Ultrathin section freezing method and novel embedding medium for freezing ultrathin section
By using a novel liquid embedding agent that is frozen into an amorphous solid in a low-temperature medium and volatilized during the rewarming process, the problems of sample deformation and residue caused by traditional embedding agents are solved, achieving efficient and pure cryogenic ultrathin sectioning and improving the sample observation effect.
Patent Information
- Application Number
- CN202410625015.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-20
- Publication Date
- 2025-11-21
AI Technical Summary
In existing cryopreservation ultrathin sectioning techniques, the interaction between traditional paraffin or resin-based embedding agents and the sample leads to changes in sample properties and structural damage, making it difficult to maintain the original morphology and making cleaning difficult, thus affecting the observation results.
A novel liquid embedding agent is used to freeze into an amorphous solid in a low-temperature medium for use as support in frozen sections. It completely evaporates during the rewarming process to avoid residue. Specific materials include water or organic solvents such as dimethyl carbonate and tetrahydrofuran.
It significantly reduces the risk of sample deformation and denaturation, obtains pure sliced samples, improves observation resolution, simplifies the cleaning process, and increases sample selection flexibility.
Smart Images
Figure CN120992302A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cryo-ultramicrosection embedding agents, and more particularly to a cryo-ultramicrosection method and a novel embedding agent for cryo-ultramicrosection. Background Technology
[0002] Transmission electron microscopy (TEM) is one of the key instruments for studying the microstructure of materials. In order to observe the microstructure of materials on a TEM and obtain good imaging quality, the sample must be very thin, usually between 10 and 100 nm thick.
[0003] Cryo-ultrathroplasty is widely used in electron microscopy sample preparation, especially for functional material samples requiring high-resolution observation. Its basic principle involves rapidly freezing the sample in liquid nitrogen or liquid ethane, then cutting it in the frozen state to obtain a sufficiently thin sample for TEM observation. Cryo-ultrathroplasty effectively preserves the original structure and morphology of biological samples, facilitating more accurate observation and analysis. In the field of functional materials, cryo-sectioning helps researchers observe minute changes and features in nanostructures. When using nitrogen as the cryocooler, the protective effect of nitrogen allows cryo-ultrathroplasty to be further applied to the sectioning of air-sensitive materials.
[0004] However, traditional cryogenic ultrathin section embedding agents, such as paraffin or resin (most commonly OCT embedding agents), may interact with the sample in practical applications, causing changes in the sample's properties. For example, they may react chemically with proteins or other biomolecules in biological samples, leading to conformational changes or loss of function. During embedding, the solidification of paraffin or resin may cause structural damage or deformation of the sample, especially for soft or fragile samples, making it difficult to maintain the original morphology during cutting. Furthermore, choosing resin or paraffin-based embedding agents not only requires additional cleaning steps for removal, but these embedding agents often produce densities and contrasts similar to the sample, increasing the difficulty of observing the sample under an electron microscope. Moreover, even with appropriate cleaning steps, residues are prone to remain, reducing the sample's resolution under the microscope and causing noise or interference during observation. All of these significantly limit the application of cryogenic sections in the field of functional materials.
[0005] Therefore, researchers urgently need to develop a slicing technique that can preserve the original shape of the sample and a new type of embedding agent that is easy to remove and leaves no residue on the sample. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for cryo-ultramicrosection and a novel embedding agent for cryo-ultramicrosection. Compared to existing technologies that use paraffin or resin-based embedding agents, the liquid embedding agent and sectioning method of this invention can produce pure samples, effectively avoid sample contamination, reduce sample deformation, simplify observation, and facilitate more precise sample characterization.
[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for cryo-ultramicrosection, wherein a novel embedding agent for cryo-ultramicrosection is used in the method; the method includes:
[0008] The sample to be sliced is placed in a fixative containing the novel embedding agent and then placed together in a low-temperature medium for freezing to obtain a quick-frozen fixed sample; wherein, the novel embedding agent is frozen into an amorphous solid in the low-temperature medium to provide support for the frozen slices.
[0009] The quick-frozen and fixed sample is placed in a cryostat at a set slicing temperature for cryosectioning; at the set slicing temperature, the novel embedding agent remains in an amorphous solid state;
[0010] The frozen sectioned sample is subjected to a rewarming treatment to allow the novel embedding agent to completely evaporate, resulting in a pure sectioned sample.
[0011] Preferably, the cryogenic medium is one of liquid nitrogen, liquid helium, liquid ammonia, and liquid ethane.
[0012] Preferably, the novel embedding agent for cryogenic ultrathin sectioning is specifically: liquid at room temperature and amorphous solid at the freezing temperature provided by the low-temperature medium; the freezing temperature range is 0°C to -160°C.
[0013] Preferably, the freezing method of placing the medium into a cryogenic medium for freezing specifically includes: immersion freezing or high-pressure freezing.
[0014] Preferably, the set slicing temperature is ±10°C of the glass transition temperature of the novel embedding agent used for cryogenic ultrathin sectioning.
[0015] Preferably, the rewarming process specifically involves rewarming to room temperature under a set pressure condition.
[0016] Secondly, embodiments of the present invention provide a novel embedding agent for the cryo-ultrathroplasty method described in the first aspect above. The novel embedding agent for cryo-ultrathroplasty is liquid at room temperature and rapidly frozen into an amorphous solid in a low-temperature medium to provide support for the frozen sections. Furthermore, the novel embedding agent completely volatilizes during the rewarming process after freezing.
[0017] Preferably, the novel embedding agent for cryogenic ultrathin sectioning specifically includes: water, or an organic solvent that is an amorphous solid at the freezing temperature provided by the low-temperature medium.
[0018] More preferably, the cryogenic medium is one of liquid nitrogen, liquid helium, liquid ammonia, and liquid ethane, and the freezing temperature ranges from 0°C to -160°C.
[0019] Preferably, the novel embedding agent for cryogenic ultrathin sections is used in the preparation of cryogenic ultrathin section samples for transmission electron microscopy (TEM).
[0020] The cryogenic ultrathin sectioning method provided in this invention significantly reduces the risk of sample deformation and denaturation by using a material that is liquid at room temperature and amorphous solid at the freezing temperature provided by the cryogenic medium as the embedding agent. Furthermore, the embedding agent can be completely evaporated without residue through rewarming, resulting in pristine and pure samples. Compared to traditional paraffin or resin-based embedding agents, the liquid embedding agent of this invention enables rapid freezing, thus significantly shortening the curing time. It also eliminates the need for additional soaking and cleaning to remove the embedding agent, improving sectioning efficiency and preventing sample contamination. In addition, this method offers greater flexibility in sample selection; solid powders or films can be embedded without additional treatment, greatly increasing the flexibility of sample selection. Attached Figure Description
[0021] Figure 1 A flowchart of the cryopreservation ultrathin sectioning method provided in an embodiment of the present invention;
[0022] Figure 2 This is a TEM image of the sliced sample obtained after cryopreservation and ultrathin sectioning according to Example 1 of the present invention;
[0023] Figure 3 This is a TEM image of the sliced sample obtained after freezing and ultrathin sectioning, provided in Comparative Example 1 of this invention. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.
[0026] This invention provides a method for cryogenic ultrathin sectioning using a novel embedding agent, which enables rapid freezing of samples and ultrathin sectioning of samples in a frozen state for observation of the internal structure of samples using an electron microscope (e.g., transmission electron microscope).
[0027] Figure 1 The flowchart below shows the frozen ultrathin sectioning method provided in the embodiments of the present invention. Figure 1 The technical solution of the present invention will be described below.
[0028] The cryopreservation ultrathin sectioning method proposed in this embodiment of the invention includes:
[0029] Step 110: Place the sample to be sliced into a fixative containing a novel embedding agent, and then place them together in a low-temperature medium for freezing to obtain a quick-frozen fixed sample.
[0030] The novel embedding agent used for cryogenic ultrathin sectioning is liquid at room temperature, rapidly freezes into an amorphous solid in a cryogenic medium, providing support for the frozen sections, and completely evaporates during the rewarming process after freezing. Specifically, it can be water or an amorphous solid organic solvent at the freezing temperature provided by the cryogenic medium. Preferably, highly volatile solvents such as dimethyl carbonate (DMC) and tetrahydrofuran are used.
[0031] The preferred range for freezing temperature is 0°C to -160°C.
[0032] The cryogenic medium can be one of the following: liquid nitrogen, liquid helium, liquid ammonia, or liquid ethane.
[0033] The fixture refers to the clamp used to fix the sample to be sliced, and it can also hold the embedding agent of this invention. Various specific structures can be implemented, and no particular limitation is made here. When the sample to be sliced is fixed in the clamp, it is immersed in the novel embedding agent of this invention. Since the height of the sample to be sliced is typically on the order of millimeters to centimeters, the novel embedding agent can be a certain amount of liquid contained in the clamp or a droplet formed by surface tension on the surface of the clamp, as long as it can completely encapsulate the sample to be sliced.
[0034] The freezing methods described above, which involve placing the medium into a cryogenic medium for freezing, specifically include: immersion freezing or high-pressure freezing.
[0035] Step 120: Place the quick-frozen and fixed sample into a cryostat at the set slicing temperature for cryosectioning;
[0036] Specifically, at the set slicing temperature, the novel embedding agent remains in an amorphous solid state.
[0037] The freezing temperature for cryogenic ultrathin sections is typically set between -60°C and -160°C; preferably, the sectioning temperature is set to ±10°C of the glass transition temperature of the novel embedding agent used for cryogenic ultrathin sections.
[0038] The specific method of slicing using a cryostat is existing technology and will not be described in detail here.
[0039] Step 130: The frozen sectioned sample is subjected to a rewarming treatment to allow the novel embedding agent to completely evaporate, resulting in a pure sectioned sample.
[0040] Specifically, the rewarming process involves rewarming the material to room temperature under a set pressure.
[0041] The set pressure conditions include atmospheric pressure, low pressure, or vacuum. The rewarming time varies depending on the pressure conditions; the lower the set pressure, the shorter the rewarming time. For rewarming under atmospheric pressure, the rewarming time is approximately 4-24 hours, depending on the volatility of the liquid used as the embedding agent.
[0042] The novel encapsulating agent is completely volatilized during the reheating process. During this process, the environmental pressure can be controlled, for example, by evacuating the surrounding atmosphere, which can further accelerate its volatilization.
[0043] The novel embedding agent used in the above-described cryogenic ultrathin sectioning method of the present invention is liquid at room temperature, rapidly frozen into an amorphous solid in a cryogenic medium, serving as a support for the frozen sections, and completely volatilizes during the rewarming process after freezing. Specifically, it can be water or an organic solvent that is amorphous solid at the freezing temperature provided by the cryogenic medium.
[0044] The novel embedding agent provided by this invention can be selected and its ratio adjusted according to the freezing temperature required by the characteristics of different materials to be sliced, so as to have a suitable freezing temperature and viscosity, and not affect the properties of the sliced material itself.
[0045] The cryogenic ultrathin sectioning method provided in this invention significantly reduces the risk of sample deformation and denaturation by using a material that is liquid at room temperature and amorphous solid at the freezing temperature provided by the cryogenic medium as the embedding agent. Furthermore, the embedding agent can be completely evaporated without residue through rewarming, resulting in pristine and pure samples. Compared to traditional paraffin or resin-based embedding agents, the liquid embedding agent of this invention enables rapid freezing, thus significantly shortening the curing time. It also eliminates the need for additional soaking and cleaning to remove the embedding agent, improving sectioning efficiency and preventing sample contamination. In addition, this method offers greater flexibility in sample selection; solid powders or films can be embedded without additional treatment, greatly increasing the flexibility of sample selection.
[0046] To better understand the technical solution of the present invention, more specific embodiments are provided below. However, it should be understood that these embodiments are merely for illustrating the technical solution of the present invention in more detail and should not be construed as limiting the present invention in any way. The embodiments are general descriptions of the materials and test methods used in the specific implementation of the solution. As will be known to those skilled in the art, in this context, unless otherwise specified, the process operation methods are well known in the art or can be obtained by those skilled in the art without creative effort.
[0047] Example 1
[0048] Step 1: Drop an appropriate amount of dimethyl carbonate (DMC) onto a fixture with an adjustable fixing groove on top. Due to surface tension, DMC forms a hemispherical droplet on the fixture. Place the sample to be sliced into the DMC and fix it through the adjustable fixing groove. Then quickly place it into liquid nitrogen for rapid freezing.
[0049] Step 2: Place the frozen and fixed sample into a cryostat and freeze-slice it at -60°C according to the standard sectioning procedure.
[0050] Step 3: The obtained sample microgrid is heated to 25°C in a vacuum environment with a vacuum degree of -0.1Mpa. DMC will evaporate during the reheating process, resulting in a pure sample with a thickness of 50nm.
[0051] Step 4: Transfer the sample to a transmission electron microscope for observation, and obtain the following results. Figure 2 The image shown.
[0052] As can be seen, the obtained sliced sample is a pure sample, with DMC completely volatilized and leaving no residue.
[0053] Comparative Example 1
[0054] This example uses a commonly used OCT embedding medium (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol), which is a resin-based embedding medium, for cryosectioning.
[0055] The sample was uniformly mixed with the embedding agent, solidified, and then transferred to a cryostat for cryosectioning to obtain sections of the same thickness as in Example 1. After thawing, the sectioned samples were transferred to a transmission electron microscope for observation, yielding results as shown below. Figure 3 The image shown.
[0056] As can be seen, the boundary between the obtained sliced sample and the resin embedding agent is not clear. If the resin embedding agent is to be removed, an additional cleaning step is required, and there may be residue that causes sample contamination.
[0057] Example 2
[0058] Step 1: Add an appropriate amount of tetrahydrofuran to the fixation container with an adjustable fixation groove inside, place the sample to be sliced into the container and immerse it in tetrahydrofuran, fix it through the adjustable fixation groove, and then quickly put it into liquid nitrogen for rapid freezing.
[0059] Step 2: Place the frozen and fixed sample into a cryostat and freeze-slice it at -110°C according to the standard slicing procedure.
[0060] Step 3: The obtained sample microgrid is heated to 25°C in a vacuum environment with a vacuum degree of -0.1 MPa. Tetrahydrofuran will evaporate during the reheating process, resulting in a pure sample with a thickness of 50 nm.
[0061] Step 4: Transfer the sample to a transmission electron microscope for observation. The observation shows that the sliced sample is a pure sample.
[0062] Example 3
[0063] Step 1: Drip an appropriate amount of pure water onto the fixture with an adjustable fixing groove on top. Due to surface tension, the pure water forms a hemispherical droplet on the fixture. Place the sample to be sliced into the pure water and fix it through the adjustable fixing groove. Then quickly place it into liquid nitrogen for rapid freezing.
[0064] Step 2: Place the frozen and fixed sample into a cryostat and freeze-slice it at -110°C according to the standard slicing procedure.
[0065] Step 3: The obtained sample microgrid is heated to 25°C in a dry atmospheric pressure environment and kept at 24h to complete the reheating process. The pure water will evaporate during the above reheating process, and a pure sample with a thickness of 50nm is obtained.
[0066] Step 4: Transfer the sample to a transmission electron microscope for observation. The observation shows that the sliced sample is a pure sample.
[0067] Example 4
[0068] Step 1: Drop an appropriate amount of ethanol onto the fixture with an adjustable fixing groove on top. Due to surface tension, the ethanol forms a hemispherical droplet on the fixture. Place the sample to be sliced into the ethanol and fix it through the adjustable fixing groove. Then quickly place it into liquid nitrogen for rapid freezing.
[0069] Step 2: Place the frozen and fixed sample into a cryostat and freeze-slice it at -120°C according to the standard sectioning procedure.
[0070] Step 3: The obtained sample microgrid is heated to 25°C in a dry atmospheric pressure environment and kept for 8 hours to complete the reheating process. Ethanol will evaporate during the above reheating process, and a pure sample with a thickness of 50 nm is obtained.
[0071] Step 4: Transfer the sample to a transmission electron microscope for observation. The observation shows that the sliced sample is a pure sample.
[0072] As can be seen from the above embodiments and comparative examples, the cryogenic ultrathin sectioning method using the novel embedding agent of the present invention is not only simple and easy to implement, but also produces pure samples, effectively avoids sample contamination, reduces sample deformation, lowers the difficulty of observation, and is more conducive to the fine characterization of samples.
[0073] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for cryopreservation ultrathin sectioning, characterized in that, The cryo-ultrathroplasty method uses a novel embedding agent for cryo-ultrathroplasty. The cryopreservation ultrathin sectioning method includes: The sample to be sliced is placed in a fixative containing the novel embedding agent and then placed together in a low-temperature medium for freezing to obtain a quick-frozen fixed sample; wherein, the novel embedding agent is frozen into an amorphous solid in the low-temperature medium to provide support for the frozen slices. The quick-frozen and fixed sample is placed in a cryostat at a set slicing temperature for cryosectioning; at the set slicing temperature, the novel embedding agent remains in an amorphous solid state; The frozen sectioned sample is subjected to a rewarming treatment to allow the novel embedding agent to completely evaporate, resulting in a pure sectioned sample.
2. The cryopreservation ultrathin sectioning method according to claim 1, characterized in that, The cryogenic medium is one of liquid nitrogen, liquid helium, liquid ammonia, and liquid ethane.
3. The method for cryopreservation ultrathin sectioning according to claim 1, characterized in that, The novel embedding agent for cryogenic ultrathin sectioning is specifically characterized as follows: it is liquid at room temperature and amorphous solid at the freezing temperature provided by the low-temperature medium; the freezing temperature range is 0℃ to -160℃.
4. The method for cryopreservation ultrathin sectioning according to claim 1, characterized in that, The freezing method of placing the medium into a cryogenic medium specifically includes: immersion freezing or high-pressure freezing.
5. The method for cryopreservation ultrathin sectioning according to claim 1, characterized in that, The set slicing temperature is ±10°C of the glass transition temperature of the novel embedding agent used for cryogenic ultrathin sections.
6. The method for cryopreservation ultrathin sectioning according to claim 1, characterized in that, The rewarming process specifically involves rewarming the temperature to room temperature under a set pressure condition.
7. A novel embedding agent for use in the cryo-ultrathroplasty method according to any one of claims 1-6, characterized in that, The novel embedding agent for cryogenic ultrathin sectioning is liquid at room temperature and rapidly frozen into an amorphous solid in a cryogenic medium, serving as a support for the cryogenic section. Furthermore, the novel embedding agent completely evaporates during the rewarming process after cryogenic sectioning.
8. The novel embedding agent according to claim 7, characterized in that, The novel embedding agent for cryogenic ultrathin sectioning specifically includes: water, or an organic solvent that is an amorphous solid at the freezing temperature provided by the low-temperature medium.
9. The novel embedding agent according to claim 8, characterized in that, The cryogenic medium is one of liquid nitrogen, liquid helium, liquid ammonia, and liquid ethane, and the freezing temperature ranges from 0°C to -160°C.
10. The novel embedding agent according to claim 7, characterized in that, The novel embedding agent for cryogenic ultrathin sections is applied to the preparation of cryogenic ultrathin section samples for transmission electron microscopy (TEM).