A preparation method for three-dimensional reconstruction of a sample by transmission electron microscope

Abstract

The present application relates to a kind of preparation methods for transmission electron microscope three-dimensional reconstruction sample, the method includes the following steps: nanometer particle dispersion liquid drop is added to porous membrane carrier net, and after air drying, carrier net is fixed on sample rod, the square grid of the porous membrane carrier net is 45 ° angle with the long axis direction of transmission electron microscope sample rod.By adjusting the position of carrier net in sample processing process, it can be guaranteed that although not in the area of carrier net center, sample is not blocked by carrier net when rotating sample rod, can expand sample selection area, so more nanoparticles are selected, improve measurement efficiency and three-dimensional reconstruction image quality.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for processing a sample for transmission electron microscopy, and in particular to a method for preparing a sample for three-dimensional reconstruction in transmission electron microscopy. BACKGROUND

[0002] Thanks to high spatial resolution, transmission electron microscopy (TEM) is considered as the most popular research equipment for studying nanoscale materials and bulk materials, and has a wide range of applications in the fields of material science, physics, chemistry, biomedical science, etc.

[0003] Transmission electron microscopy can not only provide information on the morphology and microstructure of a sample at the nanoscale, but also further reveal important information on the three-dimensional structure and composition of the sample through three-dimensional reconstruction technology. This is of great significance for in-depth understanding of the structure-property relationship of nanomaterials and the function of biological macromolecules.

[0004] Generally, the three-dimensional reconstruction method is to place the sample on a grid, select a nanoparticle located at the center of the grid, rotate the sample rod from -75 degrees to +75 degrees, and take a picture of the sample at each rotation angle. After post-processing, the three-dimensional image of the sample is obtained.

[0005] For nanoparticles, unlike bulk metal materials, there are more nanoparticle samples available for observation. Typically, the dispersed nanosample droplet is added to the metal grid, and after drying, the sample is observed.

[0006] Although there are many nanoparticle samples, it is easy to select samples for observation, but at the same time, due to the large number of particles and dispersion, it is difficult to avoid the obstruction between different particles in actual operation. This is to be avoided in three-dimensional reconstruction, and the obstruction of the grid during rotation should also be avoided.

[0007] Therefore, in the process of three-dimensional reconstruction, it is usually required to select the nanoparticle to be observed at the exact center of the grid. If other areas are selected, the sample may be obstructed by the grid during rotation. At the same time, by adjusting the concentration and volume of the dispersion liquid, it is ensured that the target nanoparticle to be observed is not obstructed by other particles during rotation. However, due to the difficulty of dispersion and other actual operations, it is difficult to ensure that the particles are perfectly monodispersed on the sample grid, and it is difficult to obtain good three-dimensional reconstruction results.

[0008] Japanese Patent P2009-70806A discloses a grid dedicated to transmission electron microscope three-dimensional observation, by improving the grid structure, including a sheet-shaped grid 110 protecting the upper structure and a support film 120 provided with dispersed nanoparticles 130 on the sheet-shaped grid 110. Labeling is performed before attaching the sample, and in the case of amorphous samples, drug samples and chemical samples, unlike the previous grid, the problem of difficult labeling and difficult three-dimensional image acquisition can be solved.

[0009] But it solves the problem of sample labeling, and cannot solve the shielding between particles in the test process. SUMMARY

[0010] In view of the problems existing in the prior art, the applicant provides a preparation method for three-dimensional reconstruction of a sample for a transmission electron microscope through repeated practice in actual operation, which can better solve the shielding problem in the three-dimensional reconstruction process of nanoparticles and obtain a precise three-dimensional reconstruction image.

[0011] The preparation method for three-dimensional reconstruction of a sample for a transmission electron microscope comprises the following steps: adding a nanoparticle dispersion liquid drop to a porous membrane carrier grid, and fixing the carrier grid on a sample rod after air drying, wherein the square grid of the porous membrane carrier grid is at an angle of 45° with the long axis direction of the transmission electron microscope sample rod.

[0012] The scheme of the present application adjusts the placement position of the carrier grid so that the included angle between the carrier grid and the long axis of the sample rod is forty-five degrees, which can expand the sample selection area, so that more nanoparticles can be selected; at the same time, it can also ensure that although the sample is not in the central area of the carrier grid, the sample will not be shielded by the carrier grid when the sample rod is rotated.

[0013] The nanoparticles in the present application can be various types of nanoparticles, and the corresponding suitable solvent can be used for dilution to prepare a dispersion liquid.

[0014] Further, the particle size of the nanoparticles is 50-200 nm.

[0015] Further, the concentration of the nanoparticle dispersion liquid is 0.02-0.03 mg / mL, and the drop volume is 2-5 μL, so that a high-quality three-dimensional reconstruction transmission electron microscope sample can be quickly prepared, and the sample preparation efficiency is greatly improved.

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

[0017] Further, the method further comprises the step of performing plasma treatment on the sample rod with the fixed support net, with parameters set as follows: O2: Ar = 1:3 in volume ratio, power 30%, time 10 seconds, and treatment times 1-3 times.

[0018] Further, the method further comprises the steps of selecting nanoparticles in the middle region of the support net, rotating the sample rod from -75 degrees to +75 degrees, taking a picture of the sample at each rotation angle, and obtaining a three-dimensional image of the sample by post-processing all the collected two-dimensional images.

[0019] Preferably, the middle region of the support net is a hexagon, which is symmetric about the center point of the support net and comprises 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 at an oblique angle of 135 degrees, the third side length extends obliquely downward from the right end of the first side length at an oblique angle of 135 degrees, the fourth side length extends obliquely downward perpendicular to the second side length, the fifth side length extends obliquely downward perpendicular to the third side length, and the sixth side length is parallel to the first side length.

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

[0021] The present application has the following advantages:

[0022] The present application adjusts the placement position of the support net in the sample processing process, so that the included angle between the support net and the long axis direction of the transmission electron microscope sample rod is 45 degrees, which can ensure that the sample will not be blocked by the support net when the sample rod is rotated, can expand the sample selection area, so that more nanoparticles can be selected, and the measurement efficiency and the quality of the three-dimensional reconstruction image are improved; further, by optimizing the dropping concentration and volume, the sample preparation efficiency can be further improved. In addition, the sample can be subjected to plasma treatment to remove surface contaminants and better imaging. The processing method of the present application has wide applicability and can be applied to different types and solvents of nanoparticle samples, and has good universality. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a schematic diagram of the fixed relationship between the support net and the sample rod of the embodiment of the present application; (a) is a picture of the support net, and (b) is a schematic diagram of the support net fixed at 45 degrees on the sample rod;

[0024] Figure 2Figure 2 is a contrast image of an observable nanoparticle region;

[0025] Figure 3 Figure 3 is a schematic diagram of an enlarged view of the right image of Figure 2; Figure 2 Figure 4 is a schematic diagram of an enlarged view of the right image of Figure 3;

[0026] Figure 4 Figure 5 is a three-dimensional reconstruction nanoparticle image of an embodiment of the present application;

[0027] Figure 5 Figure 6 is a transmission electron microscope image of Comparative Example 2;

[0028] Figure 6 Figure 7 is a transmission electron microscope image of Comparative Example 3. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of embodiments of the present application clearer, the technical solutions of the present application will be described clearly below in conjunction with embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of the present application.

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

[0031] Referring to Figure 1 The present application discloses a preparation method for a transmission electron microscope three-dimensional reconstruction sample, comprising the following steps:

[0032] The nanoparticle dispersion liquid is added dropwise to a porous membrane carrier net, and after air drying, the carrier net is fixed on a sample rod, and the square grid of the porous membrane carrier net is at an angle of 45° with the long axis direction of the sample rod.

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

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

[0035] In the above preparation method, the concentration of the nanoparticle dispersion liquid is 0.02-0.03 mg / mL, and the dropwise volume is 2-5 μL, so that a high-quality three-dimensional reconstruction transmission electron microscope sample can be quickly prepared, and the sample preparation efficiency is greatly improved.

[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, it further includes a step of plasma treatment of the sample rod with the fixed grid, with the parameters set as follows: O2:Ar = 1:3 by volume, power 30%, time 10 seconds, and 1 to 3 treatments. This can effectively remove contaminants and carbon deposits from the sample surface without damaging the three-dimensional structure of the sample. The appropriate number of treatments can be selected according to the electron irradiation resistance of different samples.

[0038] The above preparation method also includes selecting nanoparticles located in the middle region of the carrier grid, rotating the sample rod from -75 degrees to +75 degrees, taking a picture of the sample every certain angle during the rotation, and obtaining a three-dimensional image of the sample by post-processing all the acquired two-dimensional images.

[0039] Because the solution of this invention adjusts the placement of the carrier grid so that the angle between it and the long axis of the sample rod is 45°, it can expand the sample selection area, so that sample selection is no longer limited to the central position of the carrier grid (e.g., ...). Figure 2 As shown in the middle left figure, the selection area can be expanded to include the middle region of the grid used to observe particles. Figure 2 Middle right image and Figure 3 As shown, it can be a hexagon, symmetrical about the center point of the carrier grid, 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 downward at an angle of 135° from the left end of the first side length a. The third side length c extends downward at an angle of 135° from the right end of the first side length a. The fourth side length d extends downward at an angle perpendicular to the second side length b. The fifth side length e extends downward at an angle perpendicular to the third side length c. The sixth side length f is parallel to the first side length a.

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

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

[0042] Example 1

[0043] A method for preparing samples for three-dimensional reconstruction by transmission electron microscopy using gold nanoparticles, wherein the particle size of the gold nanoparticles is 50–200 nm, and the specific steps are as follows:

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

[0045] 2. Select a Quantifoil hole film with a pore diameter of 0.6 μm and a micron pore spacing of 1 μm;

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

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

[0048] 5. Place the sample rod in the plasma cleaner to treat the sample on the hole film, and the parameter settings are: volume ratio O2:Ar = 1:3, power 30%, time 10 seconds. According to the above parameters, treat different samples twice;

[0049] 6. Machine testing.

[0050] After the treatment of the gold nanoparticles is completed, the 200kv transmission electron microscope of Jihua Laboratory Test Center is used for observation and analysis. 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, magnification ×25-×10500000.

[0051] Select Figure 2 the nanoparticles in the middle area of the hexagonal grid shown in the right graph of FIG. 1, the length of the square grid of the hole film is L, the length of the first side and the sixth side is the length of the second side, the third side, the fourth side and the fifth side is 2L. Rotate the sample rod from -75 degrees to +75 degrees, and take a picture of the sample at a certain angle during the rotation. After post-processing of all the collected two-dimensional images, the three-dimensional image of the sample is obtained.

[0052] Results: The three-dimensional reconstruction image of the gold nanoparticles can be clearly obtained, as shown in FIG. 2. Figure 4

[0053] Example 2

[0054] A preparation method of a gold nanoparticle transmission electron microscope three-dimensional reconstruction sample, the particle size of the gold nanoparticles is 50-200nm, and the specific steps are as follows:

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

[0056] ​2. Select Quantifoil holey film with pore size of 0.6 pm and hole pitch of 1.6 pm;

[0057] 3. Take 3 microliters of the sample in step (1) and drop it on the holey film using a 10 microliter pipette;

[0058] 4. After air-drying, fix the grid on a sample holder, and the square grid of the grid is at an angle of 45° with the long axis of the sample holder;

[0059] 5. Place the sample holder in a plasma cleaner to treat the sample on the holey film, and the parameters are set as follows: volume ratio of O2:Ar = 1:3, power 30%, and time 10 seconds. Treat different samples for 3 times according to the above parameters;

[0060] 6. Test on the machine.

[0061] After the treatment of the gold nanoparticles is completed, observe and analyze them using a 200kv transmission electron microscope of the Jihua Laboratory Test Center. The model of the transmission electron microscope is Talos-F200X, and the parameters of the electron microscope are as follows: maximum voltage 200kv, point resolution 0.25 nm, lattice resolution 0.12 nm, and magnification ×25-×10500000.

[0062] Select Figure 2 the nanoparticles in the middle region of the hexagonal grid shown in the right part of the figure, the side length of the square grid of the holey film is L, and the side length of the first side and the sixth side is the side length of the second side, the third side, the fourth side, and the fifth side is 2L. Rotate the sample holder from -75 degrees to +75 degrees, and take a picture of the sample at every certain angle during the rotation. Obtain the three-dimensional image of the sample after post-processing of all the collected two-dimensional images.

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

[0064] Example 3

[0065] A method for preparing a three-dimensional reconstruction sample of gold nanoparticles by transmission electron microscopy, the particle size of the gold nanoparticles is 50-200 nm, and the specific steps are as follows:

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

[0067] 2. Select Quantifoil holey film with pore size of 0.6 pm and hole pitch of 1.6 pm;

[0068] 3. Take 2 microliters of the sample in step (1) and drop it on the holey film using a 10 microliter pipette;

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

[0070] 5. Place the sample rod in a plasma cleaner 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. Perform the treatment three times on different samples using the above parameters.

[0071] 6. On-machine testing.

[0072] After the gold nanoparticles were processed, they were observed and analyzed using a 200 kV transmission electron microscope (TEM) at the Ji Hua Laboratory Testing Center. The TEM model was Talos-F200X, and its parameters were as follows: maximum voltage 200 kV, point resolution 0.25 nm, lattice resolution 0.12 nm, and magnification ×25-×10,500,000.

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

[0074] Results: A clear three-dimensional reconstruction of the gold nanoparticles can be obtained.

[0075] Comparative Example 1

[0076] A method for preparing samples for three-dimensional reconstruction by transmission electron microscopy using gold nanoparticles, wherein the particle size of the gold nanoparticles is 50–200 nm, and the specific steps are as follows:

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

[0078] 2. Quantifoil porous membrane with a pore size of 0.6μm and a pore spacing of 1μm was selected;

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

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

[0081] 5. Place the sample rod in a plasma cleaner to process the sample on the porous membrane. The parameters are set as follows: volume ratio O2:Ar = 1:3, power 30%, time 10 seconds. Repeat this process once for different samples using the above parameters.

[0082] 6. On-machine testing.

[0083] After the gold nanoparticles were processed, they were observed and analyzed using a 200 kV transmission electron microscope (TEM) at the Ji Hua Laboratory Testing Center. The TEM model was Talos-F200X, and its parameters were as follows: maximum voltage 200 kV, point resolution 0.25 nm, lattice resolution 0.12 nm, and magnification ×25-×10,500,000.

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

[0085] Result: The concentration was too low, making it difficult to find a suitable sample and thus impossible to reconstruct.

[0086] Comparative Example 2

[0087] A method for preparing samples for three-dimensional reconstruction by transmission electron microscopy using gold nanoparticles, wherein the particle size of the gold nanoparticles is 50–200 nm, and the specific steps are as follows:

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

[0089] 2. Quantifoil porous membrane with a pore size of 0.6μm and a micron-sized pore spacing of 1.6μm was selected;

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

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

[0092] 5. Place the sample rod in a plasma cleaner 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. Perform the treatment three times on different samples using the above parameters.

[0093] 6. On-machine testing.

[0094] After the processing work of gold nanoparticles is completed, a 200kv transmission electron microscope of Jinhua Laboratory Test Center is used for observation and analysis. 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, magnification ×25-×10500000.

[0095] Select Figure 2 The nanoparticles in the middle region of the hexagonal support net shown in the right graph are selected, the length of the square grid of the porous membrane is L, the length of the first length and the sixth length is The length of the second length, the third length, the fourth length and the fifth length is 2L. Rotate the sample rod from-75 degrees to +75 degrees, take a picture of the sample at a certain angle during rotation, and obtain the three-dimensional image of the sample after post-processing of all the collected two-dimensional images.

[0096] Results: too high concentration, particle accumulation, as shown in Figure 5 , it is difficult to find a suitable sample and cannot be reconstructed.

[0097] Comparative example 3

[0098] A preparation method of a three-dimensional reconstruction sample of gold nanoparticles for transmission electron microscopy, the particle size of the gold nanoparticles is 50-200nm, and the specific steps are:

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

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

[0101] 3. Take 3μl of the sample in step(1) with a 10μl pipette and drop it on the porous membrane;

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

[0103] 5. Place the sample rod in the plasma cleaner to process the sample on the porous membrane, and the parameter settings are: volume ratio O2:Ar=1:3, power 30%, time 10 seconds. According to the above parameters, process different samples for 3 times;

[0104] 6. Machine testing.

[0105] After the processing work of the gold nanoparticles is completed, the 200kv transmission electron microscope of the 200kv transmission electron microscope test center of Jinhua Laboratory is used for observation and analysis. The model of the transmission electron microscope is Talos-F200X, and various parameters of the electron microscope are: the highest voltage is 200kv, the point resolution is 0.25nm, the lattice resolution is 0.12nm, and the magnification is ×25-×10500000.

[0106] Select Figure 2 The nanoparticles in the central region shown in the left graph are observed. The sample rod is rotated from-75 degrees to +75 degrees, and the sample is photographed once every certain angle during the rotation. All the collected two-dimensional images are processed to obtain the three-dimensional image of the sample.

[0107] Results: When the sample is tilted to 60°, the target nanoparticles are covered by the edge of the grid (as shown in Figure 6 ), and the target nanoparticles cannot be observed.

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

[0109] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions provided by the embodiments of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and spirits provided by the embodiments of the present application, and any modification, equivalent replacement, improvement, etc. made within the spirits and principles provided by the embodiments of the present application should be included in the protection scope provided by the embodiments of the present application.

Claims

1. A method for preparing a sample for three-dimensional reconstruction by transmission electron microscopy, comprising the steps of: adding a nanoparticle dispersion droplet onto a porous membrane support, air-drying the sample, and then fixing the support onto a sample holder, characterized in that, The square grid of the porous membrane carrier forms a 45° angle with the long axis of the sample rod; It also includes selecting nanoparticles located in the middle region of the carrier grid, rotating the sample rod from -75 degrees to +75 degrees, taking a picture of the sample every time it is rotated at a certain angle, and then processing all the acquired two-dimensional images to obtain the three-dimensional reconstruction result of the sample. The central region of the carrier net is a hexagon, symmetrical about the center point of the carrier net, and includes the first side length, the second side length, the third side length, the fourth side length, the fifth side length, and the sixth side length. The first side length is parallel to the direction of the sample rod. The second side extends downwards at an angle of 135° from the left end of the first side. The third side extends downwards at an angle of 135° from the right end of the first side. The fourth side length extends downward at an angle perpendicular to the second side length; The fifth side extends downward at an angle perpendicular to the third side. The length of the sixth side is parallel to the length of the first side; If the square mesh of the porous membrane carrier has a side length of L, then the side lengths of the first and sixth sides are: L, where the lengths of the second, third, fourth, and fifth sides are 2L.

2. The preparation method according to claim 1, characterized in that, The nanoparticles have a particle size of 50~200 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 dropping 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 1~1.6 μm.

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

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