Preparation method of three-dimensional reconstruction sample for transmission electron microscope

Abstract

The invention relates to a preparation method of a three-dimensional reconstruction sample for a transmission electron microscope, which comprises the following steps: nano particle dispersion liquid is dropwise added to a porous membrane carrying net, the carrying net is fixed on a sample rod after air drying, and a 45-degree angle is formed between the square grid of the porous membrane carrying net and the long axis direction of the transmission electron microscope sample rod. By adjusting the placement position of the carrier net in the sample treatment process, it can be ensured that the sample is not shielded by the carrier net when the sample rod is rotated although the sample rod is not in the central area of the carrier net, and the sample selection area can be expanded, so that more nano-particles can be selected, and the measurement efficiency and the three-dimensional reconstruction image quality are improved.

Description

Technical Field

[0001] The invention relates to a method for processing a transmission electron microscope sample, and in particular to a method for preparing a transmission electron microscope three-dimensional reconstruction sample. Background Art

[0002] Thanks to its high spatial resolution, transmission electron microscopy (TEM) is considered to be the most common research equipment for studying nanoscale materials and bulk materials, and has a wide range of applications in materials science, physics, chemistry, biomedicine and other fields.

[0003] Transmission electron microscopy can not only provide information on the morphology and microstructure of samples at the nanoscale, but also further reveal important information such as the three-dimensional structure and composition of the samples through three-dimensional reconstruction technology. This is of great significance for a deep understanding of the structure-performance relationship of nanomaterials and the functions of biomacromolecules.

[0004] Usually, the three-dimensional reconstruction method is to place the sample on a grid, select the nanoparticles located in the middle of the grid, rotate the sample rod from -75 degrees to +75 degrees, and take a picture of the sample every time it rotates a certain angle during the rotation process. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0005] For nanoparticles, because they are different from bulk metal materials, there are more nanoparticle samples and more particles available for observation. Usually, the dispersed nanosample dispersion is dropped onto the metal grid and observed after drying.

[0006] Although there are many nanoparticle samples, which is conducive to selecting samples for observation, it is difficult to avoid occlusion between different particles in actual operation due to the large number of particles and their dispersion. This should be avoided in three-dimensional reconstruction, and the occlusion of the carrier grid during rotation should also be avoided.

[0007] Therefore, in the 3D reconstruction process, it is usually required to select the observed nanoparticles exactly in the center of the grid. If other areas are selected, the sample may be blocked by the grid during rotation. At the same time, the concentration and volume of the dispersion are adjusted to ensure that the target nanoparticles are not blocked by other particles during rotation. However, due to practical operational difficulties such as dispersion, it is difficult to ensure that the particles can be perfectly monodispersed on the sample grid, and it is difficult to obtain good 3D reconstruction results.

[0008] Japanese patent P2009-70806A discloses a grid dedicated to three-dimensional observation of a transmission electron microscope, which improves the grid structure and includes a sheet mesh 110 for protecting the upper structure and a support film 120 for dispersing nanoparticles 130 disposed on the sheet mesh 110. The sample is marked before being attached, and when the sample is an amorphous sample, a drug sample, or a chemical sample, the problem of difficulty in attaching a mark and obtaining a three-dimensional image can be solved, unlike the previous grids.

[0009] However, it only solves the problem of sample marking, but cannot solve the occlusion between particles during the test. Summary of the invention

[0010] In response to the problems existing in the prior art, the applicant has provided a method for preparing samples for three-dimensional reconstruction of transmission electron microscopy through repeated practice in actual operations. This method can better solve the occlusion problem in the three-dimensional reconstruction process of nanoparticles and obtain accurate three-dimensional reconstructed images.

[0011] The present invention provides a method for preparing a sample for three-dimensional reconstruction of a transmission electron microscope, comprising the following steps: dropping a nanoparticle dispersion onto a porous membrane grid, fixing the grid on a sample rod after air drying, wherein the square grid of the porous membrane grid forms an angle of 45° with the long axis direction of the transmission electron microscope sample rod.

[0012] The solution of the present invention can expand the sample selection area by adjusting the placement position of the carrier grid so that the angle between it and the long axis of the sample rod is forty-five degrees, so that more nanoparticles can be selected; at the same time, it can also ensure that even if the sample is not in the center of the carrier grid, it will not be blocked by the carrier grid when the sample rod is rotated.

[0013] The nanoparticles in the present invention can be various types of nanoparticles, and a suitable solvent can be selected to dilute and prepare a dispersion.

[0014] Furthermore, the particle size of the nanoparticles is 50 to 200 nm.

[0015] Furthermore, the concentration of the nanoparticle dispersion is 0.02-0.03 mg / mL, and the drop volume is 2-5 μL, which can quickly prepare high-quality three-dimensional reconstructed transmission electron microscopy samples and greatly improve the sample preparation efficiency.

[0016] Furthermore, the porous membrane is a Quantifoil porous membrane with a pore size of 0.6 μm and a pore spacing of 1 to 1.6 μm.

[0017] Furthermore, the method further includes a step of subjecting the sample rod with the grid fixed thereon to plasma treatment, wherein the parameters are set as follows: according to the volume ratio, O2:Ar=1:3, power 30%, time 10 seconds, and treatment times 1 to 3 times. This can effectively remove pollutants and carbon deposits on the sample surface without damaging the three-dimensional structure of the sample.

[0018] Furthermore, the method includes selecting nanoparticles located in the middle area of ​​the grid, rotating the sample rod from -75 degrees to +75 degrees, photographing the sample every time it rotates a certain angle, and obtaining a three-dimensional image of the sample through post-processing of all collected two-dimensional images.

[0019] Preferably, the middle area of ​​the grid is a hexagon, symmetrical about the center point of the grid, and includes a first side length, a second side length, a third side length, a fourth side length, a fifth side length, and a sixth side length. The first side length is parallel to the direction of the sample rod, the second side length extends obliquely downward from the left end of the first side length, with an inclination angle of 135°, the third side length extends obliquely downward from the right end of the first side length, with an inclination angle of 135°, the fourth side length extends obliquely downward perpendicularly to the second side length; the fifth side length extends obliquely downward perpendicularly to the third side length; the sixth side length is parallel to the first side length.

[0020] Preferably, the side length of the porous membrane square grid is L, then the lengths of the first side and the sixth side are The second side length, the third side length, the fourth side length, and the fifth side length are 2L.

[0021] The beneficial effects of the present invention are:

[0022] The present invention adjusts the placement position of the grid during the sample processing process so that the angle between the grid and the long axis direction of the transmission electron microscope sample rod is 45 degrees, thereby ensuring that the sample will not be blocked by the grid when the sample rod is rotated, even if the area is not in the center of the grid, so that more nanoparticles can be selected, thereby improving the measurement efficiency and the quality of the three-dimensional reconstruction image; and further by optimizing the drop concentration and volume, the sample preparation efficiency can be further improved. In addition, the sample can also be subjected to plasma treatment to remove surface pollutants for better imaging. The processing method of the present invention has wide applicability and can be applied to nanoparticle samples of different types and solvents, and has good versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the fixing relationship between the grid and the sample rod according to an embodiment of the present invention; (a) is a picture of the grid, and (b) is a schematic diagram of the grid being fixed on the sample rod at 45°;

[0024] Figure 2This is a comparison diagram of the observable nanoparticle area;

[0025] Figure 3 for Figure 2 Enlarged schematic diagram of the middle right figure;

[0026] Figure 4 A three-dimensional reconstructed nanoparticle image according to an embodiment of the present invention;

[0027] Figure 5 It is the transmission electron microscope picture of comparative example 2;

[0028] Figure 6 This is the transmission electron microscope picture of Comparative Example 3. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly described below in combination with the embodiments of the present invention. Obviously, the described embodiment is a module embodiment of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels by those skilled in the art.

[0031] Reference Figure 1 The present invention discloses a method for preparing a sample for three-dimensional reconstruction of a transmission electron microscope, comprising the following steps:

[0032] The nanoparticle dispersion is dropped onto the porous membrane grid, and the grid is fixed on the sample rod after air drying, wherein the square grid of the porous membrane grid forms an angle of 45° with the long axis direction of the sample rod.

[0033] In the above preparation method, the nanoparticles can be various types of nanoparticles, and a suitable solvent can be selected to dilute and prepare a dispersion.

[0034] In the above preparation method, preferably, the particle size of the nanoparticles is 50 to 200 nm.

[0035] In the above preparation method, the concentration of the nanoparticle dispersion is 0.02-0.03 mg / mL, and the drop volume is 2-5 μL, which can quickly prepare high-quality three-dimensional reconstructed transmission electron microscopy samples and greatly improve the sample preparation efficiency.

[0036] In the above preparation method, the porous membrane is a Quantifoil porous membrane with a pore size of 0.6 μm and a pore spacing of 1 to 1.6 μm.

[0037] In the above preparation method, preferably, the step of plasma treatment of the sample rod with the grid fixed thereon is also included, and the parameters are set as follows: according to the volume ratio, O2:Ar=1:3, power 30%, time 10 seconds, and treatment times 1 to 3 times. The pollutants and carbon deposits on the sample surface can be effectively removed, and the three-dimensional structure of the sample will not be damaged. According to the degree of resistance to electron irradiation of different samples, the appropriate number of treatments can be selected.

[0038] The above preparation method also includes selecting nanoparticles located in the middle area of ​​the carrier grid, rotating the sample rod from -75 degrees to +75 degrees, photographing the sample once every certain angle during the rotation process, and obtaining a three-dimensional image of the sample through post-processing of all collected two-dimensional images.

[0039] Since the solution of the present invention adjusts the placement position of the grid so that the angle between it and the long axis of the sample rod is 45°, the sample selection area can be expanded, so that the sample selection is no longer limited to the central position of the grid (such as Figure 2 As shown in the middle left figure, you can expand the selection area and select the middle area of ​​the grid where the particles are observed. Figure 2 Middle right picture and Figure 3 As shown, it can be a hexagon, symmetrical about the center point of the carrier network, including a first side length, a second side length b, a third side length c, a fourth side length d, a fifth side length e, and a sixth side length f. The first side length a is parallel to the direction of the sample rod, the second side length b extends obliquely downward from the left end of the first side length a, and the inclination angle is 135°. The third side length c extends obliquely downward from the right end of the first side length a, and the inclination angle is 135°. The fourth side length d extends obliquely downward perpendicularly to the second side length b; the fifth side length e extends obliquely downward perpendicularly to the third side length c; and the sixth side length f is parallel to the first side length a.

[0040] Further preferably, in one embodiment, the side length of the porous membrane square grid is 1, then the side lengths of the first side length and the sixth side length are The length of the second side, the third side, the fourth side, and the fifth side is 2L.

[0041] The present invention is further described in detail below with reference to the embodiments.

[0042] Example 1

[0043] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0044] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.02 mg / ml and store it in a clean container;

[0045] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1μm;

[0046] 3. Use a 10 μl pipette to take 5 μl of the sample in step (1) and drop it onto the porous membrane;

[0047] 4. After air drying, fix the grid on the sample rod, with the square grid of the grid at a 45° angle to the long axis of the sample rod;

[0048] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, treat different samples twice;

[0049] 6. Test on the computer.

[0050] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0051] choose Figure 2 The nanoparticles in the middle area of ​​the hexagonal grid shown in the middle right figure, the side length of the porous membrane square grid is L, and the lengths of the first and sixth sides are The second side length, the third side length, the fourth side length, and the fifth side length are 2L. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0052] Results: The three-dimensional reconstruction of gold nanoparticles can be clearly obtained, such as Figure 4 shown.

[0053] Example 2

[0054] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0055] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.02 mg / ml and store it in a clean container;

[0056] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1.6μm;

[0057] 3. Use a 10 μl pipette to take 3 μl of the sample in step (1) and drop it onto the porous membrane;

[0058] 4. After air drying, fix the grid on the sample rod, with the square grid of the grid at a 45° angle to the long axis of the sample rod;

[0059] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, different samples are treated 3 times;

[0060] 6. Test on the computer.

[0061] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0062] choose Figure 2 The nanoparticles in the middle area of ​​the hexagonal grid shown in the middle right figure, the side length of the porous membrane square grid is L, and the lengths of the first and sixth sides are The second side length, the third side length, the fourth side length, and the fifth side length are 2L. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0063] Results: The three-dimensional reconstruction image of gold nanoparticles can be clearly obtained.

[0064] Example 3

[0065] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0066] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.03 mg / ml and store it in a clean container;

[0067] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1.6μm;

[0068] 3. Use a 10 μl pipette to take 2 μl of the sample in step (1) and drop it onto the porous membrane;

[0069] 4. After air drying, fix the grid on the sample rod, with the square grid of the grid at a 45° angle to the long axis of the sample rod;

[0070] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, different samples are treated 3 times;

[0071] 6. Test on the computer.

[0072] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0073] choose Figure 2 The nanoparticles in the middle area of ​​the hexagonal grid shown in the middle right figure, the side length of the porous membrane square grid is L, and the lengths of the first and sixth sides are The second side length, the third side length, the fourth side length, and the fifth side length are 2L. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0074] Results: The three-dimensional reconstruction image of gold nanoparticles can be clearly obtained.

[0075] Comparative Example 1

[0076] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0077] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.01 mg / ml and store it in a clean container;

[0078] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1μm;

[0079] 3. Use a 10 μl pipette to take 7 μl of the sample in step (1) and drop it onto the porous membrane;

[0080] 4. After air drying, fix the grid on the sample rod, with the square grid of the grid at a 45° angle to the long axis of the sample rod;

[0081] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, treat different samples once;

[0082] 6. Test on the computer.

[0083] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0084] choose Figure 2 The nanoparticles in the middle area of ​​the hexagonal grid shown in the middle right figure, the side length of the porous membrane square grid is L, and the lengths of the first and sixth sides are The second side length, the third side length, the fourth side length, and the fifth side length are 2L. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0085] Result: The concentration was too low, it was difficult to find a suitable sample, and it could not be reconstituted.

[0086] Comparative Example 2

[0087] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0088] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.04 mg / ml and store it in a clean container;

[0089] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1.6μm;

[0090] 3. Use a 10 μl pipette to take 2 μl of the sample in step (1) and drop it onto the porous membrane;

[0091] 4. After air drying, fix the grid on the sample rod, with the square grid of the grid at a 45° angle to the long axis of the sample rod;

[0092] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, different samples are treated 3 times;

[0093] 6. Test on the computer.

[0094] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0095] choose Figure 2 The nanoparticles in the middle area of ​​the hexagonal grid shown in the middle right figure, the side length of the porous membrane square grid is L, and the lengths of the first and sixth sides are The second side length, the third side length, the fourth side length, and the fifth side length are 2L. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0096] Result: Concentration is too high, particles accumulate, such as Figure 5 As shown, it is difficult to find suitable samples and cannot be reconstructed.

[0097] Comparative Example 3

[0098] A method for preparing a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, wherein the particle size of the gold nanoparticle is 50 to 200 nm, and the specific steps are:

[0099] 1. Dilute the gold nanoparticle sample to be tested with a solvent to a concentration of 0.02 mg / ml and store it in a clean container;

[0100] 2. Select Quantifoil porous membrane with pore size of 0.6μm and micropore spacing of 1.6μm;

[0101] 3. Use a 10 μl pipette to take 3 μl of the sample in step (1) and drop it onto the porous membrane;

[0102] 4. After air drying, fix the grid on the sample rod, and place the square grid of the grid and the sample rod randomly;

[0103] 5. Place the sample rod in a plasma cleaning machine to treat the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, different samples are treated 3 times;

[0104] 6. Test on the computer.

[0105] After the gold nanoparticles were processed, they were observed and analyzed using a 200kv transmission electron microscope from the Jihua Laboratory Testing Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are: maximum voltage 200kv, point resolution 0.25nm, lattice resolution 0.12nm, and magnification ×25-×10500000.

[0106] choose Figure 2 The nanoparticles in the central area shown in the middle left picture are observed. The sample rod is rotated from -75 degrees to +75 degrees. During the rotation process, the sample is photographed once every certain angle. All the collected two-dimensional images are post-processed to obtain a three-dimensional image of the sample.

[0107] Results: When the sample is tilted to 60°, the edge of the grid covers the target nanoparticles (such as Figure 6 The target nanoparticles cannot be observed.

[0108] In summary, it can be seen that the present invention provides a method for preparing high-quality three-dimensional reconstruction transmission electron microscopy samples. By optimizing and adjusting conditions such as sample preparation, pre-loading treatment, and the placement of the grid, a more ideal three-dimensional reconstruction result can be obtained. The method is simple to operate, has good repeatability, and can effectively prepare high-quality three-dimensional reconstruction transmission electron microscopy samples. This technology has broad application prospects in the fields of nanomaterials, biomacromolecules, etc., and can provide valuable characterization data for related research.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions provided according to the embodiments of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, it can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit provided according to the embodiments of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles provided according to the embodiments of the present invention should be included in the protection scope provided according to the embodiments of the present invention.

Claims

1. A method for preparing a sample for three-dimensional reconstruction of a transmission electron microscope, comprising the steps of: dropping a nanoparticle dispersion onto a porous membrane grid, and fixing the grid onto a sample rod after air drying, characterized in that: The square grid of the porous membrane grid forms an angle of 45° with the long axis direction of the sample rod.

2. The preparation method according to claim 1, characterized in that: The particle size of the nanoparticles is 50 to 200 nm.

3. The preparation method according to claim 2, characterized in that: The concentration of the nanoparticle dispersion is 0.02-0.03 mg / mL, and the drop volume is 2-5 μL.

4. The preparation method according to claim 1, characterized in that: The porous membrane is a Quantifoil porous membrane with a pore size of 0.6 μm and a pore spacing of 1 to 1.6 μm.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The method also includes the step of subjecting the sample rod with the grid fixed thereon to plasma treatment, wherein the parameters are set as follows: according to the volume ratio, O2:Ar=1:3, power 30%, time 10 seconds, and treatment times 1 to 3 times.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The method also includes selecting nanoparticles located in the middle area of ​​the grid, rotating the sample rod from -75 degrees to +75 degrees, photographing the sample every time it rotates a certain angle, and obtaining a three-dimensional reconstruction result of the sample through post-processing of all collected two-dimensional images.

7. The preparation method according to claim 6, characterized in that: The middle area of ​​the carrier net is a hexagon, symmetrical about the center point of the carrier net, and includes a first side length, a second side length, a third side length, a fourth side length, a fifth side length, and a sixth side length. The first side length is parallel to the direction of the sample rod, The second side extends obliquely downward from the left end of the first side, with an inclination angle of 135°. The third side extends obliquely downward from the right end of the first side, with an inclination angle of 135°. The fourth side length extends obliquely downwardly perpendicular to the second side length; The fifth side length extends obliquely downwardly perpendicular to the third side length; The sixth side length is parallel to the first side length.

8. The preparation method according to claim 7, characterized in that: The side length of the porous membrane square grid is L, then the lengths of the first side and the sixth side are The second side length, the third side length, the fourth side length, and the fifth side length are 2L.

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