A dual-gel system suitable for large-volume biological tissue expansion micro-ablation

Abstract

This application belongs to the field of fluorescence microscopy imaging, and more specifically, relates to a dual-gel system suitable for dilatational microscopy of large-volume biological tissues. This dual-gel system achieves uniform expansion and morphological stability of tissue samples through a dilatational gel, and then imparts high mechanical strength using a stabilizing gel. This allows millimeter-scale large-volume biological tissue samples to possess both high expansion ratio and excellent mechanical stability, achieving 3-5 times linear expansion with a Young's modulus of not less than 300 kPa. It can stably adapt to vibrational cutting operations and support cutting of 100-500 μm thick layers, with intact cut surfaces and good interlayer consistency, greatly improving the efficiency and quality of three-dimensional data acquisition. Furthermore, the hydrogel sections obtained from the cutting have intact morphology and can be used as independent slides for multidimensional analysis. This application overcomes the limitations of traditional dilatational microscopy techniques in terms of sample volume and mechanical stability, providing an efficient and reliable solution for the three-dimensional reconstruction of millimeter-scale biological tissues.

Description

Technical Field

[0001] This application belongs to the field of fluorescence microscopy imaging, and more specifically, relates to a dual-gel system suitable for large-volume biological tissue expansion micro-cutting. Background Technology

[0002] Expansion microscopy (ExM) is an imaging method developed in recent years that utilizes conventional optical microscopes to obtain nanoscale spatial resolution. The core idea of ​​this technique is to form a water-absorbing and swelling polyelectrolyte hydrogel network through in-situ polymerization. This network physically anchors and supports the spatial position of biomolecules, achieving isotropic magnification of the sample's physical scale during the water absorption and swelling process. This allows optical microscopes to resolve ultrastructures that are originally below their diffraction limit.

[0003] In specific technical approaches, ExM typically embeds highly absorbent polymeric hydrogels within biological samples and chemically links the biomolecules of interest or their markers to a polymer network. This ensures that the relative spatial relationships of the molecules maintain their positions during gel expansion, thus avoiding imaging distortion caused by non-uniform deformation. The gel solution formulation largely determines the absorbency, network structure, and mechanical properties of the polymeric hydrogel. Subsequently, the sample undergoes homogenization to disrupt endogenous structural supports or cross-linking that might limit isotropic expansion. This process is usually achieved through proteinase K digestion or chemical denaturation using surfactants combined with high-temperature treatment. Finally, the hydrogel fully absorbs water and expands in deionized water, proportionally magnifying the physical distances between the anchored biomolecules, thereby enabling optical imaging and resolution of ultrastructures.

[0004] Currently, dilatational microscopy (DIM) still faces several technical challenges in expanding to larger sample volumes. First, large-volume samples struggle to maintain stable morphology under dilatation. Existing DIM techniques are primarily developed for thin tissue sections. During homogenization, the original structural support within the tissue is disrupted, and the sample mainly relies on a low-crosslinking-density hydrogel network to maintain its overall morphology. When the sample size increases to the millimeter scale, under gravity and external disturbances, the dilatated sample is prone to macroscopic deformation, collapse, or local anisotropic distortion, making it difficult to maintain a true three-dimensional spatial structure and thus limiting the ability to accurately analyze large-volume tissues in three dimensions. Second, dilatated samples are difficult to image with incorporating tissue cutting operations to overcome the limited imaging depth. Due to objective lens working distance limitations, millimeter-scale dilatated samples typically require layer-by-layer acquisition using physical methods such as vibration cutting. However, samples processed with existing dilatation strategies have limited overall mechanical strength and weak shear resistance, easily exhibiting significant deformation or even surface tearing during cutting. This makes it difficult to maintain a stable geometric morphology under cutting stress, affecting the consistency of the interlayer structure before and after cutting and reducing the stability and repeatability of imaging results. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this application is to provide a dual-gel system suitable for large-volume biological tissue expansion microscopy cutting. This system aims to solve the problems in existing expansion microscopy techniques, such as the difficulty in adapting expanded samples to vibration cutting, the inability to achieve stable thick-layer cutting, and the tendency of the cut hydrogel sections to fall apart, curl, or break, making it difficult to maintain a stable morphology and thus limiting the application of the sections.

[0006] To achieve the above objectives, in a first aspect, this application provides a dual-gel system suitable for large-volume biological tissue expansion microablation, comprising an expansion gel prepolymer and a stabilizing gel prepolymer. The aforementioned expanding hydrogel prepolymer solution is composed of an expanding hydrogel precursor solution and an initiator stock solution. The aforementioned expanding hydrogel precursor solution contains acrylamide, sodium 2-acrylamido-2-methylpropanesulfonate, N,N'-diallyl tartaric acid diamide, and a solvent. The aforementioned stable gel prepolymer solution is composed of an expanding gel precursor solution and an initiator stock solution. The aforementioned stable gel precursor solution contains acrylamide, N,N'-methylenebisacrylamide, and a solvent.

[0007] Preferably, the concentration of acrylamide in the above-mentioned expanding gel precursor solution is 10wt%~15wt%.

[0008] Preferably, the concentration of sodium 2-acrylamido-2-methylpropanesulfonate in the above-mentioned expanding gel precursor solution is 10wt%~20wt%.

[0009] Preferably, the concentration of N,N'-diallyl tartaric acid diamide in the above-mentioned expanding gel precursor solution is 0.3wt%~1wt%.

[0010] Preferably, the concentration of acrylamide in the stabilized gel precursor solution is 10wt%~15wt%.

[0011] Preferably, the concentration of N,N'-methylenebisacrylamide in the above-mentioned stable gel precursor solution is 0.5wt%~1wt%.

[0012] Preferably, the initiators in the above-mentioned expanding gel prepolymer and the above-mentioned stable gel prepolymer are each independently a photoinitiator or a redox initiator.

[0013] More preferably, the photoinitiator is selected from Irgacure 2959 or lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the redox initiator is selected from ammonium persulfate or potassium persulfate.

[0014] Preferably, the concentration of the initiator stock solution is 6wt%~8wt%; the volume ratio of the expanding gel precursor solution to the initiator stock solution is (150~200):1; and the volume ratio of the stable gel precursor solution to the initiator stock solution is (150~200):1.

[0015] Secondly, this application provides a method for expanding microscopy of biological tissue samples using the above-mentioned dual-gel system, comprising the following steps: S1. Degrease and biomolecule anchoring treatment is performed on the initial biological tissue samples; S2. Place the anchored sample in the above-mentioned expanding gel prepolymer solution, and after permeation, a polymerization reaction will be carried out to form a gelled sample. S3. The above gelled sample is subjected to tissue homogenization treatment, and then placed in water to swell; S4. Place the expanded sample in the above-mentioned stable gel prepolymer solution, and after permeation, carry out a secondary polymerization reaction to obtain an expanded transparent sample.

[0016] Preferably, the above method can also be used for expanding micro-cutting and three-dimensional imaging of biological tissue samples, including the following steps: The above-mentioned expanded transparent biological tissue samples were fluorescently labeled, and then alternating cutting and optical imaging were performed to obtain their three-dimensional structural data, thereby achieving three-dimensional super-resolution imaging.

[0017] Preferably, in step S1, the anchoring agent used in the above-mentioned biomolecule anchoring treatment includes any one of protein anchoring agents, nucleic acid anchoring agents, and multi-molecule anchoring agents.

[0018] Preferably, in step S2, the permeation time is 12h to 24h.

[0019] Preferably, in step S3, the solution used for the tissue homogenization treatment includes sodium dodecyl sulfate, ethylenediaminetetraacetic acid, and urea.

[0020] Preferably, in step S3, the expansion time is 24h to 36h.

[0021] Preferably, in step S4, the permeation time is 24h to 48h.

[0022] Preferably, the thickness of the cut is 100μm to 500μm.

[0023] Thirdly, this application provides an expanded transparent biological tissue sample, which is obtained by the above-described method.

[0024] Preferably, the linear expansion factor of the above-mentioned expanded transparent biological tissue sample is 3 to 5 times, and the Young's modulus is not less than 300 kPa. The expanded transparent biological tissue sample can achieve three-dimensional super-resolution imaging through alternating cutting and optical imaging after being fluorescently labeled.

[0025] Fourthly, this application provides a hydrogel section, which is obtained by cutting in the manner described above, or is prepared by cutting the aforementioned expanded transparent biological tissue sample.

[0026] Preferably, the thickness of the above-mentioned hydrogel slices is 100μm~500μm, and they can maintain structural integrity and be collected individually.

[0027] Fifthly, this application provides the application of the above-mentioned hydrogel sections in immunohistochemical staining, fluorescence microscopy analysis, cell construction labeling, or dilatational microscopy three-dimensional imaging.

[0028] In summary, the technical solutions conceived in this application have the following main technical advantages compared with the prior art: (1) The dual gel system provided in this application, which is suitable for large-volume biological tissue expansion micro-cutting, forms a dual gel system with complementary functions and synergistic effect by using expansion gel prepolymer and stable gel prepolymer. It effectively overcomes the contradiction that existing gel systems cannot simultaneously achieve high expansion ratio and high mechanical strength. It achieves high expansion ratio, high mechanical stability and cutting compatibility in large-volume biological tissue samples at the millimeter scale. It is suitable for the expansion processing of large-volume tissue samples and the application of expansion microscopy technology, and provides a new solution for the application of expansion microscopy technology in millimeter-scale tissue samples.

[0029] (2) The expanded transparent sample obtained by the dual-gel system in this application has a suitable expansion ratio and excellent mechanical stability and toughness. It can stably adapt to vibration cutting operations and effectively suppress elastic rebound, local stretching and shear displacement near the cut surface during the cutting process, ensuring a high degree of consistency of the interlayer structure before and after cutting, and significantly improving the repeatability of three-dimensional imaging. Based on its high mechanical strength, the expanded transparent sample can support a relatively thick cutting thickness (cutting thickness of 100~500μm), significantly reducing the number of cutting steps while ensuring the quality of the cutting surface, and greatly improving the efficiency of three-dimensional data acquisition. In addition, the expanded transparent sample also has good optical transparency, which can achieve deeper imaging on a single exposed section, further expanding the volume range covered by a single imaging. All of the above characteristics enable this application to achieve complete acquisition of structural information of a larger volume tissue sample with fewer cutting steps in the three-dimensional imaging process of millimeter-scale tissue samples compared with existing gel systems.

[0030] (3) The hydrogel sections obtained by cutting in this application are intact in shape and can remain stable in the water environment without falling apart, curling or breaking. This allows the hydrogel sections to be effectively collected as independent slides and can be further used for subsequent processing such as staining, microscopic analysis, and re-imaging. This realizes the technical expansion of reusable hydrogel sections and multidimensional analysis.

[0031] (4) The dual-gel system and processing flow provided in this application are applicable to biological tissue samples labeled in different ways (such as fluorescent labeling, transgenic fluorescent protein labeling, etc.) and are compatible with existing automatic cutting fluorescence imaging systems, providing a universal and reliable solution for high-resolution three-dimensional structural analysis of large-volume biological samples. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process of using a dual-gel system to perform expansion microscopy on large-volume biological tissue samples in Example 1 of this application; Figure 2 This is a schematic diagram of a hydrogel slice with a thickness of 100 μm obtained by cutting the prepared expanded transparent sample in Example 1 of this application; Figure 3 This is a schematic diagram of the acquisition mode of alternating thick cutting and imaging for expanding transparent samples in Embodiment 2 of this application; Figure 4 This is a comparison image of the same tissue sample before and after expansion in Example 2 of this application; wherein content (a) is a fluorescence image before and after expansion, and content (b) is a comparison image of the long axis length of the cells before and after expansion; Figure 5This is a comparison of the imaging and features of the same spatial position of the same expanded sample before and after cutting in Embodiment 2 of this application; wherein content (a) is the result of imaging the same spatial position before and after cutting, content (b) is the horizontal feature profile before and after cutting, and content (c) is the vertical feature profile before and after cutting. Figure 6 This is a three-dimensional data reconstruction image of the mouse brain tissue block after expansion in Example 2 of this application. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0034] In the description of this application, it should be understood that the term "and / or" describes a relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The symbol " / " in this document indicates that the related objects are in an "or" relationship; for example, A / B means A or B.

[0035] In the description of the embodiments in this application, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design options. Specifically, the use of the words "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0036] In the description of the embodiments in this application, unless otherwise stated, "multiple" means two or more.

[0037] This application provides a dual-gel system suitable for large-volume biological tissue expansion micro-cutting, including an expansion gel prepolymer and a stable hydrogel prepolymer. The aforementioned expanding gel prepolymer solution is composed of an expanding gel precursor solution and an initiator stock solution. The expanding hydrogel precursor solution contains acrylamide (AM), sodium 2-acrylamino-2-methyl-1-propanesulfonic acid (AMPS-Na), N,N'-diallyl-L-tartardiamide (DATD), and a solvent. The aforementioned stable gel prepolymer solution is composed of an expanding gel precursor solution and an initiator stock solution. The aforementioned stable gel precursor solution contains acrylamide (AM), N,N'-methylenebisacrylamide (BIS), and a solvent.

[0038] This application designs a dual-gel system suitable for large-volume biological tissue expansion microablation. The absorbent AMPS-Na used in the expansion gel prepolymer formulation enhances the water absorption of the gel network by introducing a negative charge. Its molecules also strengthen the internal forces within the gel network through hydrogen bonding, improving the elasticity of the hydrogel. Simultaneously, the DATD crosslinking agent contains a vicinal diol structure in its bridging group. This provides stronger hydrophilicity at the crosslinking sites, effectively guiding water molecules into the gel network and aiding in increasing the expansion ratio. Furthermore, the additional hydroxyl groups introduced further enhance the hydrogen bonding within the gel network. The synergistic effect of AMPS-Na and DATD together endows the expansion gel network with excellent flexibility and structural stability, enabling it to better maintain sample morphology during expansion and avoid non-uniform deformation. Based on this, the high concentration of gel monomers and BIS crosslinking agents in the stabilized gel prepolymer formulation gives the stabilized gel network a high crosslinking density, which can effectively improve the stiffness and elasticity of the expanded sample, thereby giving the millimeter-scale biological tissue sample the mechanical strength required to maintain expansion and be compatible with cutting. In addition, no negative ions are introduced into the stabilized gel network, and the gel as a whole is electrically neutral and does not have the ability to absorb water, thus effectively maintaining the stability of the sample expansion ratio.

[0039] This application utilizes a dual-gel system consisting of a swelling gel and a stabilizing gel, which complement each other and synergistically enhance each other's functions. This system simultaneously achieves high expansion ratio, high mechanical stability, and cutting compatibility in large-volume biological tissue samples at the millimeter scale. It is suitable for the expansion processing of large-volume tissue samples and the application of expansion microscopy, providing a new solution for the application of expansion microscopy in millimeter-scale tissue samples.

[0040] In some embodiments, the concentration of acrylamide in the above-mentioned expanding gel precursor solution is 10wt%~15wt%, the concentration of sodium 2-acrylamido-2-methylpropanesulfonate is 10wt%~20wt%, and the concentration of N,N'-diallyl tartrate diamide is 0.3wt%~1wt%.

[0041] In some embodiments, the concentration of acrylamide in the stabilized gel precursor solution is 10wt%~15wt%, and the concentration of N,N'-methylenebisacrylamide is 0.5wt%~1wt%, resulting in a high crosslinking density in the stabilized gel network, which effectively improves the stiffness and elasticity of the expanded transparent sample. When the total concentration of monomers and crosslinking agents in the stabilized gel precursor solution is too high, the expanded transparent sample obtained by expansion microscopy of this dual-gel system has excessively high elasticity, making stable thick cutting impossible; when the total concentration of monomers and crosslinking agents is too low, the stiffness of the expanded transparent sample cannot be effectively improved, and the cut hydrogel slices are brittle and difficult to collect. When the ratio of monomers and crosslinking agents in the stabilized gel precursor solution is too high, the modulus of the expanded transparent sample cannot be effectively improved and the strain increases, making it difficult to perform smooth cutting; when the ratio of monomers and crosslinking agents in the stabilized gel precursor solution is too low, the expanded transparent sample is prone to cutting cracks during the cutting process, making it impossible to obtain structurally intact hydrogel slices.

[0042] In some embodiments, the initiators in the expanded gel prepolymer and the stabilized gel prepolymer are each independently a photoinitiator or a redox initiator. The photoinitiator may be selected from, but is not limited to, Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), etc. The redox initiator is ammonium persulfate or potassium persulfate.

[0043] In some embodiments, the concentration of the initiator mother liquor is 6wt% to 8wt%. In some embodiments, the volume ratio of the expanded gel prepolymer to the initiator mother liquor is (150 to 200):1; the volume ratio of the stabilized gel prepolymer to the initiator mother liquor is (150 to 200):1.

[0044] In some embodiments, the solvent is independently deionized water or phosphate buffer.

[0045] On the other hand, this application provides a method for biological tissue swelling microscopy using the above-mentioned dual-gel system, comprising the following steps: S1. Degrease and biomolecule anchoring treatment is performed on the initial biological tissue samples; S2. Place the anchored sample in the above-mentioned expanding gel prepolymer solution, and after permeation, a polymerization reaction will be carried out to form a gelled sample. S3. The above gelled sample is subjected to tissue homogenization treatment, and then placed in water to swell; S4. Place the expanded sample in the above-mentioned stable gel prepolymer solution, and after permeation, carry out a secondary polymerization reaction to obtain an expanded transparent sample.

[0046] In some embodiments, the above method can also be used for the expansion micro-cutting and three-dimensional imaging of biological tissue samples, including the following steps: fluorescently labeling the expanded transparent sample, and then performing alternating cutting and optical imaging to obtain its three-dimensional structural data and achieve three-dimensional super-resolution imaging.

[0047] This application provides a method for expansion microanalysis and three-dimensional imaging of biological tissues using a dual-gel system. First, an expansion gel is used to achieve gentle and uniform initial embedding and expansion. The synergistic effect between the components in the expansion gel ensures high-quality, flexible expansion and morphological stability of large-volume biological tissue samples. Then, based on this expansion, a high-crosslink density stabilizing gel is used for thorough penetration and in-situ polymerization to reinforce the expanded network, thereby imparting high mechanical strength to the sample while maintaining the expansion ratio. Through the combined effect of the formulation design of the two gels and their processing sequence, the contradiction between expansion ratio and cutting stiffness, which cannot be balanced by existing gel systems, is synergistically resolved, providing a reliable solution for expansion microanalysis and three-dimensional imaging of large-volume tissues.

[0048] In some embodiments, in step S1, the anchoring agent used in the above-mentioned biomolecular anchoring treatment includes any one of protein anchoring agents, nucleic acid anchoring agents, and multi-molecular anchoring agents. For example, the protein anchoring agent may be, but is not limited to, N-hydroxysuccinate imide methacrylate (MA-NHS), 6-propenylamyric acid succinate (AcX), etc.; the nucleic acid anchoring agent may be, but is not limited to, LabelX, MelphaX, etc.; and the multi-molecular anchoring agent may be, but is not limited to, methacrolein, etc.

[0049] In some implementations, the permeation time in step S2 is 12h to 24h.

[0050] In some embodiments, in step S3, the solution used for the tissue homogenization treatment includes sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and urea.

[0051] In some implementations, the expansion time in step S3 is 24h to 36h.

[0052] In some embodiments, in step S4, the infiltration time is 24h to 48h, so that the stable gel prepolymer can fully penetrate into the expanded sample.

[0053] In some embodiments, the thickness of the cut is 100 μm to 500 μm.

[0054] Based on the above method, this application also provides an expanded transparent biological tissue sample obtained by the above method. This expanded transparent biological tissue sample possesses high mechanical strength, capable of supporting relatively thick cutting thicknesses, reducing the number of cutting operations while maintaining stability during the cutting process, and improving overall data acquisition efficiency. Simultaneously, the expanded transparent biological tissue sample exhibits good optical transparency after absorbing water, enabling deeper optical imaging on the section exposed by a single cutting, thereby further expanding the volume range covered by a single imaging operation. These characteristics allow this application, compared to existing methods, to acquire information about a larger volume of tissue structure with fewer cutting steps during the three-dimensional imaging of millimeter-scale tissue samples.

[0055] In some embodiments, the linear expansion factor of the aforementioned expanded transparent biological tissue sample is 3 to 5 times, and the Young's modulus is not less than 300 kPa, preferably not less than 350 kPa, enabling the expanded transparent biological tissue sample to achieve three-dimensional super-resolution imaging through alternating cutting and optical imaging after fluorescent labeling. A higher Young's modulus indicates that the expanded transparent biological tissue sample possesses excellent stiffness and elasticity, which is directly related to its behavior during subsequent vibration cutting. According to materials mechanics and vibration cutting models, under the same cutting conditions, materials with higher Young's modulus typically reach their strength limit at lower strain levels. This means that the cutting process is more stable and controllable, and the cut surface is smoother and more complete. Therefore, the high Young's modulus achieved in this application ensures that the expanded transparent biological tissue sample can resist deformation and tearing during subsequent vibration cutting, thereby significantly improving cutting stability and cut surface quality. This also explains, from a mechanical perspective, why the expanded transparent biological tissue sample provided in this application can achieve effects that are difficult to achieve using methods such as ExT—that is, maintaining cut surface integrity during thick-layer cutting and ultimately obtaining independently collectable, structurally complete hydrogel sections.

[0056] The expanded transparent biological tissue sample provided in this application possesses both excellent expansion magnification and mechanical stability, enabling stable adaptation to vibration cutting operations. During the cutting process, the sample is less prone to elastic rebound, local stretching, or shear displacement near the cut surface, thus maintaining a highly consistent interlayer structure before and after cutting, significantly improving the stability and repeatability of three-dimensional imaging results. Based on this, this application provides a hydrogel section obtained by cutting using the above method, or a hydrogel section prepared from the above-mentioned expanded transparent biological tissue sample by cutting. This hydrogel section can maintain its morphological integrity in an aqueous environment, without disintegration, curling, or breakage, which is beneficial for collection and subsequent processing as an independent slide. Based on this advantage, in actual operation, those skilled in the art can use the three-dimensional structural data collected by the above-mentioned expansion micro-cutting and three-dimensional imaging to selectively collect the cut hydrogel sections for further analysis.

[0057] In some embodiments, the thickness of the above-mentioned hydrogel slices is 100μm to 500μm, and they can maintain structural integrity and be collected individually.

[0058] Based on the above advantages, this application also provides the application of the above hydrogel sections in immunohistochemical staining, fluorescence microscopy analysis, cell construction labeling or expansion microscopy three-dimensional imaging, so that the three-dimensional data acquisition process is no longer limited to single-path imaging, but has the ability to be reused and expanded for multidimensional analysis.

[0059] It should be understood that materials of the same or similar type, model, quality, properties, or function as the reagents and instruments used in the following embodiments can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0060] The following are examples and comparative examples: Example 1 This application uses a C57 mouse brain tissue block with dimensions of 2.5mm × 2.5mm × 2.5mm as a large-volume biological tissue sample. The method for expanding microscopy of large-volume biological tissue provided in this embodiment is as follows: Figure 1 As shown, it includes the following steps: (1) Degreasing treatment Large-volume biological tissue samples were immersed in CUBIC-L solution for 24 hours. The CUBIC-L solution contained 10 wt% N-butyldiethanolamine and 10 wt% Triton X-100 to disrupt the cell membrane structure within the tissue, increase tissue permeability, and reduce subsequent processing time.

[0061] (2) Biomolecular anchoring treatment After washing the defatted tissue samples with PBS solution, they were placed in MES buffer (containing 100 mM MES and 150 mM NaCl) containing 5 mM N-hydroxysuccinimide methacrylate (MA-NHS) and soaked for about 12 hours to anchor the proteins in the tissue samples.

[0062] (3) The expanded gel prepolymer solution is subjected to permeation polymerization. An aqueous solution with an AM concentration of 10 wt%, an AMPS-Na concentration of 15 wt%, and a DATD concentration of 0.3 wt% was prepared as the expandable gel precursor solution. A solution of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959) at a concentration of 6 wt% and dimethyl sulfoxide (DMSO) was prepared as the initiator stock solution. 5 mL of the expandable gel precursor solution and 20 μL of the initiator stock solution were mixed to obtain the expandable gel prepolymer solution.

[0063] The anchored tissue samples were washed with PBS solution, and the expanded gel prepolymer solution was deoxygenated for 10 min using a nitrogen blower. The samples were then immersed in the deoxygenated expanded gel prepolymer solution for 18 h. After full penetration, the samples were placed under a UV lamp for multiple short irradiations to induce photopolymerization. The total irradiation time was about 30 s, resulting in gelled samples.

[0064] (4) Homogenization and swelling treatment of tissues The gelled samples were placed in a PBS solution containing 5 wt% sodium dodecyl sulfate (SDS), 25 mM EDTA, and 8 M urea, and treated at 80°C and pH 7.5 for 36 h to homogenize the tissue. The homogenized samples were then immersed in deionized water for 24 h, with the deionized water being changed every 12 h.

[0065] (5) Stable gel prepolymer solution is used for permeation polymerization. Prepare an aqueous solution with an AM concentration of 15 wt% and a BIS concentration of 0.5 wt% to obtain the stable gel precursor solution. Mix 25 mL of the stable gel precursor solution with 100 μL of the above-mentioned initiator stock solution to obtain the stable gel prepolymer solution.

[0066] The stabilized gel prepolymer solution was deoxygenated for 10 minutes using a nitrogen blower. The expanded sample was then immersed in the deoxygenated stabilized gel prepolymer solution for 36 hours. After full penetration, the sample was irradiated under a UV lamp until complete polymerization was achieved, resulting in an expanded transparent sample. This sample was then stored in deionized water at room temperature for later use.

[0067] The initial large-volume biological tissue sample and the expanded transparent sample were placed on a grid ruler to measure their dimensions. By comparing the dimensional data under the same dimension, it was found that the large-volume biological tissue sample could achieve isotropic expansion and a linear expansion factor of up to 4 times after being expanded by the above-mentioned dual gel system.

[0068] The Young's modulus of the expanded transparent sample was tested using the compression method. The Young's modulus was taken from the slope of the linear segment of the stress-strain curve in the strain range of 0 to 15%. The experiment found that the expanded transparent sample exhibited an initial Young's modulus of about 350 kPa in the low strain range. This indicates that the above-mentioned dual-gel system can significantly improve the elastic stiffness of the expanded transparent sample by expanding microscopy, making it suitable for vibration cutting operations and more conducive to obtaining stable and continuous cutting results.

[0069] The surface of the expanded transparent sample was imaged layer by layer downwards to a depth of 1000 μm. The root mean square (RMS) value of each image was calculated and normalized. Theoretically, the image RMS value would decrease significantly with increasing imaging depth and noise. However, in this embodiment, the normalized RMS values ​​of the deep images were all greater than 0.8, indicating that the obtained images still maintained a high signal-to-noise ratio within a depth range of approximately 1000 μm from the sample surface. This shows that the dual-gel system provided in this application can achieve good overall transparency while absorbing water and expanding large-volume biological tissue samples, effectively reducing the refractive index mismatch inside the tissue and weakening light scattering, thereby improving the optical transparency of large-volume biological tissue samples and improving imaging conditions. The transparency effect introduced by the expansion can significantly reduce background signals and defocus interference during deep imaging, which is beneficial for realizing continuous deep imaging of large-volume tissue samples.

[0070] To evaluate the shear resistance of the above-mentioned expanded transparent sample, the expanded transparent sample was further fixed on a vibratory microtome and continuously cut to a thickness of 100 μm, and the resulting hydrogel sections were collected.

[0071] Figure 2 The results of collecting 100μm thick hydrogel sections obtained by cutting show that the expanded transparent samples obtained by using the dual gel system provided in this application for expansion microscopy can maintain the stability of the cutting process under vibration cutting conditions, support tissue cutting operations with thicknesses up to hundreds of micrometers, and at the same time, the hydrogel sections obtained by cutting can maintain their complete morphology.

[0072] Comparative Example 1 This comparative study investigated the cutting and collection effects of large-volume biological tissue samples obtained by using different dilatation microscopy strategies (MAP, iExM, EXT) for dilatation microscopy.

[0073] The core components of the gel system of MAP (Magnified Analysis of the Proteome) expansion microscopy are: 20 wt% ammonium acrylate (AM), 3.3 wt% sodium acrylate (SA), and 0.05 wt% N,N'-methylenebisacrylamide (BIS).

[0074] The iExM (iterative expansion microscopy) gel system consists of the following core components: a first-stage gel (for initial expansion and anchoring transfer) containing 2.5 wt% ammonium acrylate (AM), 8.625 wt% sodium acrylate (SA), and 0.2 wt% N,N'-di(hydroxyethyl)bisacrylamide (DHEBA); and a second-stage gel (for secondary expansion) containing 2.5 wt% ammonium acrylate (AM), 8.625 wt% sodium acrylate (SA), and 0.15 wt% N,N'-methylenebisacrylamide (BIS).

[0075] The core components of the gel system in ExT (Expansion Tomography) microscopy are: 10 wt% ammonium acrylate (AM), 15 wt% sodium 2-acrylamido-2-methylpropanesulfonate (AMPS-Na), and 0.1 wt% N,N'-methylenebisacrylamide (BIS).

[0076] Large-volume biological tissue samples were expanded using expansion microscopy methods known in the art and suitable for different expansion microscopy strategies. The expanded samples were then continuously cut, and the results are shown in Table 1.

[0077] Table 1. Magnification and cutting / collection of expanded samples obtained by different expansion microscopy strategies.

[0078] As shown in Table 1, when using MAP and iExM expansion microscopy strategies to expand large-volume biological tissue samples, the resulting expanded samples are too soft to be cut. When using ExT expansion microscopy strategies to expand large-volume biological tissue samples, the resulting expanded samples have a certain degree of hardness, but cannot be cut into thick layers. The hydrogel sections obtained by cutting fall apart, curl, and break, and cannot be collected individually.

[0079] Comparative Example 2 In this comparative example, the dual-gel system used is the same as in Example 1, and the method of using the dual-gel system for biological tissue expansion microscopy is the same as in Example 1. The difference is that the dual-gel system only includes the expansion gel prepolymer solution and does not include the stabilizing gel prepolymer solution.

[0080] Experiments revealed that the expanded samples obtained from this comparative treatment were relatively soft and could not maintain their shape for a long time under the influence of gravity, thus making them unsuitable for cutting on a vibratory slicer.

[0081] Comparative Example 3 In this comparative example, the dual-gel system used is the same as in Example 1, and the method of using the dual-gel system for biological tissue expansion microscopy is the same as in Example 1. The difference is that the dual-gel system only includes a stable gel prepolymer solution and does not include an expansion gel prepolymer solution.

[0082] The experiment found that, compared with the initial large-volume biological tissue sample, the sample obtained by this comparative treatment did not expand in volume, and it could not be cut when fixed on a vibratory slicer.

[0083] Comparative Example 4 In this comparative example, the dual-gel system used is the same as in Example 1, and the method of using the dual-gel system for biological tissue expansion microscopy is the same as in Example 1. The difference is that the treatment order of the stabilizing gel prepolymer and the expanding gel prepolymer is changed. That is, the stabilizing gel prepolymer is first used to perform permeation polymerization on the large-volume biological tissue sample, and then the expanding gel prepolymer is used for permeation polymerization.

[0084] Experiments revealed that the expanded samples obtained from this comparative treatment could not achieve isotropic expansion, and the mechanical strength of the expanded samples was uneven, making them unsuitable for cutting. The possible reason is that changing the processing order of the two gel prepolymer solutions made it difficult for the expanding gel prepolymer solution to fully penetrate the sample. Simultaneously, the expansion of the sample interior was constrained by the stabilizing gel, while the expansion resistance of the outer layer was relatively lower, ultimately preventing the isotropic expansion of the sample from being guaranteed.

[0085] Comparative Example 5 In this comparative example, the dual-gel system used is the same as in Example 1, and the method of using the dual-gel system for biological tissue expansion microscopy is the same as in Example 1. The difference is that the cross-linking agent DATD in the expansion gel prepolymer solution is replaced with the cross-linking agent BIS.

[0086] Experiments revealed that the expanded samples obtained from this comparative treatment could be fixed on a vibratory slicer for cutting, but hydrogel sections with a cutting thickness of 100 μm could not be obtained. The reason for this may be that after replacing the crosslinking agent in the expanded gel prepolymer with BIS, the sample expansion ratio decreased while the elasticity increased significantly. This meant that although the processed expanded samples could support vibratory cutting, they could only be cut at a lower quality (cutting thickness less than 100 μm).

[0087] Example 2 The dual-gel system provided in this embodiment is abbreviated as X1+X2+X3_Y1+Y2. The swelling gel precursor solution is uniformly named in the form X1wt% AM+X2wt% AMPS-Na+X3wt% DATD (X1, X2, and X3 represent the concentrations of AM, AMPS-Na, and DATD, respectively), and the stabilizing gel precursor solution is uniformly named in the form Y1wt% AM+Y2wt% BIS (Y1 and Y2 represent the concentrations of AM and BIS, respectively). The specific components of different dual-gel systems are shown in Table 2. Biological tissue swelling microscopy was performed according to the method provided in Example 1, and the swollen samples were fixed on a vibratory microtome for continuous cutting at a thickness of 100 μm. The cutting and slide collection results are shown in Table 2.

[0088] Table 2 Specific components of different bigel systems

[0089] As can be seen from Table 2, the expanded samples obtained by using the dual gel systems with different concentration ratios provided in this embodiment to perform expansion microscopy on large-volume biological tissue samples can all stably adapt to vibration cutting operations and support thick-layer cutting. At the same time, the hydrogel sections obtained by cutting can maintain their complete morphology.

[0090] Example 3 The method for biological tissue expansion micro-ablation and three-dimensional imaging using a dual-gel system provided in this embodiment includes the following steps: (1) Following the method provided in Example 1, a dual gel system (10+15+0.3_15+0.5) was used to expand and transparent a large volume biological tissue sample (a C57 mouse brain tissue block with a length, width and thickness of 2.5mm×2.5mm×2.5mm) to obtain an expanded and transparent sample, which was then stored in room temperature deionized water for later use.

[0091] (2) Fluorescent dye labeling The above-mentioned expanded and transparent samples were immersed in PBS solution with a concentration of 100 μg / mL of propidium iodide for 24 h for fluorescent staining, and then immersed in deionized water for 12 h.

[0092] (3) Three-dimensional fluorescence imaging Using a fluorescence microscope with automatic cutting function, such as... Figure 3 The acquisition mode, which alternates between thick cutting and imaging, first excites and images the surface layer of the fluorescently labeled, expanded, transparent sample, scanning axially layer by layer to a depth of 500 μm. Then, the imaged surface layer is mechanically cut away, and the new surface layer is imaged with fluorescence. This process is repeated until the entire sample is imaged.

[0093] Figure 4 Comparison images of the same tissue sample before and after swelling, in which... Figure 4 Content (a) shows fluorescence images of the same tissue sample before and after expansion. It can be seen that the expanded brain tissue cells maintained their original morphology well without distortion, achieving isotropic linear expansion. In practical applications, the expansion ratio can be calculated by measuring the cell or tissue structure dimensions before and after expansion. Further, in this embodiment, ten cells were randomly selected from the above fluorescence images, and the average long axis length of the brain tissue cells before expansion was calculated to be 13.06 μm, and the average long axis length of the brain tissue cells after expansion was 42.54 μm (e.g., ...). Figure 4 As shown in content (b), the dual-gel system provided in this application has excellent linear expansion ratio at the microscopic level, with a microscopic linear expansion ratio of 3.2 times.

[0094] Figure 5 The images and feature comparisons of the same spatial position of the same expanded transparent sample before and after cutting are shown. Content (a) shows the imaging results of the same spatial position before and after cutting, content (b) shows the horizontal feature profile before and after cutting, and content (c) shows the vertical feature profile before and after cutting. It can be seen that the signal distribution difference of the same spatial position of the same expanded transparent sample before and after cutting is small, and the signal distortion is small.

[0095] Figure 6 The image shows a three-dimensional reconstruction of a mouse brain tissue block after expansion, demonstrating that the expanded transparent sample obtained by the expansion microscopy process using the dual-gel system method provided in this application can stably achieve continuous cutting with a thickness of approximately 500 μm, and perform optical imaging of the newly exposed cut surface and the tissue structure within a large axial range below it after each cutting.

[0096] Compared to existing dilatational microscopy cutting strategies, the single cutting step in this application can acquire more spatial imaging information, thereby significantly reducing the number of cutting operations while ensuring the consistency of spatial structure, and improving the overall efficiency of 3D data acquisition for millimeter-scale tissue samples. Simultaneously, due to the increased cutting thickness, the cumulative number of mechanical disturbances during the alternating cutting and imaging process is reduced, which also helps to reduce morphological shifts and error accumulation introduced by repeated cutting, improving the stability and repeatability of the 3D reconstruction results.

[0097] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A dual-gel system suitable for large-volume biological tissue expansion micro-cutting, characterized in that, Including expanded gel prepolymer and stabilized gel prepolymer; The expanded hydrogel prepolymer solution is composed of an expanded hydrogel precursor solution and an initiator stock solution. The expanded hydrogel precursor solution contains acrylamide, sodium 2-acrylamido-2-methylpropanesulfonate, N,N'-diallyl tartaric acid diamide, and a solvent. The stabilized gel prepolymer solution is composed of an expanding gel precursor solution and an initiator stock solution. The stabilized gel precursor solution contains acrylamide, N,N'-methylenebisacrylamide, and a solvent.

2. The dual-gel system according to claim 1, characterized in that, In the expanded gel precursor solution, the concentration of acrylamide is 10wt%~15wt%; and / or, The concentration of the sodium 2-acrylamido-2-methylpropanesulfonate is 10wt%~20wt%; and / or, The concentration of N,N'-diallyl tartaric acid diamide is 0.3wt%~1wt%.

3. The dual-gel system according to claim 1, characterized in that, In the stabilized gel precursor solution, the concentration of acrylamide is 10wt%~15wt%; and / or, The concentration of the N,N'-methylenebisacrylamide is 0.5wt%~1wt%.

4. The dual-gel system according to claim 1, characterized in that, The initiators in the expanded gel prepolymer and the stable gel prepolymer are each independently a photoinitiator or a redox initiator; the photoinitiator is selected from Irgacure 2959 or lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the redox initiator is selected from ammonium persulfate or potassium persulfate; Preferably, the concentration of the initiator stock solution is 6wt%~8wt%; the volume ratio of the expanding gel precursor solution to the initiator stock solution is (150~200):1; and the volume ratio of the stable gel precursor solution to the initiator stock solution is (150~200):

1.

5. A method for performing swelling microscopy of biological tissue samples using the dual-gel system according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Degrease and biomolecule anchoring treatment is performed on the initial biological tissue samples; S2. Place the anchored sample in the expanded gel prepolymer solution, and after penetration, a polymerization reaction will occur to form a gelled sample. S3. The gelled sample is subjected to tissue homogenization treatment, and then placed in water to swell; S4. Place the expanded sample in the stabilized gel prepolymer solution, and after permeation, carry out a secondary polymerization reaction to obtain an expanded transparent biological tissue sample.

6. The method according to claim 5, characterized in that, The method can also be used for expansion micro-cutting and three-dimensional imaging of biological tissue samples, including the following steps: The expanded transparent biological tissue sample was fluorescently labeled, and then alternating cutting and optical imaging were performed to obtain its three-dimensional structural data, thereby achieving three-dimensional super-resolution imaging.

7. The method according to claim 5 or 6, characterized in that, In step S1, the biomolecular anchoring treatment uses an anchoring agent comprising any one of protein anchoring agents, nucleic acid anchoring agents, or multimolecular anchoring agents; and / or, In step S2, the infiltration time is 12h~24h; and / or, In step S3, the solution used for tissue homogenization includes sodium dodecyl sulfate, ethylenediaminetetraacetic acid, and urea; and / or, In step S3, the expansion time is 24h~36h; and / or, In step S4, the infiltration time is 24h~48h; and / or, The cutting thickness is 100μm~500μm.

8. An expanded, transparent biological tissue sample, characterized in that, It is obtained by processing using the method described in claim 5; Preferably, the linear expansion factor of the expanded transparent biological tissue sample is 3 to 5 times, and the Young's modulus is not less than 300 kPa. The expanded transparent biological tissue sample can achieve three-dimensional super-resolution imaging through alternating cutting and optical imaging after being fluorescently labeled.

9. A hydrogel section, characterized in that, It is obtained by cutting using the method described in claim 6, or by cutting the expanded transparent biological tissue sample described in claim 8; Preferably, the thickness of the hydrogel slice is 100μm~500μm, and it can maintain structural integrity and be collected individually.

10. The application of the hydrogel section as described in claim 9 in immunohistochemical staining, fluorescence microscopy analysis, cell construction labeling, or dilatational microscopy three-dimensional imaging.

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