., Model JSM-7800F Prime). The observation results are shown in FIGS. 10A and B. FIG. 10B is an enlarged observation image of the sample portion in FIG. 10A. Moreover, the appearance of the cured composition is shown in FIG. 10C.

From the results shown in FIGS. 10A and 10B, even in the sample in which the embedding and fixing composition of the present invention was chemically crosslinked and cured, the dark color of the light element carbon powder and the light color of the cured composition containing the heavy metal tungsten were found. It can be seen that there is a large contrast between the colors, the visibility of the observed image is improved, and fine structures down to the tens of nm level can be identified.

[Test example 2]
The cross section of a sample (vinylon+PTA) obtained by crosslinking and curing the embedding and fixing composition of Example 7 was measured by the same X-ray photoelectron spectroscopy (XPS) as in Test Example 1. The acquired C1s and W4f spectra are shown in FIGS. 6A and B and FIGS. 11A and B. Note that the scale of the peak intensity in FIGS. 11A and 11B has been adjusted for comparison.

In the C1s spectra in FIGS. 6A and 11A1 and 2, only the C--C bond spectrum appears, indicating that vinylon is formed from PVA by chemically crosslinking the embedding and fixing composition of Example 7. I know that there is. In addition, in the W4f spectra in Figures 11B1 and 2, the shape of the spectrum changes and shifts to the region of about 31 to 34 eV where the spectrum of metallic tungsten appears, indicating that WO ₃ of PTA is reduced by the addition of formaldehyde and hydrochloric acid. , it is thought that metal W or WO ₂ is formed.

[Comparative example 5]
Samples in which carbon paste powder was embedded and fixed using a PVA solution and an epoxy embedding resin were prepared in the same manner as in Comparative Examples 1 and 2. Next, FIB processing is performed using a focused ion beam device (manufactured by SII Nanotechnology Co., Ltd., model XVision 200TB) equipped with a field emission scanning electron microscope (FE-SEM) to obtain a backscattered electron image of the exposed cross section. A secondary electron image was observed. 12A1 and 2 show backscattered electron images, and FIGS. 12B1 and 2 show secondary electron images.

From the results in Figures 12A1 and 2, it can be seen that in the backscattered electron observation image, there is no contrast at all between the light element carbon powder (arrow area) and the solidified PVA and hardened epoxy embedding resin, and there is no microstructure at all. It turns out that it cannot be identified. On the other hand, from the results shown in FIGS. 12B1 and 2, it can be seen that in the secondary electron observation image, although there is a slight contrast and the outer edge can be identified, the visibility is poor and the fine structure cannot be identified.

[Example 8]
The blending ratio of polyvinyl alcohol (PVA) and phosphotungstic acid (PTA), PVA:PTA=20:1 (10g:0.5g), 10:1 (10g:1g), 3:1 (7.5g:2. Six types of embedding and fixing compositions were prepared: 5g), 1:1 (5g:5g), 1:3 (2.5g:7.5g), and 1:10 (1g:10g). The other sample preparation conditions were the same as in Example 1. Next, the surface of the sample was subjected to FIB processing in the same manner as in Comparative Example 5, and the exposed cross section was observed using FE-SEM. Observation results of samples embedded and fixed with each composition are shown in FIGS. 13A to 13F. All observed images were backscattered electron images obtained under constant detector conditions, and the contrast of the silicon wafer portion serving as the sample stage was the same for all samples.

From the results in Figures 13A to 13F, it can be seen that when the PVA:PTA blending ratio is 20:1 or 10:1, there is no contrast and the carbon powder (arrow area) is hardly visible, but from 3:1, contrast starts to appear and PTA It can be seen that as the blending ratio increases, the contrast increases in proportion. In particular, in the range of 1:1 to 1:10, there is sufficient contrast between the dark color of the carbon powder (arrow portion) and the light color of the solidified composition, improving visibility and improving the visibility of fine particles at the level of several tens of nanometers. It can be seen that the structure can be sufficiently identified. This shows that by changing the blending ratio of heavy metal salts in the embedding fixation composition depending on the constituent elements of the solid material to be observed, it is possible to optimize the contrast of the observed image and improve visibility.

For example, when observing platinum-supported carbon (Pt/C) as an electrode catalyst using an electron microscope, a small amount of PTA is blended to darken the background, corresponding to Pt, which has a large atomic number and is bright. On the other hand, when observing magnesium-supported carbon (Mg/C), an example is to mix a large amount of PTA to brighten the background, corresponding to dark Mg with a small atomic number.

There has never been a technique to control the contrast of an observed image by pre-treating the sample to be observed. This is not possible with the metal coating or negative staining mentioned above, and even with electronic staining it is extremely difficult and reproducible cannot be expected. The method of changing the compounding ratio of heavy metal salts using the embedding and fixing composition of the present invention can be achieved by simple pretreatment and has excellent reproducibility.

[Example 9]
Water-soluble heavy metal salts include ammonium molybdate ((NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O), gadolinium acetate ((CH ₃ COO) ₃ Gd·nH ₂ O), and lead acetate (Pb(CH ₃ COO)). ₂ ) was used. A total of six types of embedding and fixing compositions were prepared with the blending ratios of polyvinyl alcohol (PVA) and water-soluble heavy metal salts being 1:1 (5 g: 5 g) and 1:5 (2 g: 10 g). Other sample preparation conditions and observation conditions were the same as in Example 8. Observation results of samples embedded and fixed with each composition are shown in FIGS. 14A to 14F.

The results shown in Figures 14A to 14F show that even when water-soluble heavy metal salts other than PTA are used, there is a contrast between the dark color of the carbon powder and the bright color of the solidified composition, improving visibility and making it possible to identify the microstructure. I understand. It can also be seen that as the blending ratio of the water-soluble heavy metal salt increases, the contrast increases in proportion.

[Example 10]
1.1 mass of a complex of conductive polymer polyethylene dioxythiophene and polystyrene sulfonic acid (PEDOT/PSS) (manufactured by Sigma-Aldrich, product number 739332) was added to the same embedding and fixing composition as in Example 1. % dispersion liquid was added and mixed so as to have the blending ratio shown in Table 1 below.

Addition of PEDOT/PSS promotes gelation of the composition, so after addition, it was applied in the form of a sheet onto an aluminum sample stand and gelled. Acetylene carbon black (manufactured by STREM CHEMICALS, product number 06-0026) was sprinkled on top of the coating film to adhere and fix it. Next, the particle morphology was observed using a SEM (manufactured by FEI, model Scios) at an accelerating voltage of 1 kV without conducting conductive treatment. The observation results of sample No. 2 are shown in FIG. 15A, and Table 1 shows the presence or absence of charges at the time of observation.

Additionally, the prepared composition was applied in a linear form (width: approximately 2 mm x thickness: approximately 0.1 mm) onto a slide glass and dried. Next, the resistance value of this dried sample was measured using a tester with an electrode spacing of about 10 mm. The measurement results are also shown in Table 1.

[Comparative example 6]
A sample was prepared in the same manner as in Example 10, except that carbon black was fixed using a commercially available carbon paste (manufactured by TED PELLA, product name: Aquaduck), and carbon black particles on the carbon paste coating were fixed. The morphology was observed using SEM. The observation results are shown in FIG. 15B, and the presence or absence of charge at the time of observation is shown in Table 1. Table 1 also shows the measured resistance values of the coating films.

From the results in FIG. 15A, observation using the conductive coating film of Example 10 as a base shows that there is a large contrast between the dark color of carbon black and the light color of the gelled sheet containing tungsten as the base. It can be seen that visibility has improved. On the other hand, from the results shown in FIG. 15B, it can be seen that in observation using the conventional carbon paste as a base, visibility is poor because the main components of carbon black and carbon paste are the same and there is no contrast.

The observation method using the embedding fixation composition of the present invention shows a clear difference in image contrast obtained when observing solid materials with an electron microscope or an X-ray microscope when compared with an observation method using a conventional embedding resin. occurs. In particular, the smaller the atomic number of the elements constituting the solid material, the greater the contrast difference, allowing clearer observation. For example, it is extremely effective for solid materials composed only of carbon, such as graphite, carbon black, and diamond.

Therefore, the embedding and fixing composition of the present invention and the observation method using the same using an electron microscope etc. are useful for evaluation and analysis of materials such as carbon materials and polymer materials, and polymer electrolyte fuel cells using these materials. It is expected that this technology will be extremely useful in the industry, and it is expected that it will greatly contribute to the development of these industries.

1A1... Vertical cross section formed by FIB processing, 1A2... Cross section of Example 1 sample in which carbon powder was embedded and fixed, 1A3... Cross section of silicon wafer on sample stage, 1A4... Enlarged observation area of FIG. 1B.
2A1... Vertical cross section formed by FIB processing, 2A2... Cross section of Comparative Example 1 sample in which carbon powder was embedded and fixed, 2A3... Cross section of silicon wafer on sample stage, 2A4... Enlarged observation area of FIG. 2B.
3A1... Vertical cross section formed by FIB processing, 3A2... Cross section of Comparative Example 2 sample in which carbon powder was embedded and fixed, 3A3... Cross section of silicon wafer on sample stage, 3A4... Enlarged observation area of FIG. 3B.
4A1...Boron nitride powder aggregate, 4B1...Vertical section formed by FIB processing, 4B2... Cross section of Example 2 sample in which boron nitride powder aggregate was embedded and fixed, 4B3... Cross section of silicon wafer on sample stage.

5A1...Vertical section of the solidified Example 3 sample, 5A2... Vertical section of the silicon wafer on the sample stage.
8A1...Cross section of the solidified Example 5 sample, 8A2...Cross section of the polypropylene sheet, 8B1...Cross section of the cured Comparative Example 3 sample, 8B2...Cross section of the polypropylene sheet.
10A1: Cross-section formed by CP processing of a cross-linked and resin-embedded Example 7 sample, 10C1: Appearance of a cross-linked and resin-embedded Example 7 sample.

Claims (11)

1. An aqueous solution containing a water-soluble heavy metal salt and a water-soluble polymer and/or a derivative thereof, in which these water-soluble components are dissolved in water,
   When observing a solid material containing light elements as a main component using an electron microscope or an X-ray microscope, the visibility of the observed image is improved by the contrast between the heavy metals constituting the water-soluble heavy metal salt and the light elements that are the main components of the solid material. A composition for embedding and fixing that can improve
2. The water-soluble heavy metal salt is one or more selected from the group consisting of phosphotungstic acid (PTA), gadolinium acetate, ammonium molybdate, phosphomolybdic acid, potassium ferrocyanate, lead nitrate, and lead acetate, and the water-soluble polymer is The package according to claim 1, which is one or more selected from the group consisting of polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), polyacrylamide (PAM), polyacrylic acid (PAA) and polyethylene oxide (PEO). Composition for implantation.
3. The embedding and fixing composition according to claim 2, wherein the water-soluble heavy metal salt is phosphotungstic acid (PTA) and the water-soluble polymer is polyvinyl alcohol (PVA).
4. Any one of claims 1 to 3, which contains 0.25 to 40% by mass of a water-soluble heavy metal salt and 0.5 to 15% by mass of a water-soluble polymer and/or its derivative, based on the total amount of the composition. The embedding and fixing composition described in .
5. By providing two or more types of embedding and fixing compositions according to any one of claims 1 to 3 having different contents and/or types of water-soluble heavy metal salts, and changing the type of the embedding and fixing compositions. An observation kit for embedding and fixing compositions that allows the contrast of observation images to be adjusted.
6. By comprising the composition for embedding and fixing according to any one of claims 1 to 3 and a dispersion in which a conductive polymer is dispersed in water, and adding the dispersion to the composition for embedding and fixing. A kit for observing embedding and fixing compositions that can be imparted with conductivity.
7. The kit for observing an embedding and fixing composition according to claim 6, wherein the conductive polymer is a composite of polyethylene dioxythiophene and polystyrene sulfonic acid (PEDOT/PSS).
8. A water-soluble heavy metal salt in the form of a solid or an aqueous solution, and a water-soluble polymer in the form of a solid or an aqueous solution, wherein the water-soluble heavy metal salt and the water-soluble polymer are mixed in addition to water or mixed with each other. A kit for producing an embedding and fixing composition, which can produce the embedding and fixing composition according to any one of claims 1 to 3 by dissolving or mixing.
9. The composition for embedding and fixing according to any one of claims 1 to 3, comprising the steps of adding and dissolving a water-soluble polymer in water, and adding and dissolving a water-soluble heavy metal salt to the resulting aqueous solution. manufacturing method.
10. Impregnating a solid material with the embedding and fixing composition according to any one of claims 1 to 3, and evaporating water in the embedding and fixing composition, or a water-soluble polymer and/or a derivative thereof. An observation method using an electron microscope or an X-ray microscope, which includes a step of solidifying by crosslinking the solid material, and a step of processing the embedded and fixed solid material to expose an observation surface.
11. The method further includes the step of optimizing the contrast of the observed image by using two or more types of embedding and fixing compositions according to any one of claims 1 to 3 having different contents and/or types of water-soluble heavy metal salts. An observation method using an electron microscope or an X-ray microscope according to claim 10.

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