A hydrogel for expansion microscopy imaging, a preparation method thereof, and a sample expansion treatment method

Abstract

The present invention belongs to the field of fluorescence microscopy imaging, and particularly relates to a hydrogel for expansion microscopy imaging, a preparation method thereof, and a sample expansion treatment method. The preparation method of the hydrogel includes preparing a hydrogel precursor solution and preparing a hydrogel prepolymer solution. The sample expansion treatment method includes sample gelation and sample expansion. The hydrogel of the present invention has excellent mechanical properties and high elongation at break. It has good stability, is convenient and safe to use. Moreover, by combining the traditional biomolecule anchoring step with tissue penetration and gelation, the hydrogel is used to expand mouse brain slices, and the mouse brain slices can be isotropically expanded to the maximum size after 48 h, obtaining a linear expansion multiple of about 4.0 times. The mouse brain slices embedded with DAAM-NMA-SA hydrogel and containing green fluorescent protein labels have significantly improved imaging effects in the expansion microscopy fluorescence images compared with the fluorescence images of the original brain slices, and the resolution has been greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescence microscopy imaging, and particularly relates to a hydrogel for expansion microscopy imaging, a preparation method thereof, and a sample expansion treatment method. Background Art

[0002] Expansion microscopy (ExM) is an emerging sample embedding technology. The basic principle is to embed biological samples in water-soluble polymers, then cure the polymers through a polymerization reaction, and expand the polymer network by adding water or specific chemicals, enabling the sample to obtain images beyond the resolution limit under a conventional microscope.

[0003] However, the ExM technology also faces some challenges and limitations:

[0004] (1) Expanding the sample may cause distortion of some cell or tissue structures, especially for the observation of certain organelles and subcellular structures. The expansion process may cause deformation of the sample, especially in highly detailed structures, which may lead to spatial distortion or geometric aberration. This requires careful handling during sample preparation and microscopy imaging to minimize the impact of such distortion. The ExM technology has complexity in sample preparation, imaging, and data processing, which limits its wide use in practical applications. During the sample preparation process, multiple chemical treatment steps are required, such as anchoring, penetration, expansion, etc., and these steps need to be carefully controlled to ensure the expansion effect and sample stability.

[0005] (2) Current expansion microscopy schemes require pretreatment with reactive anchoring chemicals to attach specific labels and biomolecule classes to the gel. This step is for biomolecule anchoring. In protein-based anchoring strategies, strong protease digestion is required to achieve isotropic expansion, and this digestion method will cause a large loss of target proteins after expansion, limiting the number of detected protein structures.

[0006] (3) Optimizing and standardizing the ExM technology for different types of samples is also a challenge. Different types of tissues and cells may have different transparency, expansion properties, and stability requirements.

[0007] Therefore, there is an urgent need to further develop and optimize ExM methods applicable to different types of samples and establish standardized sample preparation and imaging procedures. Summary of the Invention

[0008] The purpose of the present invention is to overcome the disadvantages of the prior art and provide a hydrogel for expansion microscopy imaging, a preparation method thereof, and a sample expansion treatment method.

[0009] The purpose of the present invention is achieved by the following technical solutions:

[0010] The first aspect of the present invention is to provide a method for preparing a hydrogel for expansion microscopy imaging, comprising the following steps: S1. Prepare a hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide, and sodium acrylate in PBS solution to obtain a hydrogel precursor solution; wherein, the mass percentage concentrations of diacetone acrylamide, N-(hydroxymethyl) acrylamide, and sodium acrylate in the hydrogel precursor solution are denoted as X, Y, and Z respectively, X + Y is 35 - 45% and X / Y ≤ 1, Z ≤ 10%.

[0011] S2. Prepare a hydrogel prepolymer solution: Add an initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is ammonium persulfate or 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and a hydrogel for expansion microscopy imaging is obtained.

[0012] As a preferred technical solution, the concentration of diacetone acrylamide in the hydrogel precursor solution is 5 - 15 wt%, the concentration of N-(hydroxymethyl) acrylamide is 25 - 35 wt%, and the concentration of sodium acrylate is 3 - 8 wt%.

[0013] More preferably, the concentration of diacetone acrylamide in the hydrogel precursor solution is 10 wt%, the concentration of N-(hydroxymethyl) acrylamide is 30 wt%, and the concentration of sodium acrylate is 5 wt%.

[0014] As a preferred technical solution, the concentration of the initiator mother liquor is 100 - 300 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother liquor is 90 - 120:1.

[0015] The second aspect of the present invention is to provide a hydrogel for expansion microscopy imaging prepared by the above method.

[0016] The third aspect of the present invention is to provide a method for sample expansion treatment, which comprises the following steps:

[0017] (1) Sample gelation: Immerse the sample in the hydrogel for expansion microscopy imaging according to claim 5 for infiltration for 3.5 - 4.5 h, then encapsulate it with a mold, and polymerize at a temperature of 35 - 45°C for at least 3 h;

[0018] (2) Sample expansion: Demold the polymerized and encapsulated sample, take out the gelated sample and perform digestion treatment, immerse the digested sample in deionized water, and perform expansion with a gyratory shaker at 50 - 100 rpm. The expansion time is 1 - 48 h.

[0019] As a preferred technical solution, the sample is a mouse brain slice sample.

[0020] As a preferred technical solution, the specific operation of the digestion treatment is to immerse the gelled sample in the digestion buffer for 2 to 48 hours. The digestion buffer is a mixed solution of 6 to 10 U / ml proteinase K, 0.3 to 0.8% (W / V) Triton X-100 and 1×TAE buffer.

[0021] As a preferred technical solution, the mold is a glass slide with a sample groove, and the size of the sample groove matches the size of the sample.

[0022] The principle of the hydrogel of the present invention for sample swelling is as follows:

[0023] In the formula of the present invention, diacetone acrylamide monomer is used to introduce a keto group into the ExM system. First, the keto group can react with the amino group of the protein in the biological tissue sample to form a Schiff base, which can form a stable chemical bond connection, thereby fixing the biological sample; at the same time, the oxygen atom of the keto group has a lone pair of electrons, which can form hydrogen bonds with amino acids in the biological tissue, and the generation of hydrogen bonds also plays an important role in the stability and fixation of the biological sample; secondly, the double bond between the carbon atom and the oxygen atom of the keto group has strong rigidity, which may limit the rotation of certain chemical bonds and change the three-dimensional configuration of the molecule, thereby affecting the embedding mode of the biological sample in the polymer system. This spatial restriction can "lock" other molecules in specific positions, thereby improving the fixation effect. In the existing hydrogels, acrylamide has the characteristics of a cross-linking agent and can form hydrogen bonds with amino and carboxyl groups in proteins to achieve a fixation effect. Compared with the existing hydrogels, the differences are as follows:

[0024] The anchoring agent is different: In the existing simple hydrogel swelling system, hydrogen bonds are formed between the amide, hydroxyl and other groups of the hydrogel itself and the amino, carboxyl, amide and other groups in the protein to achieve the anchoring effect, which belongs to physical action and is easily damaged, and the swelling stability is insufficient. The protein anchoring agent in the more complex hydrogel swelling system is a water-insoluble reagent. Usually, the first step of protein anchoring is carried out with an organic reagent, and then the organic reagent is replaced by rehydration, and then operations such as hydrogel prepolymer solution penetration, polymerization, digestion, and swelling are carried out, and the operation is cumbersome. The anchoring agent of the present invention is water-soluble diacetone acrylamide (DAAM). The reaction of the keto group with the protein forms a Schiff base, which can form a stable chemical bond connection and can uniformly swell isotropically. The water-soluble anchoring agent DAAM can combine the anchoring step with tissue penetration, simplifying the steps of the swelling microscopy imaging technique.

[0025] The penetration method is different: The introduction of the keto group can change the permeability of the biological membrane, enabling the polymer to penetrate more effectively into the tissue or retain the biological molecules inside the tissue, thereby improving the preservation state and function of the biological tissue.

[0026] Differences in staining ability: The presence of keto groups can improve the binding ability of stains, making it easier for tissues to be stained by different types of stains and being more conducive to subsequent microscopic swelling observation.

[0027] Based on the reaction between keto groups and proteins and the high water absorbency of sodium acrylate, the present invention applies diacetone acrylamide (DAAM), N-(hydroxymethyl) acrylamide (NMA), and sodium acrylate (SA) to an expansion microscope to expand the swelling factor of the hydrogel and thereby improve the resolution of the optical microscope. The hydrogel of the present invention uses DAAM as an anchoring agent and a hydrogel monomer to provide gel complexation sites and biomolecule anchoring sites for the hydrogel network; NMA provides hydrogen bonds as the swelling agent and monomer of the hydrogel, and SA is added to enhance the swelling factor and water absorption performance of the hydrogel system. The three together constitute the DAAM-NMA-SA hydrogel system. Combining the traditional biomolecule anchoring step with tissue penetration improves the operability and simplicity of the expansion microscopy imaging technique to a certain extent.

[0028] The present invention has the following advantages: The hydrogel of the present invention uses DAAM as an anchoring agent and a hydrogel monomer to provide gel complexation sites and biomolecule anchoring sites for the hydrogel network; NMA provides hydrogen bonds as the swelling agent and monomer of the hydrogel, and SA is added to enhance the swelling factor and water absorption performance of the hydrogel system. The three together constitute the DAAM-NMA-SA hydrogel system. The hydrogel prepared by the method of the present invention has excellent mechanical properties and a high elongation at break. It has good stability, is convenient and safe to use. And by combining the traditional biomolecule anchoring step with tissue penetration, the mouse brain slices are swollen using the DAAM-NMA-SA hydrogel. The mouse brain slices can isotropically swell to the maximum size after 48 h, obtaining a linear swelling multiple of about 4.0 times. The DAAM-NMA-SA hydrogel is embedded with mouse brain slices labeled with green fluorescent protein, and the imaging effect of its expansion microscopy fluorescence image is significantly improved compared with the fluorescence image of the original brain slices, and the resolution is greatly improved. Description of the Drawings

[0029] Figure 1 It is a synthesis diagram of DAAM-NMA hydrogels with different monomer ratios. In the figure, (a-i) are hydrogels of 40-0, 35-5, 30-10, 25-15, 20-20, 15-25, 10-30, 5-35, 0-40 from left to right in sequence.

[0030] Figure 2It is the tensile stress-strain curve diagram of the hydrogel. In the figure, (a) shows the tensile stress-strain curves of different DAAM-NMA hydrogels, and (b) shows the tensile stress-strain curves of DAAM-NMA-SA hydrogels with different concentrations.

[0031] Figure 3 It is the swelling equilibrium state diagram of DAAM-NMA-SA hydrogels with different monomer concentrations in deionized water; in the figure, (a-c) show the size changes of 10-30-5, 10-30-10, and 10-30-15 hydrogels before and after swelling; (d) shows the line graph of the swelling degree of DAAM-NMA-SA hydrogels with different monomer concentrations changing with time. In the lower right corner of (a), (b), and (c): the size of the hydrogel before swelling, with a diameter of 1 cm.

[0032] Figure 4 It is the size change diagram of a mouse brain when swelling in deionized water. In the figure, (a) shows the size of the original brain slice; (b) shows the size of the brain slice after penetration; (c) shows the size of the brain slice after gelation; (d) shows the size of the brain slice after digestion; (e) shows the size of the brain slice after swelling. Scale bar: one small grid is 1 mm.

[0033] Figure 5 It is the selection diagram of the swelling multiple marker sites of a mouse brain slice. In the figure, (a) shows the brain slice after swelling; (b) shows the original brain slice; (c) shows the fluorescence image after superimposing (a) and (b). 0-3: different marker sites, scale bar: 100 m.

[0034] Figure 6 It is the fluorescence imaging analysis diagram of mouse brain neurons before and after swelling. In the figure, (a) shows the fluorescence image of mouse brain neurons before swelling; (b) shows the normalized curve of the fluorescence intensity changing with distance in the yellow line area of (a); (c) shows the fluorescence image of mouse brain neurons after swelling; (d) shows the normalized curve of the fluorescence intensity changing with distance in the yellow line area of (b). Scale bar: 10 m.

[0035] Figure 7 It is the fluorescence imaging analysis diagram of mouse brain neurons before and after swelling. In the figure, (a) shows the confocal fluorescence image of mouse brain neurons before swelling; (b) shows the confocal fluorescence image of mouse brain neurons after swelling; (c) shows the FWHM frequency distribution histogram of mouse brain neurons in the yellow line area of (a); (d) shows the FWHM frequency distribution histogram of mouse brain neurons in the yellow line area of (b); (e) shows the FWHM before and after swelling obtained from (c) and (d). n = 100, scale bar: 100 μm. Specific embodiments

[0036] The following further describes the present invention in conjunction with the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following: Embodiment 1: A preparation method of a hydrogel for expansion microscopy imaging, including the following steps:

[0037] S1. Preparation of hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in PBS solution to obtain a hydrogel precursor solution; wherein, the concentration of diacetone acrylamide in the hydrogel precursor solution is 15 wt%, the concentration of N-(hydroxymethyl) acrylamide is 25 wt%, and the concentration of sodium acrylate is 10 wt%.

[0038] S2. Preparation of hydrogel prepolymer solution: Add the initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the concentration of the initiator mother liquor is 100 mg / mL. The volume ratio of the gel precursor solution to the initiator mother liquor is 90:1 to obtain a hydrogel for expansion microscopy imaging.

[0039] Example 2: A method for preparing a hydrogel for expansion microscopy imaging, comprising the following steps:

[0040] S1. Preparation of hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in PBS solution to obtain a hydrogel precursor solution; wherein, the concentration of diacetone acrylamide in the hydrogel precursor solution is 5 wt%, the concentration of N-(hydroxymethyl) acrylamide is 30 wt%, and the concentration of sodium acrylate is 3 wt%.

[0041] S2. Preparation of hydrogel prepolymer solution: Add the initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is ammonium persulfate, and the concentration of the initiator mother liquor is 300 mg / mL. The volume ratio of the gel precursor solution to the initiator mother liquor is 90 - 120:1 to obtain a hydrogel for expansion microscopy imaging.

[0042] Example 3: A method for preparing a hydrogel for expansion microscopy imaging, comprising the following steps:

[0043] S1. Preparation of hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in PBS solution to obtain a hydrogel precursor solution; wherein, the concentration of diacetone acrylamide in the hydrogel precursor solution is 10 wt%, the concentration of N-(hydroxymethyl) acrylamide is 35 wt%, and the concentration of sodium acrylate is 8 wt%.

[0044] S2. Preparation of hydrogel prepolymer solution: Add the initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the concentration of the initiator mother liquor is 180 mg / mL. The volume ratio of the gel precursor solution to the initiator mother liquor is 90 - 120:1 to obtain a hydrogel for expansion microscopy imaging.

[0045] Example 4: A method for preparing a hydrogel for expansion microscopy imaging, comprising the following steps:

[0046] S1. Prepare a hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide, and sodium acrylate in PBS solution to obtain a hydrogel precursor solution; wherein, the concentration of diacetone acrylamide in the hydrogel precursor solution is 10 wt%, the concentration of N-(hydroxymethyl) acrylamide is 30 wt%, and the concentration of sodium acrylate is 5 wt%.

[0047] S2. Prepare a hydrogel prepolymer solution: Add an initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is ammonium persulfate, the concentration of the initiator mother liquor is 240 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother liquor is 110:1, to obtain a hydrogel for expansion microscopy imaging.

[0048] Example 5: A method for sample expansion treatment, which comprises the following steps:

[0049] (1) Sample gelation: Immerse a mouse brain slice in the hydrogel for expansion microscopy imaging prepared in Example 1 for infiltration for 3.5 h, then encapsulate it with a slide with a sample well, and polymerize at a temperature of 35 °C for at least 3 h.

[0050] (2) Sample expansion: Demold the polymerized and encapsulated sample, take out the gelated mouse brain slice and perform digestion treatment. The specific operation of the digestion treatment is to immerse the gelated sample in a digestion buffer for 2 h. The digestion buffer is a mixed solution of 6 U / ml proteinase K, 0.3% (W / V) Triton X-100, and 1×TAE buffer. The digested mouse brain slice is immersed in deionized water and expanded with a gyratory shaker at 50 rpm for 1 h.

[0051] Example 6: A method for sample expansion treatment, which comprises the following steps:

[0052] (1) Sample gelation: Immerse a mouse brain slice in the hydrogel for expansion microscopy imaging prepared in Example 3 for infiltration for 4.5 h, then encapsulate it with a slide with a sample well, and polymerize at a temperature of 45 °C for at least 3 h.

[0053] (2) Sample swelling: Demold the polymerized and encapsulated sample, take out the gelated mouse brain section and perform digestion treatment. The specific operation of the digestion treatment is to immerse the gelated sample in the digestion buffer for 48 h. The digestion buffer is a mixed solution of 10 U / ml proteinase K, 0.8% (W / V) Triton X-100 and 1×TAE buffer. After digestion treatment, the mouse brain section is immersed in deionized water and swollen with a gyratory shaker at 100 rpm for 48 h.

[0054] Example 7: A method for swelling treatment of a sample, which comprises the following steps:

[0055] (1) Sample gelation: Immerse the mouse brain section in the hydrogel prepared in Example 4 for swelling microscopy imaging for 4 h, then encapsulate it with a glass slide with a sample groove, and polymerize it at a temperature of 40 °C for at least 3 h;

[0056] (2) Sample swelling: Demold the polymerized and encapsulated sample, take out the gelated mouse brain section and perform digestion treatment. The specific operation of the digestion treatment is to immerse the gelated sample in the digestion buffer for 28 h. The digestion buffer is a mixed solution of 8 U / ml proteinase K, 0.5% (W / V) Triton X-100 and 1×TAE buffer. After digestion treatment, the mouse brain section is immersed in deionized water and swollen with a gyratory shaker at 70 rpm for 24 h.

[0057] The beneficial effects of the present invention are illustrated by the following experiments:

[0058] I. Preparation of hydrogel prepolymer solution

[0059] 1. Preparation of hydrogel precursor solution: The DAAM-NMA-SA hydrogel of the present invention is uniformly named in the form of "x% DAAM + y% NMA + z% SA" (abbreviation: x-y-z, where x, y, and z respectively represent the concentrations of DAAM, NMA, and SA). The specific preparation process is as follows (taking 100 mL of 10% DAAM + 30% NMA + 5% SA as an example):

[0060] Weigh 10 g of DAAM, 30 g of NMA, and 5 g of SA respectively in a sample bottle, dissolve them with 55 mL of PBS, and then take them out and store them in a 4 °C refrigerator after the solution is dissolved to clarity and transparency in an ultrasonic automatic cleaner. The validity period is one month.

[0061] The preparation of DAAM-NMA-SA hydrogels with other concentrations is the same as above.

[0062] 2. Preparation of hydrogel prepolymer solution: First, prepare the initiator mother liquor at a mass-volume ratio of 25% (i.e., dissolve 250 mg of AIBI in 750 μL of deionized water, and use the mother liquor immediately after preparation). Then, add the initiator mother liquor to the hydrogel precursor solution at a ratio of 100:1 (i.e., add 100 μL of the initiator mother liquor to 10 mL of the hydrogel precursor solution). (The initiator used is ammonium persulfate (APS) or 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AIBI))

[0063] 3. Hydrogel polymerization: After the hydrogel prepolymer solution is prepared according to the above operation steps, it is encapsulated in a customized glass slide with a sample slot. The diameter of the customized sample slot is 10 mm, and the depth is the thickness of the mouse brain slice. It is placed in a vacuum drying oven at 40 °C, and the hydrogel polymerization is completed after 4 h.

[0064] II. Preparation of mouse brain

[0065] Preparation of mouse brain samples: The mouse brain tissue is removed by cardiac perfusion for C57BL / 6 mice, and the mouse brain samples are fixed and preserved with 4% PFA.

[0066] The specific operation method is as follows:

[0067] 1. Intraperitoneal injection of mixed anesthetic: Anesthetize the mouse with 2.5% (wt / vol) tribromoethanol. After the mouse becomes slow-moving and deeply anesthetized, transfer it to the perfusion table in the fume hood, and perfuse 0.01 M PBS solution (RT) through the left ventricle of the mouse until the liver loses its red color. Then perfuse 4% (wt / vol) PFA solution. After the mouse becomes stiff all over, the perfusion is completed. Dissect the mouse, remove the mouse brain, immerse it in 4% PFA at 4 °C and fix it overnight, with the fixation time not exceeding 24 h. Then rinse the mouse brain with PBS solution to remove the residual PFA solution in the tissue, and the mouse brain can be stored in PBS solution at 4 °C for a short time.

[0068] 2. Pretreatment of mouse brain slices: After taking out the fixed mouse brain, gently wash it three times with PBS, then use filter paper to absorb the excess water on the surface of the mouse brain, embed the mouse brain with 4% agarose solution, and then fix it on the sample stage of the vibratome with 502 glue.

[0069] 3. Preparation of mouse brain slices: Continuously cut along the coronal plane of the mouse brain with a vibratome, and the thickness of the obtained brain slices is 300 μm. The cut mouse brain slices are encapsulated in a 24-well plate with antifreeze (15% glycerol + 40% ethanol + 45% PBS) and stored in a -20 °C refrigerator. If the mouse brain slices carry fluorescent proteins, they should be stored in the dark.

[0070] III. Embedding and imaging protocol for mouse brain tissue slices

[0071] 1. Imaging of pre-swollen mouse brain tissue sections: Take a slice of mouse brain tissue section, wash it 3 times with PBS, first fix it on a glass slide, cover it with a coverslip, and then image it with a laser scanning confocal microscope to collect fluorescent images of the pre-swollen mouse brain tissue section for comparison and correction with the fluorescent images of the swollen mouse brain tissue section.

[0072] 2. Gelation of mouse brain tissue sections: After imaging, immerse the mouse brain tissue sections in the prepolymer solution for 4 h, then encapsulate the fully penetrated mouse brain tissue sections with custom-made glass slides and transfer them to a vacuum drying oven at 40 °C for gelation treatment. After 3 h, mouse brain gels are formed.

[0073] 3. Swelling of mouse brain hydrogel samples: After taking out the mouse brain tissue sections from the vacuum drying oven after gelation, demold them. First, use a razor to peel the edges of the gel, and then carefully take out the mouse brain gel, trying to avoid possible damage to the sample during the demolding process, such as actions that can cause deformation of the sample like tearing. Then, digest the mouse brain hydrogel samples to resist internal or inter-protein interaction forces. The preparation of the digestion buffer is shown in Table 1.

[0074] Table 1 Preparation of the digestion buffer

[0075]

[0076]

[0077] Finally, place the mouse brain gel in excess deionized water and use a gyratory shaker at a speed of 70 rpm for uniform swelling to avoid anisotropic swelling caused by the gel adhering to the wall.

[0078] Imaging of swollen mouse brain tissue sections: Place the fully swollen mouse brain tissue sections on a glass slide, cover it with a coverslip (ensure that the position of the swollen mouse brain tissue section is the same as that of the pre-swollen mouse brain tissue section), and image it with a laser scanning confocal microscope. Compare and correct the obtained image with the fluorescent image of the pre-swollen mouse brain tissue section.

[0079] IV. Study on the properties of hydrogels

[0080] 1. Study on the transparency of hydrogels

[0081] The ExM imaging technique depends on the properties of hydrogels, such as light transmission properties, swelling properties, mechanical properties, etc. Hydrogels with high transparency can ensure that light can effectively penetrate the sample, thus not affecting the fluorescence imaging effect. However, since the homopolymer of DAAM hydrogel is an opaque hydrophobic porous structure, it cannot be directly applied in ExM. But the homopolymer of NMA hydrogel can make up for the defects of DAAM hydrogel, so NMA monomers are added to the DAAM hydrogel to meet the requirement of its high transparency.

[0082] First, taking 40% total monomer concentration as an example, the proportional relationship between DAAM and NMA was explored. Figure 1 The synthesis diagram of DAAM-NMA hydrogels with different monomer ratios is shown. It can be seen from the figure that when the monomer concentration of DAAM is higher than that of NMA, the DAAM-NMA hydrogels all appear milky white and hydrophobic, so the monomer concentration of DAAM should be less than or equal to the monomer concentration of NMA, that is, x / y should be less than or equal to 1.

[0083] 2. Mechanical properties

[0084] Tensile performance test, the results are as follows Figure 2 As shown, Figure 2 (a) shows the tensile stress-strain curves of 15% DAAM+25% NMA, 10% DAAM+30% NMA, and 5% DAAM+35% NMA hydrogels. As shown in Table 2, their elongations at break are 255.03%, 1587.13%, and 809.44%, respectively, and their tensile strengths are 71.63 kPa, 219.65 kPa, and 273.32 kPa, respectively. Although the tensile strength of DAAM-NMA hydrogels is improved with the addition of NMA, the elongation at break first increases and then decreases, indicating that when the concentration of NMA exceeds 30%, the elongation at break of the hydrogel reaches a critical value, so the elongation at break of 5% DAAM+35% NMA hydrogel decreases rapidly. The above research results show that the mechanical properties of 10% DAAM+30% NMA hydrogel are the best.

[0085] Figure 2 (b) shows the tensile stress-strain curves of 10% DAAM+30% NMA+5% SA, 10% DAAM+30% NMA+10% SA, and 10% DAAM+30% NMA+15% SA hydrogels, as shown in Table 2. Their elongations at break are 864.38%, 935.00%, and 782.38%, respectively, and their tensile strengths are 189.68 kPa, 167.40 kPa, and 217.72 kPa, respectively. The increase in SA concentration causes the elongation at break of DAAM-NMA-SA hydrogel to increase first and then decrease, and the tensile strength to decrease first and then increase, indicating that DAAM-NMA-SA hydrogel changes from tough to soft and then to tough with the increase in SA concentration.

[0086] Table 2 Mechanical properties of different hydrogels

[0087]

[0088] 3. Swelling properties of hydrogels

[0089] To ensure that the hydrogel expands the biological sample to the maximum extent, providing consistent sample sizes, maintaining the stability of the sample structure, and reducing the impact on fluorescence signals, thereby obtaining reliable microscopic observation results, the swelling performance of the hydrogel was tested, and the experimental results are as Figure 3 shown in Table 3.

[0090] Figure 3 (a), (b), and (c) respectively show adding 5%, 10%, and 15% sodium acrylate to the 10% DAAM + 30% NMA hydrogel. The research results show that as the concentration of sodium acrylate increases, the swelling multiple of the hydrogel continuously decreases, and its swelling factors are 5.3, 4.8, and 4.3 respectively. And when the content of sodium acrylate exceeds 15%, the hydrogel prepolymer solution is prone to precipitation, which can be attributed to the increase in the total monomer concentration of the hydrogel, resulting in the solution approaching saturation, so the gel monomers cannot be uniformly and stably dispersed in PBS. At the same time, the excessive monomer content strengthens the intermolecular chain entanglement after the hydrogel polymerization, thus causing the swelling factor to decrease. From Figure 3 (a), (b), and (c), it can be seen that when adding 5% sodium acrylate, the swelling factor of the hydrogel can reach a maximum of 5.3. From Figure 3 (d), it can be seen that after adding 5% sodium acrylate to the hydrogel, its swelling degree is the largest, reaching about 100 times. In summary, adding a small amount of sodium acrylate can significantly enhance the swelling performance of the hydrogel.

[0091] Table 3 Swelling performance of different DAAM-NMA-SA hydrogels

[0092]

[0093]

[0094] 4. Isotropic swelling of biological samples

[0095] Due to the differences in the structural characteristics of biological samples themselves, the gelated biological samples may have anisotropic swelling, resulting in errors in the final imaging results of ExM. Therefore, it is necessary to evaluate the swelling factors of the biohydrogel in different dimensions to verify that the DAAM-NMA-SA hydrogel can make the biological samples swell isotropically. This section will analyze the isotropic swelling of the mouse brain slice hydrogel from both macroscopic and microscopic perspectives.

[0096] First is the determination of the swelling factor from a macroscopic perspective. As Figure 4As shown, after the mouse brain slices were infiltrated with the hydrogel prepolymer solution, their sizes did not change significantly, but there was a slight clarification phenomenon, and they even became almost completely transparent after swelling. This is due to the influence of the swelling process and the digestion buffer on the brain tissue. After the mouse brain slices were embedded in the hydrogel, their sizes still did not change significantly. As the soaking time in deionized water increased, the sizes of the mouse brain slices continuously swelled and increased, and finally reached the swelling equilibrium state after 48 h. The swelling factors in the transverse and longitudinal directions were both about 4.0. This indicates that the mouse brain slices did not produce obvious distortion deformation and achieved isotropic swelling from a macroscopic perspective.

[0097] Further study the swelling factors of the hydrogel of biological samples from a microscopic perspective. As Figure 5 shown in and Table 4, fluorescence images of 300-μm-thick mouse brain slices obtained under a 10× microscope before and after swelling were compared using a laser scanning confocal fluorescence microscope. Figure 5 (a) and (b) respectively show the landmark sites at the same position before and after swelling. The distance between the two landmark sites increased to about 4.05 times the original distance after swelling. As can be seen from Table 4, the values of the swelling factors in different directions are basically the same, indicating that the swelling is a uniform and isotropic process and has little impact on brain neuron connections. At the same time Figure 5 (c) shows the overlapping images before and after swelling. No significant deformation of the mouse brain slices was observed in the overlapping images, which also shows that the swelling process of the DAAM-NMA-SA hydrogel is isotropic at the microscopic level, enabling the mouse brain slices fixed in it to swell uniformly at the same time.

[0098] Table 4 Linear swelling multiples of mouse brain slices

[0099]

[0100] 5. Swelling microscopy

[0101] After a series of characterization tests on the DAAM-NMA-SA hydrogel, practical applications will be carried out. Taking the mouse brain slices of C57BL / 6 mice as the research samples, the swelling microscopy effect of the DAAM-NMA-SA hydrogel was analyzed by comparing the normalized fluorescence intensity curves and FWHM at the same positions of the fluorescence images before and after swelling, as Figure 6 shown.

[0102] Figure 6 (a) and (c) are respectively enlarged fluorescence images of the same region of the mouse brain slices before and after swelling under a 10× objective lens of a confocal microscope. The images clearly changed from blurred to clear, indicating a significant imaging effect.

[0103] In addition, fluorescence intensity curve analysis was carried out on the same positions, that is, Figure 6 the yellow line regions of (a) and (b), respectively, and the results are asFigure 6 as shown in (b) and (d). The normalized fluorescence intensity curve before expansion ( Figure 6 (b)) did not show obvious changes in fluorescence intensity, while the normalized fluorescence intensity curve after expansion ( Figure 6 (b)) showed two relatively clear peaks, indicating that neurons that could not be distinguished originally could be clearly distinguished after expansion.

[0104] Then, 100 identical yellow line regions were selected in the images before and after expansion (as shown in Figure 7 (a) and (b)), and the corresponding frequency distribution histograms of the full width at half maximum (FWHM) were plotted respectively (as shown in Figure 7 (c) and (d)), as shown in Figure 7 . It can be observed that the maximum FWHM of the mouse brain slice before expansion was up to 14 μm, the minimum value was greater than 1 μm, and most of them were concentrated in the range of 3 - 5 μm. After expansion, the maximum FWHM decreased to 12 μm, the minimum value was less than 1 μm, and it was mainly concentrated in the range of 1 - 3 μm. As shown in Figure 7 (e), it can be intuitively felt that the resolution before and after expansion has been improved because the total FWHM before expansion decreased from 462 μm to 265 μm.

[0105] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all are covered by the protection scope of the present invention.

Claims

1. A preparation method of a hydrogel for expansion microscopy imaging, characterized in that, It includes the following steps: S1. Prepare a hydrogel precursor solution: Dissolve diacetone acrylamide, N-(hydroxymethyl) acrylamide, and sodium acrylate in a PBS solution to obtain a hydrogel precursor solution; wherein, the mass percentage concentrations of diacetone acrylamide, N-(hydroxymethyl) acrylamide, and sodium acrylate in the hydrogel precursor solution are denoted as X, Y, and Z respectively, X + Y is 35 - 45% and X / Y ≤ 1, Z ≤ 10%; S2. Prepare a hydrogel prepolymer solution: Add an initiator mother liquor to the hydrogel precursor solution to form a hydrogel prepolymer solution. The initiator is ammonium persulfate or 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and a hydrogel for expansion microscopy imaging is obtained.

2. The preparation method of a hydrogel for expansion microscopy imaging according to claim 1, wherein The concentration of diacetone acrylamide in the hydrogel precursor solution is 5 - 15 wt%, the concentration of N-(hydroxymethyl) acrylamide is 25 - 35 wt%, and the concentration of sodium acrylate is 3 - 8 wt%.

3. The preparation method of a hydrogel for expansion microscopy imaging according to claim 1, characterized in that, The concentration of diacetone acrylamide in the hydrogel precursor solution is 10 wt%, the concentration of N-(hydroxymethyl) acrylamide is 30 wt%, and the concentration of sodium acrylate is 5 wt%.

4. The preparation method of a hydrogel for expansion microscopy imaging according to claim 1, characterized in that, The concentration of the initiator mother liquor is 100 - 300 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother liquor is 90 - 120:

1.

5. A hydrogel for expansion microscopy imaging prepared by the method according to any one of claims 1 - 4.

6. A method for expanding a sample, characterized in that, It includes the following steps: (1) Sample gelation: Immerse the sample in the hydrogel for expansion microscopy imaging according to claim 5 for infiltration for 3.5 - 4.5 h, then encapsulate it with a mold and polymerize it at a temperature of 35 - 45°C for at least 3 h; (2) Sample expansion: Demold the polymerized and encapsulated sample, take out the gelated sample and perform digestion treatment. The digested sample is immersed in deionized water and expanded with a gyratory shaker at 50 - 100 rpm. The expansion time is 1 - 48 h.

7. A method for swelling treatment of a sample according to claim 6, characterized in that, The sample is a mouse brain slice sample.

8. A method for swelling treatment of a sample according to claim 6, characterized in that, The specific operation of the digestion treatment is to immerse the gelated sample in a digestion buffer for 2 - 48 h. The digestion buffer is a mixed solution of 6 - 10 U / ml proteinase K, 0.3 - 0.8% W / V Triton X-100, and 1×TAE buffer.

9. A method for expanding a sample according to claim 6, characterized in that, The mold is a glass slide with a sample groove, and the size of the sample groove matches the size of the sample.

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