Method for freezing and preparing sample of oocyte nucleus and application thereof

Abstract

The present application relates to the technical field of cell imaging, and particularly relates to a method for freezing and preparing a nucleus of an oocyte and application thereof. The method comprises the following steps: placing a nucleus of an oocyte on a carrier and puncturing the nucleus; removing intranuclear material without unfolding the nuclear membrane of the nucleus, the nucleus becomes a flat multilayer nuclear membrane due to the support of the intranuclear material; and fixing and freezing and preparing the flat multilayer nuclear membrane. The present application provides a new method for freezing and preparing a nucleus, which can stably and accurately prepare a nucleus sample capable of imaging the side of nuclear pores, and the sample is very clear under an electron microscope. The method provided by the present application supplements the imaging information of the side of nuclear pores which is missing in a conventional single-layer nuclear membrane sample, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of cell imaging technology, and in particular to a method for cryopreservation of oocyte nuclei and its application. Background Technology

[0002] The cell nucleus is a hallmark structure of eukaryotic cells, enclosed by a double nuclear membrane. The nuclear membrane consists of an outer nuclear membrane and an inner nuclear membrane, which fuse at specific sites to form a large multi-protein complex—the nuclear pore complex (NPC). The NPC is a crucial channel regulating the exchange of substances between the nucleus and cytoplasm, and its structural and functional dysregulation is closely related to a variety of diseases. Therefore, obtaining a high-resolution three-dimensional structure of the NPC is of great significance for understanding its working mechanism and developing related therapies.

[0003] Cryo-electron tomography (Cryo-ET) and cryo-electron microscopy (Cryo-EM) are the main techniques for in-situ analysis of the fine three-dimensional structures of large complexes such as NPCs. For these analyses, biological samples need to be frozen to preserve their native conformation as much as possible. In the structural study of NPCs, the African clawed frog (Xenopus laevis)... Xenopus laevis Oocytes are widely used as model specimens because of their large nuclei (germinal vesicles), which are easy to separate manually, and the high density of NPCs on the surface of the nuclear membrane.

[0004] A classic sample preparation method in existing technologies is the nuclear membrane spreading method: researchers first isolate the complete germinal foam, remove its contents, and then spread the remaining bilayer nuclear membrane onto an electron microscope grid for rapid freezing. The advantage of this method is that it yields a relatively flat, NPC-rich two-dimensional planar sample, which is ideal for data acquisition. However, this traditional nuclear membrane spreading method has a significant technical bottleneck: it causes all NPCs in the sample to exhibit a highly uniform orientation, meaning their central axes are almost entirely perpendicular to the supporting membrane plane of the electron microscope grid.

[0005] During data acquisition, the effective tilt range of the sample is typically limited to ±60° to ±70° due to the physical limitations of the electron microscope stage and the sharp increase in effective sample thickness at high tilt angles. When dealing with samples with highly uniform orientation, this limited tilt range prevents the acquisition of a projection image parallel to the support membrane direction, i.e., an effective side-view of the NPC (partially normalized sample). This incompleteness of the data acquisition angle information creates an information gap in the Fourier space of the 3D reconstruction, known as the "Missing Wedge." This effect introduces severe artifacts into the final 3D reconstruction results, primarily manifested as: 1) stretching and blurring of the structure along the electron beam direction (Z-axis); and 2) anisotropic resolution in the final structure, meaning the resolution along the Z-axis is significantly lower than that along the XY plane. This resolution inhomogeneity severely hinders accurate structural analysis and model building of the subunits of the NPC distributed along its central axis.

[0006] Therefore, there is an urgent need in this field for a new sample preparation scheme to increase the orientation diversity of sample particles, overcome the aforementioned problems, and achieve isotropic high-resolution three-dimensional structural analysis of the nuclear-pore complex. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a method for cryopreservation of oocyte nuclei and its application.

[0008] In a first aspect, the present invention provides a method for cryopreservation of cell nuclei, comprising: The nucleus of the oocyte is placed on a carrier, and the nucleus is punctured. Without unfolding the nuclear membrane of the cell nucleus, the intranuclear material is removed, and the cell nucleus, having lost the support of the intranuclear material, becomes a flattened multilayered nuclear membrane. The flat, multilayered nuclear membrane was fixed and frozen for sample preparation.

[0009] To observe the lateral structures of nuclear pores parallel to the nuclear membrane, current techniques often involve folding the nuclear membrane and then observing the fold where the double nuclear membrane bends, revealing a small number of lateral structures of the nuclear pores. However, because the cell nucleus is very small and the nuclear membrane is very sticky, easily adhering to tweezers or needles, it is difficult to fold it like paper, making this method extremely challenging.

[0010] To solve this problem, the present invention has developed an ingenious method through extensive research. Instead of unfolding the nuclear membrane, the contents are removed after piercing the cell nucleus, directly flattening the nucleus. In this case, the flattened nucleus adheres to the carrier, and the nuclear pore side structure parallel to the nuclear membrane direction can be directly observed at the junction of the upper and lower nuclear membranes.

[0011] Furthermore, the maximum size of the oocyte nucleus is between 50 and 1000 µm; preferably, the maximum size of the oocyte is between 400 and 600 µm.

[0012] Furthermore, the oocytes are derived from amphibians, echinoderms, ray-finned fishes, or mammals; Preferably, the oocyte is derived from an amphibian; More preferably, the oocytes are derived from Xenopus species.

[0013] The method provided by this invention can be used in a variety of amphibians with large vesicles, including salamanders and Xenopus, where the cell nucleus can be manually separated.

[0014] Furthermore, the carrier is a hydrophilicized mesh coated with a carbon film; the mesh has a pore size of 200-400 mesh and is made of one or more of copper, gold, molybdenum, nickel, or rhodium.

[0015] The lower the mesh count, the fewer the number of grids in the entire mesh, resulting in a larger field of view per grid, but poorer sample support; conversely, the higher the mesh count, the more grids in the entire mesh, resulting in a smaller field of view per grid, but better sample support. This invention preferably uses a 200-300 mesh mesh, which provides good support for nuclear membrane samples and a relatively large field of view per grid.

[0016] Further, the hydrophilication treatment includes: placing the carrier network in a mixture of hydrogen and oxygen gas for discharge treatment for 45-90 seconds, wherein the volume ratio of hydrogen to oxygen in the mixture is 1:(2-6); and / or, The carbon film is a porous carbon film; and / or, The carrier mesh is made of copper or gold.

[0017] Furthermore, the puncture of the cell nucleus is performed by using a needle with a diameter of 0.2~0.6mm to puncture the cell nucleus.

[0018] Further, the fixation includes: placing the flattened cell nuclei at -20 to 4°C and fixing them in a HEPES buffer for 5 to 20 minutes, wherein the ratio of potassium ions to sodium ions in the HEPES buffer is (4 to 6): 1, and the buffer contains 0.05 to 0.3 w / v% glutaraldehyde.

[0019] Furthermore, the cryopreservation process includes: performing water absorption and vitrification freezing on the fixed cell nuclei; Preferably, the water absorption causes the ice above the nuclear membrane of the cell nucleus to reach 100-400 nm after the vitrification freezing treatment; and / or, The vitrification process involves placing the cell nucleus directly into liquid ethane at -170 to -183°C.

[0020] In the preparation of bilayer nuclear membrane samples (compared to monolayer samples), the method of this invention allows for a thicker ice layer, preferably 300-400 nm. This is beneficial for subsequent imaging.

[0021] In a second aspect, the present invention provides a method for observing the nuclear membrane of a cell nucleus, comprising: preparing a frozen nuclear membrane sample by freezing the cell nucleus using the aforementioned method; The frozen nuclear membrane sample was observed; Preferably, the observation is performed using an electron microscope.

[0022] Thirdly, the present invention provides the application of the aforementioned method for freezing and preparing cell nuclei, or the aforementioned observation method, in observing the nuclear pore side of cell nuclei.

[0023] Preferably, the application includes screening for drugs with functions related to nuclear pores.

[0024] The method provided by this invention solves a key problem in the high-resolution structural analysis of nuclear pore complexes: the "missing cone." Besides its application in basic research for resolving the high-resolution three-dimensional structure of nuclear pore complexes, this invention also has broad and far-reaching industrial application potential. Some specific industries and application fields to which this invention is applicable are listed below: 1. The nuclear pore complex is a channel for nucleoplasmic transport, and its dysfunction is closely related to the occurrence of many diseases. For example, in antiviral drug development, many viruses (such as HIV, influenza virus, or herpesvirus) need to transport their genetic material to the cell nucleus for replication via the nuclear pore complex after infection. The method provided in this invention can resolve high-resolution lateral nuclear pore structures, precisely revealing the details of the interaction between viral proteins and the nuclear pore complex. Based on this, small molecule drugs or peptides can be designed to specifically block the entry of the viral genome into the cell nucleus, thereby developing novel broad-spectrum or specific antiviral drugs.

[0025] 2. In cancer treatment, many cancer-related signaling proteins and transcription factors, such as P53 and NF-κB, need to travel between the cell nucleus and cytoplasm. Their mislocalization is a hallmark of cancer. Some specific nucleoporins (Nups) on the nuclear pore complex are also abnormally expressed in some cancer cells. By analyzing how these key proteins pass through the nuclear pore complex, drugs can be developed to precisely intervene in the nucleoplasmic transport of cancer-related proteins. For example, tumor suppressor factors such as P53 can be "locked" inside the cell nucleus, while tumor promoters such as NF-κB can be "blocked" outside the cell nucleus, thereby achieving targeted therapy.

[0026] 3. In the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), literature indicates that functional abnormalities of the nuclear pore complex and defects in nucleoplasmic transport are key factors in disease development and progression. Patients with C9orf72 gene mutation-induced ALS (C9-ALS) exhibit abnormal distribution of NPC components. Therefore, high-resolution structural analysis of the nuclear pore complex and nucleoplasmic transport processes helps to understand how disease proteins (such as C9orf72) disrupt nuclear pore components and provides drug design targets for repairing nucleoplasmic transport.

[0027] 4. In rare diseases caused by nucleoporins, mutations in some nucleoporins can lead to rare diseases such as acute myeloid leukemia (AML). Understanding how nucleoporin mutations affect the assembly and stability of the nucleoporin complex provides a theoretical basis for the development and treatment of related diseases.

[0028] 5. In addition, it can also involve gene therapy and cell therapy, structural biology and computational biology services, and nanotechnology and biomimetic engineering.

[0029] The present invention has the following beneficial effects: This invention provides a method for cryopreservation of cell nuclei, which can stably and accurately prepare cell nuclei samples capable of imaging the nuclear pore side views. Subsequent observation under an electron microscope allows for clear observation of these nuclear pore side views, supplementing the imaging information missing from conventional nuclear membrane samples (especially images of nuclear pore side views parallel to the nuclear membrane). Ultimately, this achieves isotropic, high-resolution three-dimensional structural analysis of the nuclear pore complex. Furthermore, the method provided by this invention has a high success rate in sample preparation, enabling the acquisition of a large number of qualified frozen nuclear membrane samples in a short time, and has significant application value in the field of nuclear membrane observation of cell nuclei. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 These are Xenopus oocytes from different periods provided in Embodiment 1 of the present invention.

[0032] Figure 2 This is a scanning electron microscope image of the nucleus and nuclear membrane of a Xenopus laevis cell provided in Embodiment 1 of the present invention.

[0033] Figure 3 This is a cryo-electron microscopy image of a single-layer nuclear membrane provided in Embodiment 1 of the present invention.

[0034] Figure 4 This is an electron microscope image of the side-view of the nuclear pore provided in Embodiment 1 of the present invention.

[0035] Figure 5 This is an electron microscope image of the folding of the nuclear membrane after separation, provided in Comparative Example 1 of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. 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.

[0037] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0038] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0039] Example 1 In this embodiment, a method for cryopreservation of cell nuclei includes the following steps: 1. Prepare the experimental solutions. Prepare 2 L of sterile saline solution for amphibians (0.65% NaCl) in advance, as well as 500 ml of amphibian Ringer's solution (hereinafter referred to as AR solution: 111 mM NaCl, 1.9 mM KCl, 1.1 mM CaCl2, 2.4 mM NaHCO3), and a buffer for preserving cell nuclei (hereinafter referred to as 5:1 / HEPES buffer: 83 mM KCl, 17 mM NaCl, 10 mM HEPES, pH 7.5).

[0040] 2. First, separate the oocytes. Prepare a box of crushed ice. Take a healthy, sexually mature female African clawed frog and place it in the crushed ice for half an hour. After the frog goes into ice shock, remove it and place it abdomen up. Use a scalpel to make an incision on the lower abdomen of the frog, cutting open the skin and muscles to an opening of about 0.5-1 cm. Use forceps to remove a small portion of ovarian tissue and cut off a small piece of ovarian tissue with sterile scissors. Use sterile surgical needles and sutures to suture the muscles of the wound, and then suture the skin a second time. After all suturing is completed, place the frog in 0.65% sterile saline solution. To avoid wound infection, change the sterile saline solution every day for three to four consecutive days, and then put the frog back into the circulating water.

[0041] 3. The oocytes from the removed small piece of ovarian tissue are separated and, based on their maturity level, can be divided into stages 1-6. Figure 1 The oocytes, which have developed to stage 6, are separated from the ovarian tissue and transferred to a 5:1 / HEPES buffer for subsequent nuclear separation. Since the nucleus itself is colorless and transparent, a black silica plate can be placed under the dissecting microscope to increase contrast. The culture dish containing the solution is then pressed onto the silica plate, which makes it easier to see the nucleus clearly under the microscope.

[0042] 4. The nucleus is located at the apex of the black animal pole of the oocyte. To prevent puncturing the nucleus, avoid this area when using a needle. Use the needle of a 1 ml syringe to puncture the junction of the black and white cytoplasm of the oocyte. Then, use fine forceps to tear open the cell membrane and use a fine glass needle to peel the nucleus from the white cytoplasm at the apex of the animal pole. The nucleus itself is nearly transparent and spherical. Due to the white cytoplasm adhering to its surface, it appears as a white pearly structure under a dissecting microscope. Figure 2 (A in the middle).

[0043] 5. Using a glass tube slightly larger than the diameter of the cell nucleus (soften the glass tube by heating it with an alcohol lamp, then stretch it to obtain a thinner tube), transfer the isolated single cell nuclei into a clean 5:1 / HEPES buffer. Connect the end of this glass tube to a rubber tube and a 5 ml syringe for aspiration and respiration of the solution. Alternatively, you can control the flow of solution by blowing air directly into the tube. Use the glass pipette to aspirate and re-blow the cell nuclei in the solution, removing as much cytoplasm and other impurities as possible from the surface of the nuclei. This is sufficient for subsequent cryo-electron microscopy sample preparation. If a scanning electron microscope is used to observe the spread nuclear membrane, the nuclear pore complex structure embedded in the nuclear membrane can be seen. Nuclear pore structures observed perpendicular to the nuclear membrane are those seen from the top or bottom. On the cytoplasmic side, the observed structures are ring-shaped cytoplasmic loops, while on the nuclear-cytoplasmic side, they are nuclear basket structures. All these nuclear pores are oriented in the same direction. Figure 2 (BF in the middle).

[0044] Figure 2 Image A shows a stereomicroscopic image of a single nucleus from an isolated Xenopus oocyte, with white cytoplasmic components adhering to the surface of the nucleus. Scale bar: 500 μm.

[0045] B shows the spread-out nuclear membrane of a Xenopus oocyte observed under low magnification using a scanning electron microscope. Scale bar: 2 μm.

[0046] C represents the nucleoplasmic side of the nuclear membrane on the left and the cytoplasmic side on the right, with vesicles and cytoplasmic components on the nuclear membrane. Scale bar: 1 μm.

[0047] D represents the nuclear membrane cytoplasm surface observed under high magnification using a scanning electron microscope, revealing the cytoplasmic fiber structure of the nuclear pore complex on the nuclear membrane surface. Scale bar: 200 nm.

[0048] E represents the nuclear membrane-nuclear plane observed under high magnification using a scanning electron microscope, revealing an eight-fold symmetrical nuclear basket structure of the nuclear pore complex within the inner layer of the nuclear membrane. Scale bar: 200 nm.

[0049] F represents a single, magnified nuclear basket structure of a nuclear pore complex. Scale bar: 100 nm.

[0050] 6. Before preparing nuclear membrane cryo-electron microscopy samples, the grid needs to be hydrophilized (using a mixture of hydrogen and oxygen at a volume ratio of 1:4, with a discharge time of 60 seconds). A gold or copper grid (R2 / 1, 300 mesh, Quantifoil) coated with a carbon film is used as the grid. A fine glass pipette is used to transfer cell nuclei onto the hydrophilized grid. Testing showed that placing 2-3 cell nuclei on each grid is optimal. Placing only one cell nucleus per grid leads to grid waste, while placing too many nuclei results in insufficient grid area. Furthermore, the water absorption time needs to be extended during subsequent sample preparation, and the ice thickness on the grid becomes uneven. This can result in some cell nuclei becoming over-hydrated and denatured, while the remaining nuclei are too thickly covered with ice, preventing electrons from passing through during imaging and resulting in a completely dark field of view. Therefore, placing 2-3 cell nuclei per grid achieves a better yield and sample preparation efficiency.

[0051] 7. If preparing a single-layer nuclear membrane sample, directly tear the cell nucleus with a fine needle and glass needle, spreading the torn cell membrane as much as possible. Specifically, use a 1 ml syringe needle to puncture the nuclear membrane from the middle of the cell nucleus, then pull the nuclear membrane open and fix it onto the grid, spreading the nuclear membrane as much as possible on the grid. There will be a clump of white intranuclear material in the middle. Use a needle to separate this clump of white nucleoplasm, use tweezers to pick up the grid with the nuclear membrane, and gently rinse it in clean 5:1 / HEPES buffer to remove impurities from the nuclear membrane and cytoplasm. Then place it in 0.15% glutaraldehyde in 5:1 / HEPES buffer and fix it on ice for 10 minutes, and then freeze the sample.

[0052] 8. If preparing a double-layered nuclear membrane sample for observing the side-view structure of the nuclear pores parallel to the nuclear membrane, take a complete cell nucleus, place it on a grid, and use the needle of a 1 ml syringe (approximately 0.45 mm in diameter) to puncture the nucleus in the middle, but do not spread the nuclear membrane. After the material inside the nucleus is exposed, use a fine glass needle to gently separate the genetic material inside the nucleus. At this time, the nucleus will lose the support of the material inside the nucleus and flatten, changing from a spherical shape to a disc shape and sticking to the grid. After placing 2-3 cell nuclei on each grid, place them in a 5:1 / HEPES buffer containing 0.15% glutaraldehyde and fix on ice for 10 minutes before proceeding with subsequent freezing and sample preparation.

[0053] 9. For frozen sample preparation using the Vitrobot Mark IV (Thermo Fisher Scientific, USA), since the circular filter paper compatible with Vitrobot is highly absorbent and can easily cause nuclear membrane denaturation, a nitrocellulose membrane (NC membrane) was used instead. The NC membrane was cut into circles of the same size as the circular filter paper provided with the Vitrobot, with a small section cut out from the center. The cut NC membrane circles were then mounted on the Vitrobot machine. The humidity inside the Vitrobot chamber was set to 100%, and the temperature to 22°C. Wait time was set to 0 s, drain time to 0 s, force to 0, and blot twice. The specific absorption time was tested and adjusted according to the batch of NC membranes, typically ranging from 0.5 s, 1 s, to 2 s, with a maximum of 7 seconds. Since different batches of NC membranes have varying water absorption capacities, it is necessary to test the specific water absorption time when replacing with a new batch of NC membrane. This ensures that the ice thickness on the nuclear membrane is moderate—neither too thick to prevent electron beam penetration nor too thin to cause nuclear membrane denaturation (the ice thickness for frozen samples with a single-layer nuclear membrane is approximately 200-300 nm, and for samples with a double-layer nuclear membrane, it is approximately 300-400 nm). Once the water absorption time is determined, the water absorption time and other sample preparation parameters for samples prepared using the same batch of NC membranes are continued.

[0054] 10. After the water absorption of the net is completed, the Vitrobot machine automatically and quickly puts it into liquid ethane that has been cooled by liquid nitrogen (temperature -183℃) for freezing. The prepared samples are stored in liquid nitrogen for long-term preservation.

[0055] 11. The prepared frozen samples were observed and imaged using a Talos Arctica 200 KV transmission electron microscope for sample screening, including monolayer nuclear membrane samples (top-view). Figure 3 ) and side-view of nuclear pores in double-layered nuclear membrane samples () Figure 4 Once a sample with a suitable nuclear membrane ice layer thickness is selected, images are acquired using a Titan Krios 300 KV transmission electron microscope.

[0056] Figure 3 Image A shows a low-power cryo-electron microscopy image of the monolayer nuclear membrane of an isolated Xenopus oocyte. The black squares represent the metal mesh overlaid with the monolayer nuclear membrane. Scale bar: 50 μm.

[0057] B is a magnified view of the portion circled in red in A, showing the nuclear membrane spread in different squares. The nuclear membrane in the red box is relatively flat with more usable portion, while the nuclear membrane in other squares has more wrinkles and less usable portion. Relatively flat nuclear membranes will need to be selected during subsequent data collection. Scale bar: 50 μm.

[0058] C is a magnified view of the area circled in red in B, showing the relatively smooth nuclear membrane. Scale bar: 10 μm.

[0059] D is a magnified image of a well-laid nuclear membrane with a suitable ice thickness. The red circle represents a single nuclear pore complex particle. Because the nuclear membrane is relatively flat, almost all the nuclear pores are facing in one direction, i.e., the top-view direction. Scale bar: 100 nm.

[0060] Figure 4 Image A shows a magnified cryo-electron microscopy image of the double nuclear membrane of an isolated Xenopus oocyte. The folded edges of the double nuclear membrane clearly show the inner and outer nuclear membranes of the nucleus and the side view of the nuclear pores. The red box encloses a single nuclear pore complex particle. Scale bar: 100 μm.

[0061] B is an enlarged view of the area circled in white in A, showing a single nuclear pore complex formed by the fusion of the inner and outer nuclear membranes. Because this view is parallel to the direction of the nuclear membrane, it is a side-view of the nuclear pore. Scale bar: 100 μm.

[0062] C is the schematic diagram in B. The green lines represent the nuclear membrane, which is divided into an outer nuclear membrane and an inner nuclear membrane. The fusion of the inner and outer nuclear membranes is the nuclear pore complex structure. This schematic diagram shows the nuclear membrane and nuclear pores in the original electron microscope image. The dark part below represents the cell nucleus structure, and the light-colored area above represents the carbon mesh region.

[0063] Comparative Example 1 This invention also attempted to perform lateral imaging using a folded nuclear membrane, folding the membrane after separation, with the following results: Figure 5 As shown: This operation is quite difficult. Although there have been a few successful cases, the overall success rate is not high, and the uniformity of the samples is relatively poor. The main reasons are: 1. Because the cell nucleus itself is too small to be manipulated manually, it requires a high level of micromanipulation skills from the operator.

[0064] 2. The nuclear membrane itself is very soft and sticky, and it will adhere to the needle or glass needle used in the operation, making it difficult to bend.

[0065] 3. After tearing the nuclear membrane, try to fold it back. You need to stick the needle into the grid again to ensure that the nuclear membrane is folded and fixed to the grid. This can easily damage the carbon film on the grid, which will reduce the observable area. Figure 5 In section A, the carbon film on two sections of the grille has cracked.

[0066] 4. The area with double nuclear membrane on the grid is relatively small, and the selectable area accounts for a very small proportion of the whole. It is possible that only 1 or 2 squares in the entire grid contain folded double nuclear membrane.

[0067] 5. Due to the significant tearing of the nuclear membrane during the preceding operations, the overall structure is quite messy. Figure 5 The nuclear membrane on the carrier mesh (A) is prone to multi-layering, resulting in uneven ice thickness on the nuclear membrane during freezing preparation, with some areas having thicker ice. Figure 5 In some areas (B), the ice was too thin, causing the nuclear pores to come into contact with air, which led to protein denaturation. Figure 5 The tiny, densely packed nuclear pore particles in C and D have turned black, indicating that the protein has denatured and is unusable. Figure 5 The D-scan also showed that in some areas the nuclear membrane had overlapped due to the stretching process.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cryopreservation of cell nuclei, characterized in that, include: The nucleus of the oocyte is placed on a carrier, and the nucleus is punctured. Without unfolding the nuclear membrane of the cell nucleus, the intranuclear material is removed, and the cell nucleus, having lost the support of the intranuclear material, becomes a flattened multilayered nuclear membrane. The flat, multilayered nuclear membrane was fixed and frozen for sample preparation.

2. The method according to claim 1, characterized in that, The maximum size of the nucleus of the oocyte is between 50 and 1000 µm.

3. The method according to claim 2, characterized in that, The oocytes were derived from amphibians.

4. The method according to any one of claims 1-3, characterized in that, The carrier is a hydrophilicized mesh covered with a carbon film; the mesh has a pore size of 200-400 mesh and is made of one or more of copper, gold, molybdenum, nickel or rhodium.

5. The method according to claim 4, characterized in that, The hydrophilization treatment includes: placing the carrier network in a mixture of hydrogen and oxygen gas and discharging it for 45-90 seconds, wherein the volume ratio of hydrogen to oxygen in the mixture is 1:(2-6); and / or, The carbon film is a porous carbon film; and / or, The carrier mesh is made of copper or gold.

6. The method according to any one of claims 1-5, characterized in that, The process of puncturing the cell nucleus involves using a needle with a diameter of 0.2 to 0.6 mm to puncture the cell nucleus.

7. The method according to any one of claims 1-6, characterized in that, The fixation includes: placing the flattened cell nuclei at -20 to 4°C and fixing them in a HEPES buffer for 5 to 20 minutes, wherein the ratio of potassium ions to sodium ions in the HEPES buffer is (4 to 6): 1, and the buffer contains 0.05 to 0.3 w / v% glutaraldehyde.

8. The method according to any one of claims 1-7, characterized in that, The cryopreservation process includes: absorbing water and vitrifying the fixed cell nuclei; The water absorption causes the ice on the nuclear membrane of the cell nucleus to reach 100-400 nm after the vitrification freezing treatment; The vitrification process involves placing the cell nucleus directly into liquid ethane at -170 to -183°C.

9. A method for observing the nuclear membrane of a cell nucleus, characterized in that, include: Frozen nuclear membrane samples were obtained by cryopreservation of cell nuclei using the method described in any one of claims 1-7; The frozen nuclear membrane sample was observed.

10. The method for preparing frozen cell nuclei according to any one of claims 1-8, or the observation method according to claim 9, for observing the lateral surface of the nuclear pores of cell nuclei.

Images