Method for improving sample preparation quality of transmission electron microscope sample

Abstract

The invention relates to a method for improving sample preparation quality of a transmission electron microscope sample. The method comprises the following steps: (1) sequentially standing and cleaning a transmission electron microscope carbon film grid in chloroform, acetone and absolute ethyl alcohol; and (2) after standing and airing, using the transmission electron microscope carbon film carrying net for sample preparation. Compared with the prior art, the method has the advantages that the operation is simple and convenient, the residual organic supporting film and the organic layer can be effectively removed, the hydrophilicity of the carbon film is improved, the dispersibility of nano particles or biomacromolecules and compounds is favorably improved, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of transmission electron microscope preparation, and in particular to a method for improving the sample preparation quality of a transmission electron microscope. Background Art

[0002] A transmission electron microscope (TEM) is a technique that uses a high-energy electron beam to penetrate a sample and obtains a high-resolution microscopic image by detecting the signal after the interaction between the electrons and the sample. In TEM analysis, the specimen grid is a key component for carrying the sample, usually made of metal (such as copper, nickel, gold, etc.) and having a micron-scale grid structure. When preparing a TEM sample, the sample is firmly adsorbed on the support film of the specimen grid. The support film is an amorphous thin film, generally made of plastic (such as Formvar film) or carbon. However, the support film made of Formvar film is prone to charge accumulation under electron beam irradiation, causing sample discharge and sample drift. Therefore, in recent years, a thin carbon film has been sprayed on the Formvar film. The carbon film has high conductivity and low background noise, and can be used for the preparation of high-resolution TEM samples, suitable for observing ultra-thin samples such as nanoparticles and biological macromolecules.

[0003] Currently, commercially available carbon support films for high-resolution TEM observation mainly include ultra-thin carbon support films, micro-grid carbon support films, and regular porous support films (such as Quantifoil, C-flat, etc.) used in cryo-electron microscopy technology. These types of support films are mostly prepared by spraying a carbon film on an organic support film and then dissolving the organic support film. However, when directly using such commercially available carbon film specimen grids for TEM sample preparation and observation, there are easily problems such as unsatisfactory sample preparation quality and imaging effect. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for improving the sample preparation quality of a transmission electron microscope, with good sample dispersion and clear sample information.

[0005] The purpose of the present invention can be achieved by the following technical solutions: A method for improving the sample preparation quality of a transmission electron microscope, comprising the following steps:

[0006] (1) The TEM carbon film specimen grid is sequentially left standing and cleaned in chloroform, acetone, and absolute ethanol;

[0007] (2) After standing and air-drying naturally, the TEM carbon film specimen grid is used for sample preparation.

[0008] During the preparation of the carbon support film, it is necessary to spray a layer of carbon film on the organic support film and then dissolve the organic support film. It is required that there is no residual organic layer on the metal support grid to avoid charge accumulation in the organic layer, prevent sample particle drift, and reduce the influence of background signals. However, in actual use, due to processing problems, commercially purchased carbon film support grids are prone to the situation where the organic layer is not completely removed or there are residual organic substances on the surface of the carbon film, and this situation cannot be distinguished by the naked eye and can only be observed under an electron microscope. Although common methods such as plasma cleaning and glow discharge cleaning can remove the residual organic substances on the surface of the carbon film to a certain extent, they cannot remove the residual organic support film. The present invention cleans the carbon film support grid before use, which can complete the cleaning of the residual organic substances on the carbon film and the removal of the residual organic support film, improve the sample preparation quality, with good sample dispersion and clear sample information.

[0009] Preferably, the support grid material of the transmission electron microscope carbon film support grid includes gold (Au) or copper (Cu).

[0010] Preferably, the carbon film thickness of the transmission electron microscope carbon film support grid is 5 nm - 20 nm.

[0011] Preferably, in step (1), the carbon film side of the transmission electron microscope carbon film support grid is placed face up and left standing, and is successively left standing and cleaned in chloroform, acetone, and absolute ethanol.

[0012] More preferably, in step (1), first place the transmission electron microscope carbon film support grid with the carbon film side up in a container filled with chloroform for 20 - 30 minutes, then place the transmission electron microscope carbon film support grid after chloroform cleaning with the carbon film side up in a container filled with acetone for 20 - 30 minutes, and finally place the transmission electron microscope carbon film support grid after acetone cleaning with the carbon film side up in a container filled with absolute ethanol for 20 - 30 minutes.

[0013] Even more preferably, before filling the container with chloroform, acetone, or absolute ethanol in step (1), clean it with absolute ethanol, acetone, and absolute ethanol successively for 10 - 30 seconds each.

[0014] Even more preferably, the container in step (1) includes a petri dish, and the liquid levels of chloroform, acetone, or absolute ethanol in the petri dish are always higher than the transmission electron microscope carbon film support grid during the cleaning process;

[0015] Even more preferably, in step (1), the migration of the transmission electron microscope carbon film support grid between the containers is achieved by using tweezers to pick it up, and the edge of the transmission electron microscope carbon film support grid is picked up with tweezers.

[0016] Preferably, step (1) includes the following steps:

[0017] Clean the petri dish successively with absolute ethanol, acetone, and absolute ethanol;

[0018] After all the above solvents have evaporated, lay a weighing paper on it.

[0019] Pour chloroform into a petri dish. Use forceps to pick up the transmission electron microscope carbon film carrier grid to be cleaned and place it in the petri dish, with the carbon film facing up and let it stand still.

[0020] Use forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with chloroform and place it in a second petri dish containing acetone, with the carbon film facing up and let it stand still.

[0021] Use forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with acetone and place it in a third petri dish containing absolute ethanol, with the carbon film facing up and let it stand still.

[0022] Use forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with absolute ethanol and place it in a fourth dry petri dish covered with a weighing paper to air dry for standby.

[0023] Further preferably, the step of cleaning the petri dish with absolute ethanol, acetone, and absolute ethanol in sequence includes:

[0024] First, clean the petri dish with absolute ethanol for 10 - 30 seconds.

[0025] Second, clean the petri dish with acetone for 10 - 30 seconds.

[0026] Finally, clean the petri dish with absolute ethanol for 10 - 30 seconds.

[0027] Further preferably, the amount of chloroform poured into the petri dish is at least enough to submerge the carrier grid and will not cause the carrier grid to be exposed to air due to evaporation during the cleaning process.

[0028] Further preferably, when pouring chloroform into the petri dish and using forceps to pick up the transmission electron microscope carbon film carrier grid to be cleaned and place it in the petri dish, the time for it to stand still with the carbon film facing up is 20 - 30 minutes.

[0029] Further preferably, when using forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with chloroform and place it in a second petri dish containing acetone, the amount of acetone is at least enough to submerge the carrier grid and will not cause the carrier grid to be exposed to air due to evaporation during the cleaning process.

[0030] Further preferably, when using forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with chloroform and place it in a second petri dish containing acetone, the time for it to stand still with the carbon film facing up is 20 - 30 minutes.

[0031] Further preferably, when using forceps to pick up the transmission electron microscope carbon film carrier grid cleaned with acetone and place it in a third petri dish containing absolute ethanol, the amount of absolute ethanol is at least enough to submerge the carrier grid and will not cause the carrier grid to be exposed to air due to evaporation during the cleaning process.

[0032] Further preferably, use tweezers to pick up the transmission electron microscope carbon film carrier grid that has been cleaned with acetone and place it in a third petri dish containing absolute ethanol. Let it stand with the carbon film facing up for 20 - 30 minutes.

[0033] Further preferably, standing and air-drying in a dry petri dish means standing and naturally air-drying in a fume hood.

[0034] Preferably, the method for preparing the transmission electron microscope carbon film carrier grid includes covering an organic support film on the carrier grid, spraying a carbon film, and then dissolving the organic support film.

[0035] Further preferably, the material of the organic support film is Fanghua film or collodion film.

[0036] A transmission electron microscope carbon film carrier grid is processed through the following steps: sequentially stand and wash the transmission electron microscope carbon film carrier grid in chloroform, acetone, and absolute ethanol, and then stand and air-dry.

[0037] An application of the above-mentioned transmission electron microscope carbon film carrier grid, using the transmission electron microscope carbon film carrier grid in a transmission electron microscope to detect nanoparticle or biological macromolecule and complex samples.

[0038] Preferably, the transmission electron microscope includes a cryo-transmission electron microscope.

[0039] Preferably, the application of the transmission electron microscope carbon film carrier grid includes the following steps:

[0040] S1: Place the transmission electron microscope carbon film carrier grid with the carbon film facing up, place 2 - 4 μL of the sample solution on the carbon film, adsorb it with filter paper for 2 - 4 s, then quickly freeze it in liquid ethane, and finally transfer it to liquid nitrogen for storage to prepare a frozen sample;

[0041] S2: Transmit the prepared frozen sample to a cryo-transmission electron microscope and image it at the liquid nitrogen temperature.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention can effectively remove the residual organic support film and keep the edges of the inner holes of the carrier grid neat, which helps to prepare a high-quality cryo-electron microscope sample carrier grid.

[0044] 2. The present invention can effectively improve the hydrophilicity of the carbon film carrier grid, which is beneficial to improving the dispersion of nanoparticles or biological macromolecules and complexes.

[0045] 3. The present invention is convenient to operate, can quickly clean the residual organic matter on the surface of the carrier grid, and has a simple process and high efficiency.

[0046] 4. The present invention uses chloroform, acetone, and absolute ethanol for cleaning in sequence. Chloroform can dissolve non-polar or weakly polar organic substances and will not damage the carbon film. Acetone can dissolve polar organic substances and is completely miscible with chloroform. Absolute ethanol can remove the residual solvent and further clean the surface. Thus, the residual organic support film in the mesh holes of the metal support grid is removed, and the residual organic layer on the carbon film is cleaned, which can reduce the influence of the background signal, improve the conductivity of the support grid, reduce sample drift, and improve the detection accuracy.

[0047] 5. The present invention adopts static cleaning and strictly controls the static cleaning time in chloroform, acetone, and absolute ethanol, so that the carbon film on the metal support grid is hardly damaged and the breakage rate is low.

[0048] 6. The present invention can complete the cleaning of the residual organic substances on the carbon film and the removal of the residual organic support film at one time, and also has the advantages of simple operation and low breakage rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic flow chart of the cleaning method of the transmission electron microscope carbon film support grid of the present invention;

[0050] Figure 2 It is a schematic diagram of the transmission electron microscope inspection of the carbon film support grid before cleaning in Example 1;

[0051] Figure 3 It is a schematic diagram of the transmission electron microscope inspection of the carbon film support grid after cleaning in Example 1;

[0052] Figure 4 It is a schematic diagram of the contact angle measurement of the carbon film support grid before cleaning in Example 1;

[0053] Figure 5 It is a schematic diagram of the contact angle measurement of the carbon film support grid after cleaning in Example 1;

[0054] Figure 6 It is a schematic diagram of the protein particles prepared by the carbon film support grid inspected by cryo-electron microscopy in Example 1;

[0055] Figure 7 It is a schematic diagram of the protein fibers prepared by the carbon film support grid inspected by cryo-electron microscopy in Example 1;

[0056] Figure 8 It is a schematic diagram of the transmission electron microscope inspection of the carbon film support grid before cleaning in Example 2;

[0057] Figure 9 It is a schematic diagram of the transmission electron microscope inspection of the carbon film support grid after cleaning in Example 2;

[0058] Figure 10 It is a schematic diagram of the transmission electron microscope inspection of the carbon film support grid after glow discharge cleaning in Comparative Example 1;

[0059] Figure 11 Schematic diagram of the transmission electron microscope inspection of the carbon film grid after cleaning in Comparative Example 2. Detailed implementation mode

[0060] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation modes and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0061] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0062] Example 1

[0063] See Figure 1 , the method for cleaning the transmission electron microscope grid in this embodiment includes:

[0064] Step 01: Clean the petri dish with anhydrous ethanol, acetone, and anhydrous ethanol in sequence. First, clean the petri dish with anhydrous ethanol for 10 seconds; secondly, clean the petri dish with acetone for 10 seconds; finally, clean the petri dish with anhydrous ethanol for 10 seconds.

[0065] Step 02: After all the above solvents have evaporated, lay a weighing paper;

[0066] Step 03: Pour chloroform into the petri dish, and the amount poured is at least enough to cover the grid and the grid will not be exposed to the air due to evaporation during the cleaning process; use forceps to pick up the transmission electron microscope carbon film grid to be cleaned and put it into the petri dish, with the carbon film facing up and let it stand for 30 min;

[0067] Step 04: Pour acetone into the second petri dish, and the amount poured is at least enough to cover the grid and the grid will not be exposed to the air due to evaporation during the cleaning process; use forceps to pick up the transmission electron microscope carbon film grid cleaned with chloroform solution and put it into the petri dish containing acetone, with the carbon film facing up and let it stand for 20 min;

[0068] Step 05: Pour anhydrous ethanol into the third petri dish, and the amount poured is at least enough to cover the grid and the grid will not be exposed to the air due to evaporation during the cleaning process; use forceps to pick up the transmission electron microscope carbon film grid cleaned with acetone solution and put it into the petri dish containing anhydrous ethanol, with the carbon film facing up and let it stand for 20 min;

[0069] Step 06: Use forceps to pick up the transmission electron microscope carbon film grid cleaned with anhydrous ethanol solution and put it into the fourth dry petri dish with a weighing paper laid, and let it stand in a fume hood to dry naturally for standby.

[0070] Figure 2 , 3 Transmission electron microscope inspection of the carbon film grid (model: Quantifoil Cu 300mesh R1.2 / 1.3) before and after cleaning. Figure 2 Inspection of the carbon film grid before cleaning under a transmission electron microscope, where residual organic support film can be seen at the pore edges. Figure 3 Inspection of the carbon film grid after cleaning under a transmission electron microscope, where the residual organic support film at the pore edges disappears, and the pore edges are clean and flat.

[0071] Figure 4 , 5 Schematic diagram of the contact angle measurement of the carbon film grid before and after cleaning. After cleaning, the contact angle of the carbon film grid changes from 99.4° to 78.5°, and the hydrophilicity of the carbon film grid increases.

[0072] Transmission electron microscope tests were performed using the carbon film grid finally prepared in this example. The samples were apoferritin protein particles and protein fibers, including the following steps:

[0073] (1) Use FEI Vitrobot Mark IV for plunge-freezing sample preparation. Add 3 μL of protein sample onto the front side of the film, adsorb with filter paper for 3 s, then plunge into liquid ethane for rapid freezing, and finally transfer to liquid nitrogen for storage;

[0074] (2) The prepared frozen samples were transferred to a Thermofisher Glacios 200kV cryo-transmission electron microscope using an Autoloader automatic sample loading system for low electron dose imaging at liquid nitrogen temperature.

[0075] The measured results are as Figure 6 Cryo-electron microscopy inspection of apoferritin protein particles prepared with the carbon film grid of this example, Figure 7 Cryo-electron microscopy inspection of protein fibers prepared with the carbon film grid of this example. It can be seen that the carbon film grid of this example has good dispersibility for the prepared samples and clear sample particles.

[0076] Example 2

[0077] In this example, the same method as in Example 1 was used to clean the carbon film grid (model: Ultrathin C Film on Lacey Carbon Support film, 400mesh, Cu).

[0078] Figure 8 , 9 Transmission electron microscope inspection of the carbon film grid before and after cleaning. Figure 8The inspection of the carbon film grid before cleaning under a transmission electron microscope shows that there are large areas of residual organic matter on the continuous carbon film, and the background signal seriously affects the sample information. Figure 9 The inspection of the carbon film grid after cleaning under a transmission electron microscope shows that the residual organic matter has disappeared and the sample information is clear.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is that the transmission electron microscope carbon film grid is cleaned by glow discharge, which specifically includes the following steps:

[0081] (1) Place the carbon film grid with the film side up on the glow discharge holder;

[0082] (2) Use a glow discharge instrument (model: PELCO easiGlow TM Glow Discharge 91000) to clean the carbon film grid, with parameters of 25 mA and 60 s.

[0083] The transmission electron microscope inspection of the cleaned carbon film grid is as Figure 10 shown. It can be seen that the glow discharge cleaning fails to remove the residual organic support film at the pore edge, and the flatness of the pore edge is poor.

[0084] It can be known that common plasma cleaning, glow discharge cleaning and other methods can remove the residual organic layer on the carbon film surface, but fail to remove the residual support film.

[0085] Comparative Example 2

[0086] The difference between this comparative example and Example 1 is that the transmission electron microscope carbon film grid is cleaned with tetrahydrofuran, acetone, and ethanol, which specifically includes the following steps:

[0087] (1) Clean the petri dish with anhydrous ethanol, acetone, and anhydrous ethanol in sequence. First, clean the petri dish with anhydrous ethanol for 10 seconds; second, clean the petri dish with acetone for 10 seconds; finally, clean the petri dish with anhydrous ethanol for 10 seconds;

[0088] (2) After all the above solvents have evaporated, lay a weighing paper;

[0089] (3) Place the transmission electron microscope grid with the carbon film side facing up in the petri dish, pour in tetrahydrofuran, and the pouring amount is at least enough to cover the grid and prevent the grid from being exposed to air due to evaporation during the cleaning process; let it stand for 20 min;

[0090] (4) Pour acetone into the second petri dish in an amount sufficient to at least cover the EM grid and ensure that the grid is not exposed to air due to evaporation during the cleaning process. Use forceps to pick up the TEM carbon film grid that has been cleaned with chloroform solution and place it in the petri dish containing acetone, with the carbon film facing up, and let it stand for 20 minutes.

[0091] (5) Pour absolute ethanol into the third petri dish in an amount sufficient to at least cover the EM grid and ensure that the grid is not exposed to air due to evaporation during the cleaning process. Use forceps to pick up the TEM carbon film grid that has been cleaned with acetone solution and place it in the petri dish containing absolute ethanol, with the carbon film facing up, and let it stand for 20 minutes.

[0092] (6) Use forceps to pick up the TEM carbon film grid that has been cleaned with absolute ethanol solution and place it in the fourth dry petri dish lined with weighing paper, and let it air dry naturally in the fume hood for standby.

[0093] The inspection of the cleaned carbon film grid by transmission electron microscopy is as Figure 11 shown. It can be seen that the breakage rate of the carbon film on the metal grid is very high, and tetrahydrofuran is very destructive to the carbon film.

[0094] A method for improving the quality of TEM sample preparation provided by the present invention has the advantages of simple operation, which can effectively remove the residual organic support film and organic layer, improve the hydrophilicity of the carbon film, and is beneficial to improving the dispersibility of nanoparticles or biological macromolecules and complexes.

[0095] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A method for improving the sample preparation quality of a transmission electron microscope, characterized in that, It includes the following steps: (1) The carbon film grid of the transmission electron microscope is sequentially left standing and cleaned in chloroform, acetone, and absolute ethanol; (2) After leaving it to dry by standing, the carbon film grid of the transmission electron microscope is used for sample preparation.

2. The method for improving the quality of TEM sample preparation according to claim 1, wherein The grid material of the carbon film grid of the transmission electron microscope includes gold or copper, and the thickness of the carbon film is 5 nm - 20 nm.

3. The method for improving the sample preparation quality of a transmission electron microscope according to claim 1, wherein In step (1), the carbon film side of the carbon film grid of the transmission electron microscope is placed facing up and left standing, and it is sequentially left standing and cleaned in chloroform, acetone, and absolute ethanol.

4. The method for improving the sample preparation quality of a transmission electron microscope according to claim 3, characterized in that, In step (1), first place the carbon film grid of the transmission electron microscope with the carbon film side facing up and leave it standing in a container filled with chloroform for 20 - 30 min, then place the carbon film grid of the transmission electron microscope after chloroform cleaning with the carbon film side facing up and leave it standing in a container filled with acetone for 20 - 30 min, and finally place the carbon film grid of the transmission electron microscope after acetone cleaning with the carbon film side facing up and leave it standing in a container filled with absolute ethanol for 20 - 30 min.

5. The method for improving the quality of TEM sample preparation according to claim 4, characterized in that Before the container in step (1) is filled with chloroform, acetone, or absolute ethanol, it is sequentially cleaned with absolute ethanol, acetone, and absolute ethanol for 10 - 30 seconds each.

6. The method for improving the sample preparation quality of a transmission electron microscope according to claim 4, characterized in that The container in step (1) includes a petri dish, and the liquid levels of chloroform, acetone, or absolute ethanol in the petri dish are always higher than the carbon film grid of the transmission electron microscope during the cleaning process; in step (1), the carbon film grid of the transmission electron microscope is transferred between each container by using the method of clamping with tweezers.

7. The method for improving the quality of TEM sample preparation according to claim 1, wherein The preparation method of the carbon film grid of the transmission electron microscope includes covering an organic support film on the grid, spraying a carbon film, and then dissolving the organic support film; the material of the organic support film is formvar film or collodion film.

8. A transmission electron microscope carbon film support grid, characterized in that, It is processed through the following steps: The carbon film grid of the transmission electron microscope is sequentially left standing and cleaned in chloroform, acetone, and absolute ethanol, and left to dry by standing.

9. An application of the transmission electron microscope carbon film support grid according to claim 8, characterized in that, The carbon film grid of the transmission electron microscope is used for a transmission electron microscope to detect nanoparticle or biological macromolecule and complex samples.

10. The application of the transmission electron microscope carbon film support grid according to claim 9, wherein, It includes the following steps: S1: Place the carbon film grid of the transmission electron microscope with the carbon film side facing up, place 2 - 4 μL of the sample solution on the carbon film, adsorb it with filter paper for 2 - 4 s, then quickly freeze it in liquid ethane, and finally transfer it to liquid nitrogen for storage to prepare a frozen sample; S2: Transfer the prepared frozen sample to a cryo - transmission electron microscope and perform imaging at the liquid nitrogen temperature.

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