A hydrogel continuous expansion method for super-resolution imaging and related kits

Abstract

The present application relates to a kind of hydrogel continuous expansion method and related kit for super-resolution imaging, the method comprises the following steps: S1: biological sample is carried out protein anchoring treatment;S2: the biological sample of S1 protein anchoring treatment is carried out first round hydrogel embedding and once expansion, S3: the biological sample of S2 processing is carried out protein denaturation treatment;S4: the biological sample of S3 denaturation processing is embedded in neutral glue formed by ATMS (B) solution, S5: the biological sample embedded in neutral glue in step S4 is carried out second round hydrogel embedding and twice expansion.The method of the present application can be twice expanded to cell or tissue sample, so that total linear expansion multiple reaches 40-50 times, and mechanical strength is moderate, and various microscopes can be combined to realize the visualization of subcellular structure, nano structure and single molecule.

Description

Technical Field

[0001] This application relates to the field of hydrogel technology, specifically to a method for continuous expansion of hydrogels and related reagent kits, and particularly to a technological innovation aimed at improving the expansion factor of hydrogels, with the goal of improving resolution issues in spatial analysis. Background Technology

[0002] Expandable hydrogel technology is a method that enhances the spatial resolution of tissue or cell samples by utilizing the expansion properties of hydrogel materials. With the continuous development of space biology and histology research, researchers increasingly need high-resolution techniques to accurately resolve the microscopic structures and their spatial distribution within cells and tissues. Traditional histological techniques, such as staining and microscopic observation, are often limited by sample size and resolution, making it difficult to capture minute subcellular structures, such as mitochondria and endoplasmic reticulum.

[0003] Expandable hydrogel technology has emerged as a space enhancement technique that effectively improves spatial resolution by embedding tissue samples within expandable hydrogels, allowing the tissue to expand without altering its morphology. This technology is widely used in biomedicine, pathology, and tissue engineering, playing a crucial role, particularly in high-resolution biological research such as spatial proteomics and RNAomics. While expandable hydrogel technology can significantly magnify the volume of tissue samples, current techniques typically only achieve limited expansion folds, usually between several and a dozen times. When studying minute subcellular structures (such as mitochondria and endoplasmic reticulum), existing expansion folds are still insufficient for high-resolution analysis. This means that even with increased sample volume, precise spatial analysis at finer scales remains challenging.

[0004] There are few reports on improving the expansion factor of hydrogels. The current mainstream method involves using two hydrogels with different formulations for two rounds of embedding and expansion. In this method, the cross-linking agent used in the first hydrogel layer can be destroyed by strong acids or bases. That is, after initial expansion, the first hydrogel layer can be dissolved by strong acids or bases, and the sample expands again through the second hydrogel polymer network formed in the newly opened space from the first expansion, thus achieving a higher expansion factor. Techniques such as iExM and "unclear microscopy" can linearly expand samples by 20 times through secondary expansion. However, because these techniques require strong chemical conditions such as strong acids or bases, these extreme chemical conditions may damage biomolecules such as proteins and DNA in the sample, thus affecting data quality, especially in high-throughput analyses such as proteomics, where sample loss and analytical errors are significant.

[0005] In addition, there is an "expansion revealing (ExR)" technique that uses the same hydrogel for two rounds of embedding and expansion, which can also linearly expand the sample by 20 times. However, the mechanical strength of the first gel is extremely low, making it difficult to manipulate. There is also a dilatation microscopy technique called "20ExM," which achieves about 20 times expansion in a single expansion step, enabling resolution of less than 20 nanometers on a conventional microscope and achieving iterative expansion performance. However, its maximum linear expansion factor is limited to only about 20 times. Summary of the Invention

[0006] This invention aims to provide a method and related kit for continuous expansion of hydrogels for super-resolution imaging, which does not require the use of any strong chemical conditions such as strong acids or strong bases, and can achieve a linear expansion factor of about 40-50 times.

[0007] On one hand, the present invention provides a method for continuous expansion of hydrogels for super-resolution imaging, the method comprising the following steps:

[0008] S1: Protein anchoring treatment of biological samples;

[0009] S2: The biological sample, after being treated with S1 protein anchoring, was added to ATMS(A) solution for gelation to complete the first round of hydrogel embedding.

[0010] The ATMS(A) solution contains:

[0011] Monomers, including sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), and pentaerythritol allyl ether (PAE), and

[0012] coagulant,

[0013] The molar ratio of monomer SMA:DMAA:PAE is 1:4:(0.0008-0.01), and the concentration of SMA in the ATMS(A) solution is 0.08-0.1 g / mL.

[0014] S3: Perform protein denaturation treatment on biological samples that have undergone the first round of hydrogel embedding in S2;

[0015] S4: Place the hydrogel after S3 denaturation into pure water and incubate it until it expands to its maximum size.

[0016] S5: The biological sample that has undergone one expansion in S4 is added to ATMS(B) solution for gelation, so that the denatured biological sample is embedded in the neutral gel formed by ATMS(B) solution.

[0017] The ATMS(B) solution contains: monomer DMAA, and a coagulant.

[0018] In the ATMS(B) solution, the concentration of monomer DMAA was 0.9-2.7 M.

[0019] S6: The biological samples embedded with neutral gel in S5 were subjected to a second round of hydrogel embedding using the same ATMS(A) solution as in S2 and incubated in pure water for secondary expansion.

[0020] In some embodiments, the biological sample may refer to a tissue block (e.g., a whole tissue), a tissue section (e.g., a paraffin section, a vibrating section, a frozen section), or a cell sample. In some embodiments, the biological sample is a complete mouse brain or a tissue section thereof.

[0021] In a specific implementation, in step S1, the biological sample for protein anchoring is a tissue section with a thickness of 1μm-200μm, such as a tissue section with a thickness of 50μm.

[0022] In a specific embodiment, in step S1, protein anchoring buffer is added to the rat brain vibration slice, incubated at room temperature, and then washed with anchoring termination buffer.

[0023] In a specific embodiment, the protein anchoring buffer is prepared using a protein anchoring reagent selected from N-succinimide acrylate (NSA), N-(allyloxycarbonyloxy)-succinimide (NAS), or combinations thereof. In a specific embodiment, in step S1, the protein anchoring buffer is 0.1 mg / mL NSA in 100 mM MES; the anchoring termination buffer is a 100 mM MOPS buffer solution.

[0024] In a specific embodiment, in step S2, the biological sample is air-dried, placed on a glass slide, and sufficient ATMS(A) solution is added to the surface of the biological sample. Under a moist environment, it is incubated at 2-6°C, for example, 4°C, for 8-16 hours. Then, a coverslip is placed on top, and the sample is gelled under anaerobic conditions at 30-40°C, for example, 37°C.

[0025] In a specific embodiment, in step S3, denaturing agents such as SDS, proteinase K, trypsin, collagenase, intracellular protease, urea, thiourea, methanol or ethanol can be used. Alternatively, an autoclave, microwave oven or other similar device can be used for denaturation, and the denaturing agent can be expanded to its maximum size with pure water.

[0026] In a specific embodiment, in step S4, the hydrogel after the denaturation in S3 is placed in pure water and incubated at room temperature for 15 min to 1 h, for example, for 30 min, to expand once until the maximum multiple is reached.

[0027] In a specific implementation, in step S5, the biological sample after one expansion is immersed in ATMS(B) solution, incubated on a shaker, the liquid on the surface of the hydrogel is aspirated, a coverslip is placed on it, and the gel is formed under anaerobic conditions. After the gel is formed, it is washed with 1×PBS or stored overnight in 1×PBS.

[0028] In a specific embodiment, in the ATMS(A) solution, the molar ratio of monomer SMA:DMAA:PAE is 1:4:0.0008-0.01, and the concentration of SMA is 0.08-0.1 g / mL.

[0029] In a specific embodiment, the concentration of monomer DMAA in the ATMS(B) solution is 0.9-2.7M.

[0030] In a specific embodiment, the coagulant in the ATMS(A) solution and the ATMS(B) solution is selected from one or more of potassium persulfate (KPS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and 2,2'-azobisisobutylamidine dihydrochloride (V50). Preferably, the coagulant is ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). Preferably, in the ATMS(A) solution, the concentration of APS is 0.001-0.005 g / mL, and the concentration of TEMED is 0.001-0.005 g / mL; in the ATMS(B) solution, the concentration of APS is 0.0004-0.0006 g / mL, and the concentration of TEMED is 0.0004-0.0006 g / mL. More preferably, in the ATMS(A) solution, the concentration of APS is 0.003 g / mL and the concentration of TEMED is 0.002 g / mL; in the ATMS(B) solution, the concentration of APS is 0.0005 g / mL and the concentration of TEMED is 0.0005 g / mL.

[0031] In a specific embodiment, in step S6, the biological sample embedded with the neutral gel is immersed again in the ATMS(A) solution, incubated on a shaker, covered with a coverslip, and gelled at 30-40°C, for example, 37°C under anaerobic conditions. After gelation, pure water is left overnight to expand to the maximum expansion factor.

[0032] In specific implementations, depending on different needs, biological samples before expansion, after one expansion, after embedding in neutral gel, and after two expansions can be stained for super-resolution imaging.

[0033] The staining can be performed using conventional methods or reagents in the art. For example, for nuclear staining, staining reagents such as DAPI, Hoechst series, Propidium Iodide, Ethidium Bromide, Methylene Blue, Crystal Violet, Feulgen Reagent, SYTO series, Laminin-labeled dyes, and Chromomycin A3 can be used; for whole protein staining, the SYPRO series can be used, such as SYPRO Ruby, SYPRO Orange, SYPRO Red, SYPRO Tangerine, SYPRO Cyanine 5Cy5, and SYPRO Green; immunohistochemistry includes indirect immunohistochemistry, direct immunohistochemistry, HRP, ALP, immunofluorescence, etc.; and immunofluorescence detection achieves high sensitivity, multiplex labeling, and high-resolution imaging of the target antigen through different antibody combinations (e.g., primary antibody + fluorescently labeled secondary antibody, primary antibody + secondary antibody + fluorescently labeled triple antibody, primary antibody + Nano secondary antibody) and fluorescent labeling strategies, such as Q-dot and DNA paint.

[0034] In a specific implementation, during immunofluorescence detection, different organelles and structures can be fluorescently labeled as needed for immunofluorescence detection. Specifically, biological samples before swelling, after one swelling, after embedding in neutral gel, and after a second swelling are incubated in primary antibody blocking solution at room temperature for 1 hour, followed by overnight incubation with primary antibody at 2-6°C, for example, 4°C. After washing with 1×PBS, secondary antibody is incubated overnight at 2-6°C, for example, 4°C. The cell nuclei are stained with DAPI staining solution and / or mitochondria are labeled with IraZolve-Mito, followed by microscopic imaging.

[0035] In a specific implementation, S5 and S6 in the method can be repeated once or multiple times as a combination, depending on actual needs, to provide the linear expansion factor required for the specific application.

[0036] On the other hand, the present invention provides a kit for continuous hydrogel expansion for super-resolution imaging, the kit comprising:

[0037] Anchoring reagents: N-succinimide acrylate (NSA) and / or N-(allyloxycarbonyloxy)-succinimide (NAS) and MES buffer;

[0038] Anchoring termination reagent: MOPS buffer;

[0039] Gel reagents: sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), pentaerythritol allyl ether (PAE).

[0040] In a specific embodiment, the kit also includes an instruction manual explaining how to use the kit.

[0041] In a specific embodiment, the kit also includes a coagulant.

[0042] In a specific embodiment, the coagulant is selected from one or more of potassium persulfate (KPS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and V50.

[0043] On the other hand, this application provides the application of the above-mentioned methods in antigen detection, protein-protein interaction studies, proteomics, and tissue or cell imaging.

[0044] Beneficial effects

[0045] This invention provides a method for continuous (more than two expansions) expansion of hydrogels for super-resolution imaging. Specifically, this invention provides a neutral gel formulation. Using this neutral gel, the hydrogel can undergo secondary expansion of cell or tissue samples without any strong acid or alkali treatment, resulting in a total linear expansion factor of 40-50 times or more, with moderate mechanical strength. When combined with optical microscopes (transmission light microscopes, phase contrast microscopes, fluorescence microscopes, polarizing microscopes), electron microscopes (transmission electron microscopes, scanning electron microscopes), scanning probe microscopes (atomic force microscopes, scanning tunneling microscopes), confocal microscopes, super-resolution microscopes, laser confocal microscopes, and other microscopes, it can achieve visualization of subcellular structures, nanoscale structures, and single molecules. Attached Figure Description

[0046] Figure 1 A schematic diagram of the method flow of this application is shown.

[0047] Figure 2 The following diagram shows the results of nuclear imaging of rat brain vibration sections before and after expansion according to the method of the present invention: A: Nuclear imaging before expansion; B: Nuclear imaging after expansion; C: Neural synapse after one expansion; D: Neural synapse after two expansions. Detailed Implementation

[0048] The technical solutions of this application are described in detail below through specific embodiments to enable those skilled in the art to better understand the content of this application; however, these embodiments are not intended to limit the scope of this application.

[0049] In this application, "super-resolution imaging" of expandable hydrogels refers to a method combining hydrogel expansion technology with microscopic imaging. This method embeds tissue samples within expandable hydrogels, causing the sample volume to increase uniformly through physical expansion, thereby stretching and magnifying subcellular structures. This expansion effectively improves the spatial resolution of the image without altering the resolution of the optical microscope, making previously difficult-to-observe details clearly visible. Materials used in the following examples:

[0050]

[0051] 1×PBS: NaCl: 8g / L, Na2HPO4: 1.44g / L, KCL: 0.2g / L, KH2PO4: 2.4g / L.

[0052] Preparation of each reagent in the methods described in the following examples:

[0053] 1. NSA (anchor) stock solution: 10 mg / mL, 10 mL

[0054] Dissolve 100 mg NSA in anhydrous DMSO to a final volume of 10 mL and store in a 4°C freezer.

[0055] 2. Protein anchoring buffer: MES (195.2 g / mol) solution, 100 mM (pH 6.0), 50 mL

[0056] Mix 25 mL of 0.2 M MES stock solution with 25 mL of ddH2O to make a final volume of 50 mL, test the pH to see if it is 6.0, and store in a 4°C refrigerator.

[0057] 3. Protein anchoring solution (0.1 mg / mL NSA in 100 mM MES), 50 μL-100 μL per tissue, freshly prepared.

[0058] reagents Final concentration (%) Required amount NSA mother liquor 1％ 2μL Protein anchoring buffer 99％ 198μL

[0059] 4. Anchoring termination buffer: MOPS (209.2633 g / mol) solution, 100 mM (pH 7.0), 10 mL

[0060] Mix 2 mL of 0.5 M MOPS stock solution with 8 mL of ddH2O to make a final volume of 10 mL, test the pH to see if it is 7.0, and store in a 4°C refrigerator.

[0061] 5. Monomer solution: SMA:DMAA:PAE (molar ratio) = 1:4:0.0008, aqueous solution, pH adjusted to approximately 7 with 10% HCl. The total volume prepared is approximately 8.5 ml.

[0062] reagents Required amount DMAA 3mL SMA 0.8624g PAE mother liquor 15.4μL <![CDATA[ddH2O]]> 4.635mL 10% HCl 350μL

[0063] 6. Neutral monomer solution: 1-3M DMAA aqueous solution.

[0064] reagents Required amount DMAA 300μl <![CDATA[ddH2O]]> 1294μL

[0065] 7. APS stock solution, 10% w / v, 1 mL

[0066] Dissolve 0.1g of APS in ddH2O to make a final volume of 1mL, prepare fresh and store in a 4°C refrigerator.

[0067] 8. TEMED stock solution, 10% w / v, 1 mL

[0068] Dissolve 0.1g TEMED (approximately 0.1mL) in ddH2O to make a final volume of 1mL. Prepare fresh and store in a 4°C refrigerator.

[0069] 9. Protein denaturation buffer: 5.8%-20% SDS.

[0070] 10. ATMS(A), 50μL-100μL per tissue, freshly prepared and kept on ice before use.

[0071] reagents Final concentration Required amount Monomer solution 90％ 900μL APS stock 3％ 30μL TEMED stock 2％ 20μL <![CDATA[ddH2O]]> 5％ 50μL

[0072] (Monomer solution: APS stock: TEMED stock = 90:3:2 volume ratio)

[0073] 11. ATMS(B): Neutral adhesive layer, freshly prepared, placed on ice before use.

[0074] reagents Final concentration Required amount neutral monomer solution 90％ 900μL APS stock 0.5％ 5μL TEMED stock 0.5％ 5μL <![CDATA[ddH2O]]> 9％ 90μL

[0075] (Neutral monomer solution: APS stock: TEMED stock = 90:0.5:0.5 volume ratio)

[0076] The hydrogel continuous expansion method for super-resolution imaging in this application can be achieved through... Figure 1 The steps shown are as follows: slice sample, protein anchoring, first hydrogel embedding, protein denaturation, neutral gel embedding, second hydrogel embedding, and immunofluorescence staining.

[0077] Example 1:

[0078] The gel continuous expansion method for super-resolution imaging of this application is exemplarily operated as follows.

[0079] 1. Sliced ​​sample:

[0080] • Vibratory sectioning: After fresh tissue samples are taken out and chemically fixed (usually overnight with 4% PFA fixative), the samples are cut into sections of about 50-100μm using a vibratory microtome.

[0081] • Paraffin sections: Dewaxing is required. The specific process is as follows:

[0082] Place the slices into a dehydration container.

[0083] Add heptane to the container and discard the heptane after 10 minutes.

[0084] Repeat the heptane dewaxing step once.

[0085] The dewaxed sample was subjected to gradient hydration with ethanol, as shown below:

[0086] Add 100% ethanol to the container, keep it for 5 minutes, and then discard the liquid.

[0087] Add 90% ethanol to the container, keep it for 5 minutes, and then discard the liquid.

[0088] Add 75% ethanol to the container, keep for 5 minutes, then discard the liquid. Set the expansion coefficient of the biological sample before expansion to 1×.

[0089] 2. Protein anchoring:

[0090] • Add 50-150 μl of protein anchoring solution to a 50 μm rat brain vibration section (fixed with 4% PFA), incubate at room temperature for 3 h, and wash 3 times with anchoring termination buffer.

[0091] 3. First round of hydrogel embedding:

[0092] • Allow the slides to air dry completely in a fume hood. Attach the cut plastic frame to the slide and add sufficient ATMS(A) solution to the sample surface. Then place the sample in a pipette tip box filled with water at the bottom (keeping the sample out of contact with water) and incubate overnight at 4°C.

[0093] Cover with a coverslip and allow to gel at 37°C under oxygen-free conditions for 2 hours;

[0094] • Remove the coverslip and incubate at 95°C for 4-8 hours for protein denaturation.

[0095] 4. Primary expansion:

[0096] • Place the denatured hydrogel into a petri dish containing pure water and incubate at room temperature for 30 minutes to allow it to expand to its maximum size.

[0097] 5. Embedding with neutral adhesive:

[0098] • Immerse the hydrogel after one expansion in ATMS(B) solution and incubate on a shaker at room temperature for 1 hour. Blot the surface of the hydrogel dry with clean paper, cover with a coverslip, and gel at 37°C under anaerobic conditions for 2 hours. After gelation, wash three times with 1×PBS for 15 minutes each time, or store overnight in 1×PBS.

[0099] 6. Second round of hydrogel embedding:

[0100] • Immerse the hydrogel embedded in the neutral gel again in ATMS(A) solution and incubate on a shaker at room temperature for 1 hour. Cover with a coverslip and incubate at 37°C under anaerobic conditions for 2 hours to form a gel.

[0101] 7. Secondary expansion:

[0102] • After gelation, leave the product in pure water overnight to allow it to expand to its maximum expansion ratio.

[0103] 8. Staining:

[0104] The swollen hydrogel was incubated in primary antibody blocking solution at room temperature for 1 hour, followed by overnight incubation with primary antibody at 4°C. After washing three times with 1×PBS, the hydrogel was incubated with secondary antibody at 4°C overnight. Cell nuclei were stained with DAPI staining solution, washed three times with 1×PBS, and then swollen with pure water to maximum magnification for microscopic imaging.

[0105] Microscopic imaging results before and after expansion are as follows Figure 2 As shown. From Figure 2 It can be seen that before the expansion, the diameter of the mouse brain cell nucleus was about 5-10 μm. Figure 2 (A in the text); After expansion, the cell nucleus size can reach 520 μm ( Figure 2 In B), linear expansion is nearly 50 times; in one step ( Figure 2 C in the middle) and second-order ( Figure 2 The expanded neural synapse (D) can be visualized at the nanoscale using immunofluorescence staining.

Claims

1. A method for continuous expansion of a hydrogel for super-resolution imaging, the method comprising the following steps: S1: Protein anchoring treatment of biological samples; S2: The biological sample, after being treated with S1 protein anchoring, was added to ATMS(A) solution for gelation to complete the first round of hydrogel embedding. The ATMS(A) solution contains: Monomers, including sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), and pentaerythritol allyl ether (PAE), and coagulant, The molar ratio of monomer SMA:DMAA:PAE is 1:4:(0.0008-0.01), and the concentration of SMA in the ATMS(A) solution is 0.08-0.1 g / mL. S3: Perform protein denaturation treatment on biological samples that have undergone the first round of hydrogel embedding in S2; S4: Place the hydrogel after S3 denaturation in pure water and incubate it once until it expands to its maximum size; S5: The biological sample that has undergone one expansion in S4 is added to ATMS(B) solution for gelation, so that the denatured biological sample is embedded in the neutral gel formed by ATMS(B) solution. The ATMS(B) solution contains: monomer DMAA, and a coagulant. In the ATMS(B) solution, the concentration of monomer DMAA was 0.9-2.7 M. S6: The biological samples embedded with neutral gel in S5 were subjected to a second round of hydrogel embedding using the same ATMS(A) solution as in S2 and incubated in pure water for secondary expansion.

2. The method according to claim 1, wherein, The biological sample is a tissue block (e.g., a whole tissue), or a tissue section (e.g., a paraffin section, a vibrating section, a frozen section), or a cell sample, for example, a complete mouse brain or a tissue section thereof.

3. The method according to claim 1, wherein, In step S1, the biological sample for protein anchoring is a tissue section with a thickness of 1μm-200μm, for example, a tissue section with a thickness of 50μm; and / or In step S1, protein anchoring buffer was added to the rat brain vibration slices, and after incubation at room temperature, the slices were washed with anchoring termination buffer. Preferably, the protein anchoring buffer is prepared from a protein anchoring reagent selected from N-succinimide acrylate (NSA), N-(allyloxycarbonyloxy)-succinimide (NAS), or a combination thereof. More preferably, in step S1, the protein anchoring buffer is 0.1 mg / mL NSA in 100 mM MES; and the anchoring termination buffer is 100 mM MOPS buffer solution.

4. The method according to claim 1, wherein, In step S2, the biological sample is air-dried, placed on a glass slide, and sufficient ATMS(A) solution is added to the surface of the biological sample. Under a moist environment, it is incubated at 2-6°C, for example, 4°C, for 8-16 hours. Then, a coverslip is placed on top, and the sample is gelled under anaerobic conditions at 30-40°C, for example, 37°C.

5. The method according to claim 1, wherein, In step S3, denaturation is performed using a denaturing agent and / or an autoclave, microwave oven, or similar device, followed by expansion with pure water to the maximum extent. Optionally, the denaturing agent is selected from SDS, proteinase K, trypsin, collagenase, intracellular protease, urea, thiourea, methanol, and ethanol; and / or In step S4, the hydrogel after S3 denaturation is placed in pure water and incubated at room temperature for 15 min to 1 h, for example, 30 min, to allow it to expand once until it reaches its maximum expansion.

6. The method according to claim 1, wherein, In step S5, the biological sample after one expansion is immersed in ATMS(B) solution, incubated on a shaker, the liquid on the surface of the hydrogel is aspirated, a coverslip is placed on, and gelation is carried out under anaerobic conditions. After gelation, the sample is washed with 1×PBS or stored overnight in 1×PBS.

7. The method according to claim 1, wherein, In step S2, in the ATMS(A) solution, the molar ratio of monomer SMA:DMAA:PAE is 1:4:0.0008-0.01, and the concentration of SMA is 0.08-0.1 g / mL; In step S5, the concentration of monomer DMAA in the ATMS(B) solution is 0.9-2.7M.

8. The method according to claim 1, wherein, In both ATMS(A) and ATMS(B) solutions, the coagulant is one or more selected from potassium persulfate (KPS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and 2,2'-azobisisobutylamidine dihydrochloride (V50). Preferably, the coagulant is ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). Preferably, in the ATMS(A) solution, the concentration of APS is 0.001-0.005 g / mL and the concentration of TEMED is 0.001-0.005 g / mL; in the ATMS(B) solution, the concentration of APS is 0.0004-0.0006 g / mL and the concentration of TEMED is 0.0004-0.0006 g / mL. More preferably, in the ATMS(A) solution, the concentration of APS is 0.003 g / mL and the concentration of TEMED is 0.002 g / mL; in the ATMS(B) solution, the concentration of APS is 0.0005 g / mL and the concentration of TEMED is 0.0005 g / mL.

9. The method according to claim 1, wherein, In step S6, the biological sample embedded in the neutral gel is immersed again in the ATMS(A) solution, incubated on a shaker, covered with a coverslip, and gelled under anaerobic conditions at 30-40°C, for example, 37°C. After gelation, it is placed in pure water overnight to expand to the maximum expansion factor.

10. The method according to claim 1, wherein, The biological samples before expansion, after one expansion, after embedding in neutral gel, and after two expansions in the above method are stained and then subjected to super-resolution imaging. For example, the staining is immunofluorescence staining.

11. The method according to claim 1, wherein, S5 and S6 in the method can be repeated once or multiple times as a combination to provide the desired linear expansion factor.

12. A kit for continuous hydrogel expansion for super-resolution imaging, the kit comprising: Anchoring reagents: N-succinimide acrylate (NSA) and / or N-(allyloxycarbonyloxy)-succinimide (NAS) and MES buffer; Anchoring termination reagent: MOPS buffer; Gel reagents: Sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), pentaerythritol allyl ether (PAE). Optionally, the kit also includes instructions for explaining how to use the kit. Optionally, the kit also includes a coagulant. Optionally, the coagulant is one or more selected from potassium persulfate (KPS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and 2,2'-azobisisobutylammonium dihydrochloride (V50).

13. The application of the method as described in any one of claims 1-11 in antigen detection, protein-protein interaction studies, proteomics, tissue or cell imaging.

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