Hydrogel for expansion microscope imaging, preparation method thereof and sample expansion treatment method

Abstract

The invention belongs to the field of fluorescence microscopic imaging, and particularly relates to hydrogel for expansion microscope imaging, a preparation method of the hydrogel and a sample expansion treatment method. The preparation method of the hydrogel comprises the steps of preparing a hydrogel precursor solution and preparing a hydrogel prepolymerization solution. The sample expansion treatment method comprises sample gelation and sample expansion. The hydrogel disclosed by the invention is excellent in mechanical property and high in elongation at break. The device has good stability and is convenient and safe to use. And the traditional biomolecule anchoring step is combined with tissue permeation and gelation, hydrogel is used for expanding the mouse brain slice, the mouse brain slice can be isotropically expanded to the maximum size after 48 hours, and the linear expansion multiple of about 4.0 times is obtained. The DAAM-NMA-SA hydrogel is used for embedding a mouse brain slice marked by green fluorescent protein, the imaging effect of an expanded microscopic fluorescence image of the DAAM-NMA-SA hydrogel is remarkably improved compared with that of a fluorescence image of an original brain slice, and the resolution ratio is greatly increased.

Description

Technical Field

[0001] The invention belongs to the field of fluorescence microscopic imaging, and in particular relates to a hydrogel used for expansion microscopic imaging, a preparation method thereof, and a sample expansion processing 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 solidify the polymers through polymerization reactions, and expand the polymer network by adding water or specific chemicals, so that images of the samples can be obtained beyond the resolution limit under traditional microscopes.

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

[0004] (1) Swelling the sample may cause distortion of certain 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 cause spatial distortion or geometric distortion. This requires careful handling during sample preparation and microscopy imaging to minimize the impact of this distortion. ExM technology is complex in sample preparation, imaging, and data processing, which limits its widespread use in practical applications. During sample preparation, multiple chemical treatment steps are required, such as anchoring, infiltration, expansion, etc. These steps need to be carefully controlled to ensure the expansion effect and sample stability.

[0005] (2) Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to attach specific markers and biomolecule classes to the gel. This step is called biomolecule anchoring, and in protein-based anchoring strategies, strong protease digestion is required to achieve isotropic expansion. This digestion method will cause a large loss of target proteins after expansion, limiting the number of protein structures that can be detected.

[0006] (3) Optimization and standardization of ExM techniques for different types of samples is also a challenge. Different types of tissues and cells may have different transparency, swelling properties, and stability requirements.

[0007] Therefore, there is an urgent need to further develop and optimize ExM methods suitable for different types of samples and to establish standardized sample preparation and imaging steps. 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 microscope imaging, a preparation method thereof and a sample expansion treatment method.

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

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

[0011] S2. Preparation of hydrogel prepolymer solution: Adding initiator mother solution to hydrogel precursor solution to form hydrogel prepolymer solution, wherein the initiator is ammonium persulfate or azobisisobutylimidazoline hydrochloride, to obtain a hydrogel for expansion microscopy imaging.

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

[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 solution is 100-300 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother solution is 90-120:1.

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

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

[0017] (1) Sample gelation: immersing the sample in the hydrogel for expansion microscopy imaging according to claim 5 for 3.5 to 4.5 hours, then encapsulating it in a mold, and polymerizing it at a temperature of 35 to 45° C. for at least 3 hours;

[0018] (2) Sample expansion: demold the encapsulated sample after polymerization, take out the gelled sample and perform digestion treatment, immerse the digested sample in deionized water, and expand it on a cyclotron oscillator at 50 to 100 rpm, and the expansion time is 1 to 48 hours.

[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 a digestion buffer for 2 to 48 hours, wherein 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 sample expansion by the hydrogel of the present invention is:

[0023] The diacetone acrylamide monomer is used in the formula of the present invention 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 carries a lone pair of electrons, which can form hydrogen bonds with amino acids in biological tissues, and the generation of hydrogen bonds also plays an important role in the stability and fixation of biological samples; 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 method of the biological sample in the polymer system. This spatial restriction can make other molecules be "locked" in a specific position, thereby improving the fixation effect. Acrylamide in the existing hydrogel has the characteristics of a cross-linking agent, which can form hydrogen bonds with the amino and carboxyl groups in the protein to achieve a fixation effect. Compared with the existing hydrogel, it has the following differences:

[0024] Different anchoring agents: The existing simple hydrogel expansion system uses the amide, hydroxyl and other groups of the hydrogel itself to form hydrogen bonds with the amino, carboxyl, amide and other groups in the protein to achieve the anchoring effect. This is a physical effect, which is easily destroyed and has insufficient expansion stability. The protein anchoring agent of the more complex hydrogel expansion system is a water-insoluble reagent. It is usually necessary to use an organic reagent to carry out the first step of protein anchoring, and then further rehydrate and replace the organic reagent, and then perform hydrogel prepolymerization liquid penetration, polymerization, digestion, expansion and other operations, which are cumbersome to operate. The anchoring agent of the present invention is water-soluble diacetone acrylamide (DAAM). The reaction of the ketone group with the protein forms a Schiff base, which can form a stable chemical bond connection and can expand uniformly and isotropically. The water-soluble anchoring agent DAAM can combine the anchoring step with tissue penetration, simplifying the steps of the expansion microscopy technology.

[0025] Different penetration modes: The introduction of ketone groups can change the permeability of biological membranes, allowing polymeric substances to penetrate into tissues more effectively or maintain biological molecules inside tissues, thereby improving the preservation state and function of biological tissues.

[0026] Different staining abilities: The presence of ketone groups can improve the binding ability of the dye, making the tissue easier to be stained by different types of dyes, which is more conducive to subsequent microscopic expansion observation.

[0027] Based on the reaction between ketone groups and proteins and the high water absorption of sodium acrylate, the present invention applies diacetone acrylamide (DAAM), N-(hydroxymethyl) acrylamide (NMA) and sodium acrylate (SA) to expansion microscopy to expand the expansion factor of the hydrogel, thereby improving the resolution of the optical microscope. The hydrogel of the present invention uses DAAM as an anchoring agent and hydrogel monomer to provide gel complexing sites and biomolecule anchoring sites for the hydrogel network; NMA provides hydrogen bonds as a swelling agent and monomer for the hydrogel, and SA is added to improve the swelling factor and water absorption performance of the hydrogel system. The three together constitute a DAAM-NMA-SA hydrogel system. Combining the traditional biomolecule anchoring step with tissue penetration improves the operability and simplicity of the expansion microscopy technology solution 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 a gel complexing site and a biomolecule anchoring site for the hydrogel network; NMA provides hydrogen bonds as a swelling agent and monomer of the hydrogel, and SA is added to improve the swelling factor and water absorption performance of the hydrogel system, and the three together constitute a DAAM-NMA-SA hydrogel system. The hydrogel prepared by the method of the present invention has excellent mechanical properties and high elongation at break. It has good stability, is easy to use and safe. In addition, the traditional biomolecule anchoring step is combined with tissue penetration, and the DAAM-NMA-SA hydrogel is used to expand the rat brain slice, and the rat brain slice can be isotropically expanded to the maximum size after 48 hours, and a linear expansion multiple of about 4.0 times is obtained. The DAAM-NMA-SA hydrogel embeds the rat brain slice containing the green fluorescent protein marker, and the expansion microscopic fluorescence image thereof is significantly improved compared with the fluorescence image of the original brain slice, and the resolution is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Synthesis diagram of DAAM-NMA hydrogels with different monomer ratios. In the figure, (ai) from left to right are 40-0, 35-5, 30-10, 25-15, 20-20, 15-25, 10-30, 5-35, and 0-40 hydrogels.

[0030] Figure 2Graphs showing the tensile stress-strain curves of hydrogels, including (a) the tensile stress-strain curves of different DAAM-NMA hydrogels, and (b) the tensile stress-strain curves of DAAM-NMA-SA hydrogels with different concentrations.

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

[0032] Figure 4 This is a diagram showing the size changes of the mouse brain when it swells in deionized water. In the figure, (a) the original brain slice size; (b) the brain slice size after infiltration; (c) the brain slice size after gel; (d) the brain slice size after digestion; (e) the brain slice size after swelling. Scale bar: one small grid is 1 mm.

[0033] Figure 5 The following are the selected landmarks of the expansion multiples of mouse brain slices. In the figure, (a) brain slice after expansion; (b) original brain slice; (c) fluorescence image after superposition of (a) and (b). 0-3: different landmarks, scale bar: 100m.

[0034] Figure 6 Fluorescence imaging analysis of rat brain neurons before and after expansion. In the figure, (a) fluorescence image of rat brain neurons before expansion; (b) normalized curve of fluorescence intensity in the yellow line area in (a) as a function of distance; (c) fluorescence image of rat brain neurons after expansion; (d) normalized curve of fluorescence intensity in the yellow line area in (b) as a function of distance. Scale bar: 10m.

[0035] Figure 7 Fluorescence imaging analysis of mouse brain neurons before and after expansion. In the figure, (a) confocal fluorescence image of mouse brain neurons before expansion; (b) confocal fluorescence image of mouse brain neurons after expansion; (c) FWHM frequency distribution histogram of mouse brain neurons in the yellow line area in Figure (a); (d) FWHM frequency distribution histogram of mouse brain neurons in the yellow line area in Figure (b); (e) FWHM before and after expansion obtained from Figures (c) and (d). n = 100, scale bar: 100μm. DETAILED DESCRIPTION

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

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

[0038] S2. Preparation of hydrogel prepolymer solution: adding initiator mother solution to the hydrogel precursor solution to form a hydrogel prepolymer solution, wherein the initiator is azobisisobutylimidazoline hydrochloride, the concentration of the initiator mother solution is 100 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother solution 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. Preparing a hydrogel precursor solution: dissolving diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in a PBS solution to prepare a hydrogel precursor solution; wherein the concentration of diacetone acrylamide in the hydrogel precursor solution is 5wt%, the concentration of N-(hydroxymethyl) acrylamide is 30wt%, and the concentration of sodium acrylate is 3wt%;

[0041] S2. Preparation of hydrogel prepolymer solution: adding initiator mother solution to hydrogel precursor solution to form hydrogel prepolymer solution, wherein the initiator is ammonium persulfate, the concentration of the initiator mother solution is 300 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother solution is 90 to 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. Preparing a hydrogel precursor solution: dissolving diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in a PBS solution to prepare a hydrogel precursor solution; wherein the concentration of diacetone acrylamide in the hydrogel precursor solution is 10wt%, the concentration of N-(hydroxymethyl) acrylamide is 35wt%, and the concentration of sodium acrylate is 8wt%;

[0044] S2. Preparation of hydrogel prepolymer solution: adding initiator mother solution to the hydrogel precursor solution to form a hydrogel prepolymer solution, wherein the initiator is azobisisobutylimidazoline hydrochloride, the concentration of the initiator mother solution is 180 mg / mL, and the volume ratio of the gel precursor solution to the initiator mother solution is 90 to 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. Preparing a hydrogel precursor solution: dissolving diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in a PBS solution to prepare a hydrogel precursor solution; wherein the concentration of diacetone acrylamide in the hydrogel precursor solution is 10wt%, the concentration of N-(hydroxymethyl) acrylamide is 30wt%, and the concentration of sodium acrylate is 5wt%;

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

[0048] Embodiment 5: A method for sample expansion treatment, comprising the following steps:

[0049] (1) Sample gelation: The mouse brain slices were immersed in the hydrogel prepared in Example 1 for expansion microscopy imaging for 3.5 h, then sealed with a glass slide with a sample groove, and polymerized at 35° C. for at least 3 h;

[0050] (2) Sample expansion: The encapsulated sample after polymerization was demolded, and the gelled rat brain slices were taken out and digested. The specific operation of the digestion treatment was to immerse the gelled sample in a digestion buffer for 2 hours. The digestion buffer was a mixed solution of 6U / ml proteinase K, 0.3% (W / V) Triton X-100 and 1×TAE buffer. The digested rat brain slices were immersed in deionized water and expanded on a 50rpm cyclotron for 1 hour.

[0051] Embodiment 6: A sample expansion treatment method, comprising the following steps:

[0052] (1) Sample gelation: The mouse brain slices were immersed in the hydrogel prepared in Example 3 for expansion microscopy imaging for 4.5 h, then sealed with a glass slide with a sample groove, and polymerized at 45° C. for at least 3 h;

[0053] (2) Sample expansion: The encapsulated sample after polymerization was demolded, and the gelled rat brain slices were taken out and digested. The specific operation of the digestion treatment was to immerse the gelled sample in a digestion buffer for 48 hours. The digestion buffer was a mixed solution of 10U / ml proteinase K, 0.8% (W / V) Triton X-100 and 1×TAE buffer. The digested rat brain slices were immersed in deionized water and expanded on a 100 rpm cyclotron for 48 hours.

[0054] Embodiment 7: A sample expansion treatment method, comprising the following steps:

[0055] (1) Sample gelation: The mouse brain slices were immersed in the hydrogel prepared in Example 4 for expansion microscopy imaging for 4 h, then sealed with a glass slide with a sample groove, and polymerized at 40° C. for at least 3 h;

[0056] (2) Sample expansion: The encapsulated sample after polymerization was demolded, and the gelled rat brain slices were taken out and digested. The specific operation of the digestion treatment was to immerse the gelled sample in a digestion buffer for 28 hours. The digestion buffer was a mixed solution of 8U / ml proteinase K, 0.5% (W / V) Triton X-100 and 1×TAE buffer. The digested rat brain slices were immersed in deionized water and expanded on a 70rpm cyclotron for 24 hours.

[0057] The beneficial effects of the present invention are described below by experiments:

[0058] 1. 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" (abbreviated as: xyz, where x, y, z represent the concentrations of DAAM, NMA and SA, respectively). The specific configuration process is as follows (taking 100 mL of 10% DAAM + 30% NMA + 5% SA as an example):

[0060] Use an electronic balance to weigh 10 g DAAM, 30 g NMA and 5 g SA respectively into a sample bottle, dissolve them with 55 mL PBS, and then dissolve the solution in an ultrasonic automatic cleaning instrument until it is clear and transparent. Then take it out and store it in a 4°C refrigerator. The validity period is one month.

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

[0062] 2. Preparation of hydrogel prepolymer solution: First, prepare the initiator mother solution at a mass volume ratio of 25% (i.e., 750 μL of deionized water dissolves 250 mg of AIBI, and the mother solution is prepared and used immediately), and then add the initiator mother solution to the hydrogel precursor solution at a ratio of 100:1 (i.e., 10 mL of hydrogel precursor solution plus 100 μL of initiator mother solution). (The initiator uses ammonium persulfate (APS) or azobisisobutylimidazoline hydrochloride (AIBI))

[0063] 3. Hydrogel polymerization: After the hydrogel prepolymerization solution is prepared according to the above steps, it is encapsulated in a customized glass slide with a sample groove, where the customized sample groove has a diameter of 10 mm and a depth equal to 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 hours.

[0064] 2. Preparation of Mouse Brain

[0065] Preparation of mouse brain samples: Brain tissues were removed from C57BL / 6 mice by cardiac perfusion, and the mouse brain samples were fixed and preserved using 4% PFA.

[0066] The specific operation method is as follows:

[0067] 1. Intraperitoneal injection of mixed anesthetics: 2.5% (wt / vol) tribromoethanol anesthetizes mice. When the mice become slow in movement and deeply anesthetized, transfer them to the perfusion table in the fume hood and perfuse 0.01M PBS solution (RT) through the left ventricle of the mice until the liver loses its blood color from red. Then perfuse 4% (wt / vol) PFA solution. After the mice become stiff, the perfusion is completed. Dissect the mice, remove the mouse brains, immerse them in 4% PFA at 4°C, and fix them overnight. Fixation should not exceed 24 hours. Then rinse the mouse brains with PBS solution to remove the residual PFA solution in the tissues. The mouse brains can be stored in PBS solution at 4°C for a short period of time.

[0068] 2. Pre-treatment of mouse brain slices: After the fixed mouse brain is taken out, it is gently washed three times with PBS, and then the excess water on the surface of the mouse brain is absorbed with filter paper, and the mouse brain is embedded with 4% agarose solution, and then fixed on the sample holder of the vibration slicer with 502 glue.

[0069] 3. Preparation of rat brain slices: Use a vibrating slicer to continuously cut along the coronal plane of the rat brain, and the thickness of the obtained brain slices is 300 μm. The cut rat brain slices are packaged in a 24-well plate with antifreeze (15% glycerol + 40% ethanol + 45% PBS) and stored in a -20°C refrigerator. If the rat brain slices contain fluorescent protein, keep them away from light.

[0070] 3. Embedding and Imaging Protocol for Mouse Brain Tissue Sections

[0071] 1. Imaging of pre-expanded rat brain tissue slices: Take a piece of rat brain slice and wash it three times with PBS, fix it on a glass slide first, cover it with a coverslip and then use a laser scanning confocal microscope to image it, and collect the fluorescence image of the pre-expanded rat brain tissue slice to facilitate comparison and correction of the fluorescence image of the expanded rat brain tissue slice.

[0072] 2. Gelation of rat brain slices: After imaging, the rat brain slices were immersed in the prepolymer solution for 4 hours, and then the completely infiltrated rat brain slices were encapsulated with customized glass slides and moved into a vacuum drying oven at 40°C for gelation. After 3 hours, rat brain gel was formed.

[0073] 3. Expansion of rat brain hydrogel samples: After taking out the rat brain slices from the vacuum drying oven, demold them. First, peel off the edge of the gel with a razor, and then carefully remove the rat brain gel. Here, try to avoid possible damage to the sample during the demolding process, such as tearing and other actions that may cause deformation of the sample. Then digest the rat brain hydrogel sample to resist intrinsic or protein-protein interaction forces. The configuration of the digestion buffer is shown in Table 1.

[0074] Table 1 Digestion buffer configuration

[0075]

[0076]

[0077] Finally, the mouse brain gel was placed in excess deionized water and swollen evenly using a gyroscopic oscillator at a rotation speed of 70 rpm to avoid anisotropic swelling caused by gel adhesion to the wall.

[0078] Imaging of mouse brain tissue slices after expansion: Place the fully expanded mouse brain slices on a glass slide, cover with a coverslip (make sure the position of the expanded mouse brain slices is consistent with the position of the pre-expanded mouse brain slices), and use a laser scanning confocal microscope to image. The obtained images are compared and corrected with the fluorescent images of the pre-expanded mouse brain tissue slices.

[0079] 4. Study on hydrogel properties

[0080] 1. Hydrogel transparency study

[0081] ExM imaging technology relies on the properties of hydrogels, such as light transmittance, swelling properties, and mechanical properties. Highly transparent hydrogels can ensure that light can effectively penetrate the sample, thereby not affecting the fluorescence imaging effect. However, since DAAM hydrogel homopolymers are opaque hydrophobic porous structures, they cannot be directly used in ExM. However, NMA hydrogel homopolymers can make up for the defects of DAAM hydrogels, so NMA monomers are added to DAAM hydrogels to meet their high transparency requirements.

[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] In order to ensure that the hydrogel can expand the biological sample to the maximum extent, provide a consistent sample size, maintain the stability of the sample structure, and reduce the impact on the fluorescence signal, so as to obtain reliable microscopy observation results, the swelling performance of the hydrogel was tested. The experimental results are shown in Figure 2. Figure 3 And as shown in Table 3.

[0090] Figure 3 (a), (b), and (c) show the addition of 5%, 10%, and 15% sodium acrylate to the 10% DAAM + 30% NMA hydrogel, respectively. The results show that with the increase in the concentration of sodium acrylate, the hydrogel expansion multiple continues to decrease, and the expansion 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. This can be attributed to the increase in the total monomer concentration of the hydrogel, which causes the solution to approach a saturated state. Therefore, the gel monomer cannot be evenly and stably dispersed in PBS. At the same time, the excessively high monomer content strengthens the intermolecular chain entanglement of the hydrogel after polymerization, thereby reducing the expansion factor. Figure 3 (a), (b), and (c) show that when 5% sodium acrylate is added, the maximum expansion factor of the hydrogel can reach 5.3. Figure 3 (d) It can be seen that after adding 5% sodium acrylate, the hydrogel has the largest expansion degree, which can reach about 100 times. In summary, adding a small amount of sodium acrylate can significantly enhance the expansion performance of the hydrogel.

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

[0092]

[0093]

[0094] 4. Isotropic expansion of biological samples

[0095] Due to the differences in the structural properties of biological samples, the gelled biological samples may have anisotropic expansion, which may cause errors in the final ExM imaging results. Therefore, it is necessary to evaluate the expansion factors of biological hydrogels in different dimensions to verify that DAAM-NMA-SA hydrogels can make biological samples expand isotropically. This section will analyze the isotropic expansion of mouse brain slice hydrogels from both macroscopic and microscopic perspectives.

[0096] The first is the determination of the expansion factor from a macroscopic perspective. Figure 4As shown in the figure, after the mouse brain slices were infiltrated by the hydrogel prepolymer solution, there was no obvious change in size, but there was a slight transparency phenomenon, and even after expansion, they were almost completely transparent. This is due to the influence of the expansion process and the digestion buffer on the brain tissue. After the mouse brain slices were embedded in the hydrogel, the size still did not change significantly. As the immersion time in deionized water increased, the size of the mouse brain slices continued to expand and eventually reached a swelling equilibrium state after 48 hours. The horizontal and vertical expansion factors were measured to be around 4.0. This shows that the mouse brain slices did not produce obvious distortion and deformation, and achieved isotropy of expansion from a macroscopic perspective.

[0097] Further study the expansion factor of biological sample hydrogel at a microscopic perspective. Figure 5 As shown in Table 4, fluorescence images of 300 μm thick mouse brain slices before and after expansion were compared using a laser scanning confocal fluorescence microscope under a 10× microscope. Figure 5 (a) and (b) show the landmarks at the same position before and after expansion, respectively. After expansion, the distance between the two landmarks increases to about 4.05 times the original distance. As shown in Table 4, the values ​​of the expansion factor in different directions are basically the same, which indicates that expansion is a uniform and isotropic process and has little effect on brain neuron connections. Figure 5 (c) shows the overlapping images before and after expansion. No significant deformation of the mouse brain slices was observed in the overlapping images, which also shows that the expansion process of DAAM-NMA-SA hydrogel is isotropic at the microscopic level, so that the mouse brain slices fixed in it can also expand uniformly at the same time.

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

[0099]

[0100] 5. Expansion Microscopy

[0101] After a series of characterization tests on DAAM-NMA-SA hydrogel, it will be put into practical application. Using brain slices of C57BL / 6 mice as research samples, the expansion microscopic imaging effect of DAAM-NMA-SA hydrogel was analyzed by comparing the normalized fluorescence intensity curve and FWHM at the same position of the fluorescence image before and after expansion. Figure 6 shown.

[0102] Figure 6 (a) and (c) are magnified fluorescence images of mouse brain slices before and after expansion of the same area under a 10× confocal microscope. The image obviously changes from blurry to clear, indicating that the imaging effect is significant.

[0103] In addition, the same position is Figure 6 The yellow line area of ​​(a) and (b) was analyzed for fluorescence intensity curves. The results are shown in Figure 6 (b) and (d). 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) shows two clearer peaks, indicating that the neurons that were originally indistinguishable can be clearly distinguished after expansion.

[0104] Then select 100 identical yellow line areas in the images before and after expansion (such as Figure 7 (a) and (b)), and the corresponding half-peak width (FWHM) frequency distribution histograms are drawn (as shown in Figure 7 (c) and (d)), as shown in Figure 7 As shown in the figure, it can be observed that the FWHM of the mouse brain slices before expansion is as high as 14μm, the minimum value is greater than 1μm, and most of them are concentrated in the range of 3-5μm. After expansion, the maximum value of FWHM decreases to 12μm, the minimum value is less than 1μm, and most of them are concentrated in the range of 1-3μm. Figure 7 As shown in (e), the improvement in resolution before and after expansion can be intuitively felt, because the total FWHM before expansion dropped from 462μm to 265μm.

[0105] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel for expansion microscopy imaging, characterized in that: The following steps are involved: S1. Preparing a hydrogel precursor solution: dissolving diacetone acrylamide, N-(hydroxymethyl) acrylamide and sodium acrylate in a PBS solution to prepare 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. Preparation of hydrogel prepolymer solution: Adding initiator mother solution to hydrogel precursor solution to form hydrogel prepolymer solution, wherein the initiator is ammonium persulfate or azobisisobutylimidazoline hydrochloride, to obtain a hydrogel for expansion microscopy imaging.

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

1.

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

6. A sample expansion processing method, characterized in that: It includes the following steps: (1) Sample gelation: immersing the sample in the hydrogel for expansion microscopy imaging according to claim 5 for 3.5 to 4.5 hours, then encapsulating it in a mold, and polymerizing it at a temperature of 35 to 45° C. for at least 3 hours; (2) Sample expansion: demold the encapsulated sample after polymerization, take out the gelled sample and perform digestion treatment, immerse the digested sample in deionized water, and expand it on a cyclotron oscillator at 50 to 100 rpm, and the expansion time is 1 to 48 hours.

7. A sample expansion processing method according to claim 6, characterized in that: The sample is a mouse brain slice sample.

8. A sample expansion processing method according to claim 6, characterized in that: The specific operation of the digestion treatment is to immerse the gelled sample in a digestion buffer for 2 to 48 hours, wherein 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.

9. A sample expansion processing method 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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