Polymer and manufacturing method thereof

Abstract

According to the polymer and the preparation method thereof provided by the invention, the hydrogel is modified by HAMA, and the modified hydrogel has the characteristic of difficulty in fragmentation, has moderate strength and can be used for cutting scanning. The modified hydrogel is matched with an iterative expansion microscopy (iExM) technology, so that the amplification factor can reach more than 60 times. Therefore, the modified and amplified hydrogel sample can be cut for layer-by-layer microscopic observation, so that the application of the expansion microscopy technology is not limited by the condition that the sample is too large and cannot be operated in a limited working distance, and the application range of the expansion microscopy technology is expanded; furthermore, the novel iExM modified hydrogel system can further reach the analytic scale of nearly tens of nanometers for the biological tissue, so that the microstructure and secret of more biological tissues can be known, and the biological structure and physiology can be deeply known.

Description

Technical Field

[0001] The present invention relates to a method for fabricating polymers with polymers, and particularly to a polymer and its fabrication method. Background Art

[0002] Expansion microscopy (ExM) is a super-resolution imaging technique. This method fixes fluorescent molecules on colloids, and then takes advantage of the property of hydrogel to absorb water and swell, expanding the distance between fluorescent molecules in the sample, thereby breaking through the diffraction limit and achieving the effect of improving resolution.

[0003] However, due to the working distance limitation of fluorescence microscopes, hydrogel samples beyond the working distance of fluorescence microscopes cannot be observed, which further limits the application of ExM in large tissues and iterative expansion microscopy (iExM). And currently, most of the hydrogels used for ExM on the market lack rigidity and are prone to fragmentation or have too high elasticity, resulting in difficulty in cutting. Therefore, how to develop hydrogel materials suitable for cutting and scanning in expansion microscopy technology is an important topic. Summary of the Invention

[0004] In view of this, the present disclosure provides a polymer and its fabrication method, which is used as a hydrogel for fabricating samples to be used within the limited working distance of a fluorescence microscope.

[0005] The present disclosure provides a method for fabricating a polymer, including: performing immunostaining on a first sample to obtain a second sample; treating the second sample with an anchoring agent to obtain a third sample; placing the third sample in a first swollen hydrogel solution for infiltration to obtain a first polymer embedding the biological sample; placing the first polymer gel in a polymer solution for embedding to obtain a second polymer embedding the biological sample; and placing the second polymer in a second swollen hydrogel solution to obtain a third polymer embedding the biological sample, wherein the second swollen hydrogel solution contains Hyaluronic acid Methacryloyl (HAMA).

[0006] In an embodiment of the present disclosure, the step of placing the third sample in the first swollen hydrogel solution for infiltration to obtain the first polymer embedding the biological sample further includes: placing the first polymer in the first swollen hydrogel solution at least twice to swell the first polymer.

[0007] In one embodiment of the present disclosure, the step of placing a third sample in a first swollen hydrogel solution for permeation to obtain a first polymer embedding a biological sample further includes: placing the first polymer in a digestion buffer, wherein the digestion buffer includes Proteinase K or SDS.

[0008] In one embodiment of the present disclosure, the first sample is a biological sample, and the first sample includes cells, organs or tissues.

[0009] In one embodiment of the present disclosure, the immunostaining includes immunofluorescence staining.

[0010] In one embodiment of the present disclosure, the anchoring agent includes a reagent having a biomolecule-reactive chemical group and a hydrogel-reactive chemical group.

[0011] In one embodiment of the present disclosure, the anchoring agent includes 6-((acryloyl)amino)hexanoic acid N-hydroxysuccinimide ester (Acryloyl-X), N-hydroxysuccinimide methacrylate (MA-NHS), or methacrolein.

[0012] In one embodiment of the present disclosure, the first swollen hydrogel solution includes any one or a combination of N,N-dimethylacrylamide (DMAA), acrylamide (AA), sodium acrylate (SA), N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), and hyaluronic acid methacryloyl (HAMA).

[0013] In one embodiment of the present disclosure, the polymer solution for embedding includes any one or a combination of acrylamide (AA), sodium acrylate (SA), and N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA).

[0014] In one embodiment of the present disclosure, the first swollen hydrogel solution and the polymer solution for embedding further include a polymerization activator, and the polymerization activator includes ammonium persulfate (APS), potassium persulfate (KPS), or tetramethylethylenediamine (TEMED).

[0015] In one embodiment of the present disclosure, the second swollen hydrogel solution includes any one or a combination of N,N-dimethylacrylamide (DMAA), sodium acrylate (SA), hyaluronic acid methacryloyl (HAMA), and N,N'-methylenebisacrylamide (MBAA).

[0016] In one embodiment of the present disclosure, the composition of the second swollen hydrogel solution includes: 10% to 40% of N,N-dimethylacrylamide (DMAA), 10% to 30% of sodium acrylate (SA), 0.01% to 2.0% of hyaluronic acid methacrylate (HAMA), and 0.001% to 0.5% of N,N'-methylenebisacrylamide (MBAA).

[0017] In one embodiment of the present disclosure, the molecular weight of hyaluronic acid methacrylate is from 0.1 kDa to 2000 kDa.

[0018] In one embodiment of the present disclosure, the molecular weight of hyaluronic acid methacrylate is from 50 kDa to 80 kDa.

[0019] In one embodiment of the present disclosure, the third polymerization is for microscopic analysis.

[0020] In one embodiment of the present disclosure, before the step of immunostaining the first sample to obtain the second sample, it further includes: fixing the first sample with a fixative, where the fixative contains paraformaldehyde (PFA).

[0021] The present disclosure also provides a polymer for embedding biological samples and can be obtained by the foregoing manufacturing method.

[0022] In one embodiment of the present disclosure, the embedded biological sample is for microscopic analysis.

[0023] In one embodiment of the present disclosure, the microscopic analysis includes application to expansion microscopy.

[0024] Based on the above, the present disclosure modifies the hydrogel with HAMA through a polymer manufacturing method. The modified hydrogel has the characteristics of being not easily fragmented, and its strength is moderate enough to be used for cutting and scanning. The modified hydrogel combined with the iExM technology can achieve a magnification of sixty times. In this way, the modified hydrogel can make the application of expansion microscopy no longer limited by the working distance, improve the application range of expansion microscopy, and further advance in the development of large tissues and iExM, so as to understand more about the microstructures and mysteries of biological tissues, and thus gain a deeper understanding of the structure and physiology of organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flowchart of the polymer manufacturing method of the present disclosure.

[0026] Figure 2 is a schematic diagram of the means for solving problems in the present disclosure.

[0027] Figure 3 is a schematic diagram of the composition of the polymer of the present disclosure.

[0028] Figure 4 It is a figure obtained by observing the polymer of the present disclosure in a biological sample under a microscope.

[0029] Figure 5 It is a schematic diagram of the scanned image after cutting a biological sample with an ultrasonic scalpel in the present disclosure.

[0030] Description of the Reference Numerals

[0031] S110, S120, S130, S140, S150: Processes

[0032] 210, 220, 230, 240: Phenomena

[0033] 211, 212, 221: Distances

[0034] 301, 302, 303, 304, 310, 311, 320, 321, 322, 330, 340, 341, 350, 360, 370, 380: Molecules

[0035] 501, 502, 511, 512, 513, 514, 515, 516, 517, 518: Distances

[0036] 503: Analyte Detailed Description of the Embodiments

[0037] Reference will now be made in detail to exemplary embodiments of the present disclosure, and examples of the exemplary embodiments will be described in the accompanying drawings. The terms "first", "second", etc. mentioned throughout the specification of this case (including the claims) are used to name components or to distinguish different embodiments or scopes, and are not used to limit the upper or lower limits of the number of components, nor to limit the order of components. Additionally, wherever possible, components / elements denoted by the same reference numerals in the drawings and embodiments represent the same or similar parts.

[0038] Please refer to Figure 1 , Figure 1 It is a flowchart of the method for fabricating the polymer of the present disclosure. In process S110, a first sample is immunostained to obtain a second sample. In process S120, the second sample is treated with an anchoring agent to obtain a third sample. In process S130, the third sample is placed in a first swelling hydrogel solution for permeation to obtain a first polymer embedding the biological sample. In process S140, the first polymer is placed in a polymer solution for embedding to obtain a second polymer embedding the biological sample. In process S150, the second polymer is placed in a second swelling hydrogel solution to obtain a third polymer embedding the biological sample, wherein the second swelling hydrogel solution contains hyaluronic acid methacrylate.

[0039] Specifically, the hydrogel polymer obtained through this disclosure has the characteristics of being not easily broken and having moderate elasticity, such that the biological sample produced using the hydrogel polymer of this disclosure is easy to cut and not prone to breaking during cutting. In this way, the size of the hydrogel can be kept within the working distance of the fluorescence microscope to perform fluorescence scanning of the biological sample.

[0040] Figure 2 It is a schematic diagram of the means for solving the problem in this disclosure. Please refer to Figure 2 , in phenomenon 210, the working distance 211 of the microscope is less than the height 212 of the biological sample after being embedded in the hydrogel, resulting in the biological sample being unable to be placed under the microscope for observation. Therefore, if a cut sample is obtained by cutting the biological sample embedded in the hydrogel, as shown in phenomenon 220, there is a gap 221 between the bottom of the microscope lens and the top of the cut sample, and at this time, the cut sample can be observed through the microscope.

[0041] Continuing from the previous paragraph, however, the biological sample made of the conventionally used hydrogel has the problem of being prone to breaking during cutting. As shown in phenomenon 230, if the sample is prone to breaking, it cannot be cut into a thickness suitable for microscopic observation. Therefore, in phenomenon 240, if a high molecular monomer, hyaluronic acid methacrylate (HAMA), is added to the hydrogel through this disclosure as a cross-linking agent to improve the hydrogel composition commonly used in expansion microscopy, the biological sample is not easily broken during cutting and is suitable for expansion microscopy cutting and scanning.

[0042] In the embodiment of this disclosure, first, the hydrogel polymer is prepared. Then, the prepared hydrogel polymer is subjected to an embedding operation with the biological sample.

[0043] In the embodiment of this disclosure, specifically, first, the sample (the first sample) is immunostained to obtain the immunostained sample (the second sample). Next, the immunostained sample is treated with an anchoring agent to obtain the anchored immunostained sample (the third sample). Then, the third sample is placed in the first swelling hydrogel solution for penetration to obtain the first polymer, and the first polymer is placed in the embedding polymer solution to obtain the second polymer. Finally, the second polymer is placed in the second swelling hydrogel solution including hyaluronic acid methacrylate to obtain the third polymer. This third polymer is the final hydrogel product of this disclosure. The aforementioned sample is a biological sample, including cells, organs, or tissues. The aforementioned immunostaining includes immunofluorescence staining.

[0044] In one embodiment of the present disclosure, before the step of immunostaining the sample to obtain the immunostained sample, it further includes fixing the first sample with a fixative, and the fixative contains paraformaldehyde (PFA).

[0045] Furthermore, in one embodiment of the present disclosure, the process of obtaining the aforementioned first polymer further includes placing the first polymer in a first swollen hydrogel solution twice to obtain the first polymer. That is, the first polymer can be first placed in the first swollen hydrogel solution for a period of time, then the first swollen hydrogel solution is updated, and then the first polymer that has been immersed in the first swollen hydrogel solution once is immersed in the updated first swollen hydrogel solution. Finally, the first polymer immersed in the first swollen hydrogel solution twice is obtained.

[0046] Continuing from the previous paragraph, since the first swollen hydrogel solution includes any one or a combination of N,N-dimethylacrylamide (DMAA), acrylamide (AA), sodium acrylate (SA), N,N'-(1,2-dihydroxyethylene) bisacrylamide (DHEBA), and hyaluronic acid methacrylate (HAMA), if immersed only once, there is a possibility that the above-mentioned molecules cannot completely replace the functional groups of the first polymer. Therefore, through the soaking process twice or more, the above-mentioned molecules can be significantly attached to the functional groups of the first polymer.

[0047] In one embodiment of the present disclosure, the polymer solution for embedding includes any one or a combination of acrylamide (AA), sodium acrylate (SA), and N,N'-(1,2-dihydroxyethylene) bisacrylamide (DHEBA).

[0048] In other embodiments of the present disclosure, the aforementioned first swollen hydrogel solution may include a polymerization activator, and the polymer solution for embedding may also include a polymerization activator. The polymerization activator includes ammonium persulfate (APS), potassium persulfate (KPS), or tetramethylethylenediamine (TEMED). Through the polymerization activator, the added DMAA, AA, SA, or DHEBA can be more easily attached to the functional groups of the first polymer.

[0049] In an embodiment of the present disclosure, the second swollen hydrogel solution includes any one or a combination of N,N-dimethylacrylamide (DMAA), sodium acrylate (SA), hyaluronic acid methacrylate (HAMA), and N,N'-methylenebisacrylamide (MBAA). The composition of the second swollen hydrogel solution may include 10% to 40% of N,N-dimethylacrylamide (DMAA), 10% to 30% of sodium acrylate (SA), 0.01% to 2.0% of hyaluronic acid methacrylate (HAMA), and 0.001% to 0.5% of N,N'-methylenebisacrylamide (MBAA).

[0050] Please refer to Figure 3 , Figure 3 which is a schematic diagram of the composition of the polymer disclosed herein. In the hydrogel, 301 is, for example, a polymer chain of AA / SA, 302 is, for example, the crosslinking of MBAA, 303 is the branch of DMAA, and 304 is, for example, the crosslinking of HAMA. The detailed molecular structure of HAMA is shown as molecule 310, the detailed molecular structure of DMAA is shown as molecule 320, the detailed molecular structure of SA is shown as molecule 330, and the detailed molecular structure of MBAA is shown as molecule 340.

[0051] Continuing from the previous paragraph, molecule 310 can form HAMA crosslinks through crosslinking, as shown in molecule 311. Since HAMA can form crosslinks, the molecular weight of the HAMA crosslinks can be determined by the number of crosslinking times. The molecular weights of different HAMA crosslinks can affect the final magnification factor, which will be described in detail in the subsequent paragraphs.

[0052] Continuing from the previous paragraph again, molecule 350 is a polymer chain of DMAA / SA, which can be formed by crosslinking DMAA and SA. In addition, molecule 321 and molecule 322 are, respectively, DMAA crosslinks and DMAA branches, and molecule 341 is an MBAA crosslink. As the method for fabricating the polymer disclosed herein, the aforementioned molecules can be respectively added to the first swollen hydrogel solution, the polymer solution for embedding, or the second swollen hydrogel solution, so that when performing processes S130 to S150, the functional groups of the polymer can be replaced, thereby achieving the magnification effect of the hydrogel polymer.

[0053] Please continue to refer to Figure 3 , molecule 360 is a HAMA / DMAA crosslink, molecule 370 is a HAMA / DMAA branch, and molecule 380 is a HAMA / DMAA / SA polymer chain. In the polymer with HAMA crosslinks, molecule 360, molecule 370, and molecule 380 can replace the functional groups on the polymer with HAMA crosslinks, thereby achieving the magnification effect of the hydrogel polymer after adding HAMA.

[0054] In another embodiment of the present disclosure, the process of obtaining the aforementioned first polymer further includes placing the first polymer in a digestion buffer, and the digestion buffer may include Proteinase K or SDS.

[0055] In one embodiment of the present disclosure, the anchoring agent includes a reagent having biomolecule-reactive chemical groups and hydrogel-reactive chemical groups. Among them, the anchoring agent may further include 6-((acryloyl)amino)hexanoic acid N-succinimidyl ester (Acryloyl-X), N-hydroxysuccinimidyl methacrylate (MA-NHS), or methacrolein. Through the anchoring agent, the functional groups on the obtained first polymer can be more stably attached thereto.

[0056] In the embodiments of the present disclosure, different from the contents added to the polymer in the past, the present disclosure also adds hyaluronic acid methacrylate. Further, the molecular weight of hyaluronic acid methacrylate can also affect the subsequent amplification of the sample and the clarity of the sample. In one embodiment of the present disclosure, the molecular weight of hyaluronic acid methacrylate is from 0.1 kDa to 2000 kDa. In other embodiments of the present disclosure, the molecular weight of hyaluronic acid methacrylate is from 50 kDa to 80 kDa.

[0057] Please refer to Figure 4 , Figure 4 which is a diagram observed under a microscope when the polymer of the present disclosure is applied to a biological sample. As Figure 4 shown, when the molecular weight of hyaluronic acid methacrylate (HAMA) added to the hydrogel is 80 kDa, it can be seen from (a1), (b1), and (c1) observed under the microscope before addition and (a2), (b2), and (c2) observed after the hydrogel is added with HAMA and amplified. In the diagrams at a scale of 1 mm for (a1), (b1), and (c1) and a scale of 1 cm for (a2), (b2), and (c2), the size of the tissue is almost the same. That is to say, if (a2), (b2), and (c2) are continuously amplified to a scale of 1 mm, more detailed diagrams of the tissue can be further observed. In the present disclosure, the magnification obtained by adding HAMA with a molecular weight of 80 kDa to the hydrogel is 5.87 times. Therefore, a magnification of 58.7 times can be obtained by multiplying the 10-fold magnification of the microscope lens by the 5.87-fold magnification of the hydrogel.

[0058] Continuing from the previous paragraph, when the molecular weight of hyaluronic acid methacrylate (HAMA) added to the hydrogel is 5 kDa, the sizes of the tissues are almost the same in the images of (d1), (e1), and (f1) observed under the microscope before addition and (d2), (e2), and (f2) observed after adding HAMA to the hydrogel and magnifying. That is, in the images of (d1), (e1), and (f1) at a scale of 1 mm and (d2), (e2), and (f2) at a scale of 1 cm. This means that if (d2), (e2), and (f2) are continuously magnified to a scale of 1 mm, more detailed images of the tissue can be further observed. In this disclosure, the magnification obtained by adding HAMA with a molecular weight of 5 kDa to the hydrogel is 6.18 times. Therefore, a magnification of 61.8 times can be obtained by multiplying the 10-fold magnification of the microscope lens by the 6.18-fold magnification of the hydrogel.

[0059] As can be seen from the above, by adding HAMA to the hydrogel, the original sample can be magnified by about 6 times, and different molecular weights of HAMA can affect the final magnification. The implementers of this disclosure can adjust the molecular weight of HAMA according to actual needs to obtain the required magnification. Furthermore, it can be further known from the above that after adding HAMA, the magnified tissue is not damaged and is suitable for observation.

[0060] In the embodiments of this disclosure, the third polymer obtained through the polymer production method can be used for embedding biological samples and further for microscopic observation and subsequent analysis. In addition, the aforementioned microscopic analysis can further include application to expansion microscopy.

[0061] Please refer to Figure 5 , Figure 5 which is a schematic diagram of the scanned image of a biological sample after cutting with an ultrasonic scalpel in this disclosure. In Figure 5 , a microscope lens with a 10-fold magnification is used, and the hydrogel polymer made as in Figure 3 is used to magnify the sample by 6 times, for a total magnification of 60 times (10 multiplied by 6). Since the sample is embedded after being cut with an ultrasonic scalpel, the microscope lens can be at a distance of 501 from the sample. This distance can be, for example, 2500 μm, and the focusing distance 502 for each scan can be, for example, 1000 μm, and within this distance 502, the sample includes a part of the tissue of the analyte 503. At this time, the observer can observe the state of the tissue of the analyte within the distance 502 through the microscope and record the state, for example, in the form of a fluorescence staining image within the distance 511 in the scanned tissue structure 510 by taking pictures. The observer can repeat the above actions, and each time the observation is made, the focusing position is moved downward by a distance with respect to the sample. In this way, the tissue of each layer of the sample of the analyte 503 corresponding to each observation can be recorded from the distance 511 to the distance 518.

[0062] Continuing from the previous paragraph, since the hydrogel improved through the present disclosure has the characteristics of making the sample easy to cut and not easy to break, the observer can also cut the embedded sample into layers with a height of 1000 μm each by an ultrasonic knife, and perform observation records separately to stack them as shown in the tissue structure 510.

[0063] In summary, the present disclosure modifies the hydrogel with HAMA through a polymer production method. The modified hydrogel has the property of not being easily fragmented, and its strength is moderate enough to be used for cutting and scanning. The modified hydrogel combined with the iExM technology can achieve a magnification of sixty times. In this way, the modified hydrogel can make the application of expansion microscopy no longer limited by the working distance, improve the application range of expansion microscopy, and further advance in the development of large tissues and iExM, so as to understand more microstructures and mysteries of biological tissues, and thus have a deeper understanding of the structure and physiology of organisms.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fabricating a polymer, characterized in that, Comprising: Immunostaining a first sample to obtain a second sample; Treating the second sample with an anchoring agent to obtain a third sample; Placing the third sample in a first swollen hydrogel solution for penetration to obtain a first polymer embedding the biological sample; Placing the first polymer gel in an embedding polymer solution to obtain a second polymer embedding the biological sample; and Placing the second polymer in a second swollen hydrogel solution to obtain a third polymer embedding the biological sample, wherein the second swollen hydrogel solution contains hyaluronic acid methacryloyl (HAMA).

2. The method for manufacturing a polymer according to claim 1, characterized in that The step of placing the third sample in the first swollen hydrogel solution for penetration to obtain the first polymer embedding the biological sample further comprises: Placing the first polymer in the first swollen hydrogel solution at least twice to swell the first polymer.

3. The method for producing the polymer according to claim 1, characterized in that, The step of placing the third sample in the first swollen hydrogel solution for penetration to obtain the first polymer embedding the biological sample further comprises: Placing the first polymer in a digestion buffer, wherein the digestion buffer comprises proteinase K or SDS.

4. The method for making a polymer according to claim 1, wherein, The first sample is a biological sample, wherein the first sample comprises cells, organs or tissues.

5. The method for producing a polymer according to claim 1, wherein, The immunostaining comprises immunofluorescence staining.

6. The method for producing the polymer according to claim 1, characterized in that, The anchoring agent comprises a reagent having a biomolecule-reactive chemical group and a hydrogel-reactive chemical group.

7. The method for manufacturing a polymer according to claim 1, characterized in that, The anchoring agent comprises 6-((acryloyl)amino)hexanoic acid succinimidyl ester (Acryloyl-X), N-hydroxysuccinimidyl methacrylate (MA-NHS) or methacrolein.

8. The method for manufacturing a polymer according to claim 1, wherein, The first swollen hydrogel solution comprises any one or a combination of N,N-dimethylacrylamide (DMAA), acrylamide (AA), sodium acrylate (SA), N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), hyaluronic acid methacryloyl (HAMA).

9. The method for manufacturing a polymer according to claim 1, wherein The embedding polymer solution comprises any one or a combination of acrylamide (AA), sodium acrylate (SA), N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA).

10. The method for producing a polymer according to claim 1, wherein The first swollen hydrogel solution and the embedding polymer solution further comprise a polymerization activator, wherein the polymerization activator comprises ammonium persulfate (APS), potassium persulfate (KPS) or tetramethylethylenediamine (TEMED).

11. The method for manufacturing a polymer according to claim 1, wherein, The second swollen hydrogel solution comprises any one or a combination of N,N-dimethylacrylamide (DMAA), sodium acrylate (SA), hyaluronic acid methacryloyl (HAMA), N,N'-methylenebisacrylamide (MBAA).

12. The method for fabricating a polymer according to claim 1, wherein, The composition of the second swelling hydrogel solution includes: 10% to 40% of N,N-dimethylacrylamide (DMAA), 10% to 30% of sodium acrylate (SA), 0.01% to 2.0% of hyaluronic acid methacrylate (HAMA), and 0.001% to 0.5% of N,N'-methylenebisacrylamide (MBAA).

13. The method for producing a polymer according to claim 1, characterized in that, The molecular weight of the hyaluronic acid methacrylate is 0.1 kDa to 2000 kDa.

14. The method for manufacturing a polymer according to claim 1, characterized in that, The molecular weight of the hyaluronic acid methacrylate is 50 kDa to 80 kDa.

15. The method for manufacturing a polymer according to claim 1, characterized in that, The third polymerization is for microscopic analysis.

16. The method for producing a polymer according to claim 1, wherein, Before immunostaining the first sample to obtain the second sample, it further includes: Fixing the first sample with a fixative, where the fixative contains paraformaldehyde (PFA).

17. A polymer for embedding a biological sample, the polymer is prepared by the method as described in claims 1-16.

18. The polymer according to claim 17, characterized in that, The embedded biological sample is for microscopic analysis.

19. The polymer according to claim 18, wherein, The microscopic analysis includes application to expansion microscopy.

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