A method for preparing a transmission electron microscope sample of a hydrogel material

Abstract

The present application belongs to the technical field of transmission electron microscope sample preparation, and provides a preparation method of a hydrogel material transmission electron microscope sample. The preparation method comprises: fixing the structure of the hydrogel material by high-pressure freezing, freezing sectioning the hydrogel material after the structure is fixed to obtain a hydrogel ultrathin section; pulling the hydrogel ultrathin section to a support grid to make the hydrogel ultrathin section adhere to the support grid; placing the support grid in liquid nitrogen for storage, and then sending the support grid into a cryogenic transmission electron microscope through a cryogenic transmission system for microstructure characterization. The present application fixes the structure of the hydrogel by high-pressure freezing to retain the original structure of the hydrogel; the ultrathin sectioning is performed under the condition of freezing, and the hydrogel does not need to be embedded in resin; the sectioning is directly performed under the condition of low-temperature freezing and the section is adhered to the support grid, the support grid is sent into the cryogenic transmission electron microscope through the cryogenic transmission system, and the structure characterization is performed under a low electron dose, so that the accuracy of the microstructure of the hydrogel material and the distribution information of the nanofiller is improved.

Description

Technical Field

[0001] This invention relates to the field of transmission electron microscopy (TEM) sample preparation technology, and more particularly to a method for preparing TEM samples of hydrogel materials. Background Technology

[0002] Hydrogels are polymer materials with a three-dimensional network structure. They are soft, rich in water, highly malleable, and possess excellent biocompatibility, adjustable mechanical properties, and ease of functionalization. By changing the polymer type, synthesis method, and crosslinking mechanism, a variety of hydrogels with different physicochemical properties can be prepared. These hydrogels have wide applications in drug delivery carriers, tissue engineering, biomimetic materials, flexible sensors, and smart devices. The network structure of hydrogels lacks long-range order and exhibits heterogeneity at different scales. The physical properties of hydrogels largely depend on the polymer structure at different scales. Therefore, observing the network structure of hydrogels at the nano and micro scales is crucial for understanding and predicting the physical properties of these materials and for their rational design.

[0003] Currently, X-ray / neutron / light scattering are the main methods for characterizing the physical structure of hydrogels at different scales from nanometers to micrometers. However, the average structural information provided by these characterization methods is often several orders of magnitude larger than the actual mesh size (which is typically 10–100 nm). Scanning electron microscopy (SEM) and cryo-scanning electron microscopy (cryo-SEM), as important techniques for directly characterizing hydrogel network structures, are mainly used to characterize the micrometer-scale network structure of hydrogels.

[0004] With the deepening of hydrogel research, the demand for precise characterization of the hydrogel network structure and the distribution of nanofillers within it is increasing. Transmission electron microscopy (TEM) is one of the most effective methods for directly imaging material structures at nanoscale resolution. Currently, the sample processing methods for TEM characterization of hydrogel materials mainly include dehydration and drying, and ultrathin sectioning, specifically: 1) Drying the hydrogel according to its characteristics using methods such as natural air drying, critical point drying, and freeze-drying; 2) Embedding the dried hydrogel material in resin and waiting for the resin to polymerize and solidify (usually requiring more than 48 hours); 3) Freezing and sectioning the embedded hydrogel material to obtain ultrathin sections with a thickness of 50-100 nm; 4) Using a slide-collecting ring dipped in reagents such as sucrose to transfer the sections to a grid; 5) Drying the grid at room temperature, followed by TEM observation. This technique has certain limitations: ① During the drying process, the morphology of the hydrogel will change significantly, and the corresponding network structure will also change, making it difficult to observe the accurate microscopic network morphology and structure of the hydrogel. Hydrogels become shriveled after natural air drying, and their microscopic network structure often collapses. Hydrogels treated with critical point drying show a significant reduction in volume, and the sample surface becomes wrinkled. Hydrogels treated with freeze-drying increase in volume due to the formation of ice crystals from internal water during freezing, resulting in a corresponding increase in the size of the microscopic network. ② Resin-embedded hydrogels easily reduce the imaging contrast of the hydrogel structure, affecting the observation of the microstructure. Dried hydrogels usually have a certain degree of toughness and require resin infiltration and embedding before they can be held and fixed for ultrathin sections. However, resin often has a similar electron density to the polymer chains in the hydrogel network, leading to a lack of contrast in the network structure under an electron microscope, making structural characterization impossible. To improve contrast, staining the hydrogel network is often used, but this method easily causes polymer deformation, failing to obtain true microscopic structural information of the hydrogel. Furthermore, during resin embedding and polymerization, the hydrogel material may also undergo chemical reactions with the resin, causing changes in the material's surface structure. ③ The characterization of the distribution of nanofillers in the hydrogel network structure is limited. With the advancement of functionalization and refinement research on hydrogels, hydrogel materials loaded with nanoparticles have become a research hotspot. The demand for observing the microstructure of hydrogels has gradually shifted from network structure to the distribution of nanofillers within the network structure. However, current TEM characterization methods for hydrogels require drying. During the drying and dehydration process, the distribution morphology of nanofillers dispersed in the hydrogel network structure changes as water is lost, making it impossible to determine the true distribution of nanofillers in the hydrogel using electron microscopy. ④ The resin curing time is relatively long, increasing the sample pretreatment time. ⑤ When transferring sections from a frozen environment to a room temperature environment, the hydrogel is soft and elastic, and the process of transferring to room temperature involves water evaporation, which may alter the microstructure.⑥ Sections retrieved and transferred using solutions such as sucrose are randomly attached to the grid. The sections may be obscured by the grid support or located at the edge of the grid, which is detrimental to electron microscopy observation. ⑦ During electron microscopy observation, hydrogel materials are often damaged by electron beam irradiation, leading to structural artifacts.

[0005] Based on this, a method for processing hydrogel material transmission electron microscopy samples is provided to observe the microstructure of hydrogels that is closest to their natural state, which is of great significance for the characterization and application of hydrogels. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing hydrogel material transmission electron microscopy samples to address the shortcomings of existing technologies.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing transmission electron microscopy (TEM) samples of hydrogel materials, comprising the following steps: 1) The hydrogel material is structurally fixed by high-pressure freezing; 2) After the structure is fixed, the hydrogel material is cryosectioned to obtain ultrathin hydrogel sections; 3) Pull the hydrogel ultrathin section onto the support grid to make the hydrogel ultrathin section adhere to the support grid; 4) The grid was stored in liquid nitrogen and then sent to a cryo-transmission electron microscope via a cryo-transmission system for microstructural characterization.

[0008] Preferably, the pressure of the high-pressure freezing in step 1) is 2000~2500 bar, and the temperature of the high-pressure freezing is -196~-140℃.

[0009] Preferably, the temperature of the frozen slices in step 2) is -180~-100℃.

[0010] Preferably, the thickness of the hydrogel ultrathin slice in step 2) is 50~100nm.

[0011] Preferably, during the freezing and slicing process in step 2), the carrier mesh is held by micro-manipulation and pre-cooled.

[0012] Preferably, both the freezing sectioning and precooling are performed in a cryo-microtome.

[0013] Preferably, step 3) is performed in a cryo-microtome.

[0014] The beneficial effects of this invention are: This invention uses high-pressure freezing to fix the hydrogel structure, ensuring no crystallization ice is formed, preserving the original structure of the hydrogel, and guaranteeing no change in the hydrogel network structure. Ultrathin sections are directly prepared under freezing conditions, eliminating the drying step that could lead to hydrogel structural changes. Furthermore, there is no need for resin embedding, avoiding the influence of resin on the imaging contrast of the hydrogel network structure. The preparation cycle is shortened by eliminating the need to wait for resin curing. Under low-temperature freezing conditions, a stylus pen is used to directly pick up the slides and pull them onto the grid. The slides are placed at the center of the sample wells on the grid, and the charge-discharge mode of an electrostatic generator assists in attaching the slides to the grid, ensuring observation of the slides within a suitable field of view. The grid-supported slides are preserved in liquid nitrogen and transported to a cryo-transmission electron microscope (CREM) via a cryo-transmission system for microstructural characterization, avoiding deformation due to moisture evaporation during the recovery process from frozen to room temperature. The low-electron-dose mode of CREM is used for structural characterization, reducing the exposure time of the sample to electron irradiation, avoiding electron irradiation damage, and contributing to obtaining more realistic and natural structural information, effectively improving the accuracy of the microstructure of the hydrogel material's network and the distribution of nanofillers. Attached Figure Description

[0015] Figure 1 This is a transmission electron microscope image of Example 1; Figure 2 This is a stereomicroscope image of the slices attached to the grid in Example 1; Figure 3 This is a transmission electron microscope image of Example 2; Figure 4 This is a transmission electron microscope image of Example 3; Figure 5 This is a transmission electron microscope image of Example 4; Figure 6 This is a transmission electron microscope image of Comparative Example 1; Figure 7 This is a transmission electron microscope image of Comparative Example 2; Figure 8 This is a transmission electron microscope image of Comparative Example 3; Figure 9 This is a transmission electron microscope image of Comparative Example 4. Detailed Implementation

[0016] This invention provides a method for preparing transmission electron microscopy (TEM) samples of hydrogel materials, comprising the following steps: 1) The hydrogel material is structurally fixed by high-pressure freezing; 2) After the structure is fixed, the hydrogel material is cryosectioned to obtain ultrathin hydrogel sections; 3) Pull the hydrogel ultrathin section onto the support grid to make the hydrogel ultrathin section adhere to the support grid; 4) The grid was stored in liquid nitrogen and then sent to a cryo-transmission electron microscope via a cryo-transmission system for microstructural characterization.

[0017] In this invention, the pressure of the high-pressure freezing in step 1) is preferably 2000~2500 bar, more preferably 2100~2400 bar, and even more preferably 2200~2300 bar; the temperature of the high-pressure freezing is preferably -196~-140℃, more preferably -180~-150℃, and even more preferably -170~-160℃.

[0018] In this invention, the high-pressure freezing in step 1) is preferably carried out in a high-pressure freezer, which can complete the high-pressure freezing within 30ms.

[0019] In this invention, the temperature of the frozen slices in step 2) is preferably -180~-100℃, more preferably -160~-120℃, and even more preferably -150~-140℃.

[0020] In this invention, the thickness of the hydrogel ultrathin slice in step 2) is preferably 50~100nm, more preferably 60~90nm, and even more preferably 70~80nm.

[0021] In this invention, during the freezing and slicing process described in step 2), it is preferable to hold the carrier mesh by micro-manipulation and pre-cool the carrier mesh.

[0022] In this invention, the freezing sectioning and precooling are preferably performed in a cryo-microtome.

[0023] In this invention, step 3) is preferably performed in a cryo-microtome.

[0024] In this invention, step 3) preferably involves using the charge-discharge mode of an electrostatic generator to attach the hydrogel ultrathin slices to the carrier mesh.

[0025] In this invention, the microstructure characterization described in step 4) is preferably performed using low-dose techniques of cryo-transmission electron microscopy. Conventional methods involve directly magnifying or reducing the target area step by step before taking pictures, constantly exposing the target sample area to electron irradiation, resulting in electron irradiation damage. Low-dose microstructure characterization, on the other hand, involves first focusing at a higher target magnification using the lowest possible electron dose on the sample's edge region, then switching to a low-magnification, large-field-of-view location to find the target area, and finally returning to high magnification for imaging. This switching between different magnifications avoids electron irradiation of the target area of ​​the sample, thereby reducing electron irradiation damage and improving the accuracy of microstructure characterization.

[0026] In this invention, the dose range of the low-dose technology is preferably determined based on the tolerance of the hydrogel material to electron irradiation.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] The Xizhilang jelly was characterized by transmission electron microscopy, specifically as follows: The hydrogel material was placed on the sample stage of a high-pressure cryostat and then transferred into the cryostat for structural fixation. The high-pressure cryostat was set at 2100 bar and -140°C, with freezing completed within 30 ms. The fixed hydrogel material was then transferred to the cold chamber of a pre-cooled cryomicrotome, with the cold chamber temperature set to -140°C. The hydrogel was then cryosectioned within the cold chamber to obtain hydrogel ultrathin sections approximately 100 nm thick. Simultaneously, a micromanipulation system of the cryomicrotome was used to hold a grid, which was then placed into the cold chamber for pre-cooling. After cryosectioning, an eyelash pen was used to guide the hydrogel ultrathin section to the center of the grid's sample well. The section was then adhered to the grid using a charge-discharge mode of an electrostatic generator. The adhered grid was transferred to liquid nitrogen for storage and then placed on the sample stage of a cryotransmission microscope (CTEM) via the cryotransmission system. The microscopy was performed at approximately 25°C. - TEM characterization was performed in a low-dose mode at / Ų.

[0030] Figure 1 This is a transmission electron microscope image of Example 1.

[0031] Figure 2 This is a stereomicroscope image of the slices attached to the grid in Example 1. Figure 2 As can be seen, the hydrogel ultrathin sections are mainly distributed in the central region of the grid, making them easy to observe. Figure 2 The red arrow in the middle indicates a hydrogel ultrathin section.

[0032] Example 2

[0033] The polyelectrolyte hydrogel (prepared according to Zhu J, Qiu S, Duan M, et al. Polyelectrolytegradient hydrogels for efficient solar evaporation[J]. Advanced Functional Materials, 2026, 36(3): e12350) was characterized by transmission electron microscopy. The specific method differed from that in Example 1 in that the cold cavity temperature was set to -140℃ and the thickness of the hydrogel ultrathin section was about 90nm.

[0034] Figure 3This is a transmission electron microscope image of Example 2.

[0035] Example 3

[0036] The collagen hydrogel was prepared by preparing a 0.5% (w / v) polypeptide aqueous solution and sonicating it for 30 min to ensure uniform dissolution. Collagen was then dissolved in PBS buffer solution and mixed with an equal volume of the polypeptide aqueous solution. The mixture was then incubated overnight at 37°C to obtain a self-assembled peptide-collagen composite hydrogel.

[0037] The self-assembled peptide-collagen complex hydrogel was characterized by transmission electron microscopy. The specific method differed from that in Example 1 in that the cold chamber temperature was set to -150℃ and the thickness of the hydrogel ultrathin section was about 80nm.

[0038] Figure 4 This is a transmission electron microscope image of Example 3.

[0039] Example 4

[0040] The polyelectrolyte hydrogel containing gold nanoparticles (prepared according to Zhu J, Qiu S, Duan M, et al. Polyelectrolyte gradient hydrogels for efficient solar evaporation[J]. Advanced Functional Materials, 2026, 36(3): e12350) was characterized by transmission electron microscopy, using the same method as in Example 1.

[0041] Figure 5 This is a transmission electron microscope image of Example 4.

[0042] Comparative Example 1

[0043] The Xizhilang jelly was characterized by transmission electron microscopy, specifically as follows: After drying the jelly, it was frozen and sectioned at -60°C and observed under a conventional electron microscope.

[0044] Figure 6 This is a transmission electron microscope image of Comparative Example 1.

[0045] Comparative Example 2

[0046] The polyelectrolyte hydrogel (prepared according to Zhu J, Qiu S, Duan M, et al. Polyelectrolytegradient hydrogels for efficient solar evaporation[J]. Advanced Functional Materials, 2026, 36(3): e12350) was characterized by transmission electron microscopy, using the same method as Comparative Example 1.

[0047] Figure 7 This is a transmission electron microscope image of Comparative Example 2.

[0048] Comparative Example 3

[0049] The collagen hydrogel was prepared by preparing a 0.5% (w / v) polypeptide aqueous solution and sonicating it for 30 min to ensure uniform dissolution. Collagen was then dissolved in PBS buffer solution and mixed with an equal volume of the polypeptide aqueous solution. The mixture was then incubated overnight at 37°C to obtain a self-assembled peptide-collagen composite hydrogel.

[0050] The self-assembled peptide-collagen complex hydrogel was characterized by transmission electron microscopy, using the same method as Comparative Example 1.

[0051] Figure 8 This is a transmission electron microscope image of Comparative Example 3.

[0052] Comparative Example 4

[0053] The polyelectrolyte hydrogel with added gold nanoparticles (prepared according to Zhu J, Qiu S, Duan M, et al.Polyelectrolyte gradient hydrogels for efficient solar evaporation[J].Advanced Functional Materials, 2026, 36(3): e12350) was characterized by transmission electron microscopy, using the same method as Comparative Example 1.

[0054] Figure 9 This is a transmission electron microscope image of Comparative Example 4.

[0055] Comparison Figure 1 , 6 , Figure 3 , 7 , Figure 4 , 8 and Figure 5 , 9It can be seen that, compared to the hydrogel structure images obtained by high-pressure freezing combined with low-temperature observation, samples that are conventionally dried and then frozen sections are almost impossible to observe under a conventional transmission electron microscope, and may even show obvious traces of electron irradiation damage (such as...). Figure 6 , 8 The distribution of nanoparticles in the hydrogel is no longer uniform (e.g.) Figure 9 ).

[0056] As can be seen from the above embodiments, the present invention provides a method for preparing transmission electron microscopy (TEM) samples of hydrogel materials. The hydrogel structure is fixed by high-pressure freezing, followed by slicing in a cryo-microtome and transferring the slices to a pre-cooled support grid. The cryo-microtome slices adhere to the grid and are stored in liquid nitrogen. They are then transported to a cryo-TEM via a cryo-transfer system for microstructural characterization in low-dose mode. The method of the present invention is performed entirely under low-temperature freezing conditions, which preserves the original structure of the hydrogel and avoids structural deformation. Microstructural characterization at low doses reduces damage to the hydrogel sample from electron irradiation, helps to obtain a more natural and realistic microstructure, and improves the accuracy of information on the microstructure network structure and nanofiller distribution of the hydrogel material.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a transmission electron microscopy (TEM) sample of a hydrogel material, characterized in that, It includes the following steps: 1) The hydrogel material is structurally fixed by high-pressure freezing; 2) After the structure is fixed, the hydrogel material is cryosectioned to obtain ultrathin hydrogel sections; 3) Pull the hydrogel ultrathin section onto the support grid to make the hydrogel ultrathin section adhere to the support grid; 4) The grid was stored in liquid nitrogen and then sent to a cryo-transmission electron microscope via a cryo-transmission system for microstructural characterization.

2. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 1, characterized in that, Step 1) The pressure of the high-pressure freezing is 2000~2500 bar, and the temperature of the high-pressure freezing is -196~-140℃.

3. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 1 or 2, characterized in that, Step 2) The temperature of the frozen slices is -180~-100℃.

4. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 3, characterized in that, Step 2) The thickness of the hydrogel ultrathin slice is 50~100nm.

5. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 4, characterized in that, In step 2), during the frozen sectioning process, the carrier mesh is held in place by micro-manipulation and pre-cooled.

6. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 5, characterized in that, Both the freezing sectioning and precooling were performed in a cryo-microtome.

7. The method for preparing a transmission electron microscopy sample of hydrogel material according to claim 1, characterized in that, Step 3) is performed in a cryo-microtome.

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