Ultrathin slice copper mesh supporting film for transmission electron microscope and preparation method of ultrathin slice copper mesh supporting film

Abstract

The invention discloses an ultrathin slice copper mesh supporting film for a transmission electron microscope and a preparation method of the ultrathin slice copper mesh supporting film. The method comprises the following steps: preparing a dichloroethane solution of Piloform Powder; pretreating the glass slide and the copper mesh; fixing the glass slide in a refitted centrifugal device, dropwise adding a film-adding solution, and then carrying out high-speed short-time centrifugation to form a uniform film; immersing the coated glass slide into ultrapure water at 55-60 DEG C to peel and float the film; and putting the copper net on a floating film, fishing up by using a sealing film, and drying to obtain the copper net. The centrifugal device is formed by modifying a common centrifugal machine into a TCT cell slide preparation clamp and additionally installing an anti-splashing structure. The prepared supporting film is uniform in thickness, resistant to bombardment, high in success rate, low in device cost, easy to popularize and suitable for the field of transmission electron microscope sample preparation.

Description

Technical Field

[0001] This invention relates to the field of transmission electron microscopy (TEM) sample preparation technology, specifically to an ultrathin copper mesh support film for TEM and its preparation method. Background Technology

[0002] In transmission electron microscopy (TEM) sample preparation, ultrathin sections of samples need to be supported on a very thin and robust support film, which is attached to a metal mesh (usually a copper mesh). Aromatic films are generally used as the copper mesh support film. Traditional methods for preparing the support film typically involve directly dipping a glass slide or glass strip into a chloroform solution, allowing the solvent to evaporate and form a thin film, then directly applying the film with cold water, followed by peeling and retrieval via a water bath.

[0003] However, traditional methods have many drawbacks: 1) The film thickness is uneven and greatly affected by ambient temperature, humidity and operation techniques, resulting in poor repeatability; 2) The film thickness is difficult to control precisely. If it is too thick, it will affect electron microscopy observation, and if it is too thin, it will easily break; 3) Manual operation has a low success rate and is prone to wrinkles, damage and other problems, wasting materials and time.

[0004] Therefore, there is an urgent need in this field for an automated or semi-automated method to prepare ultrathin support films with uniform thickness, high success rate, and good repeatability. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ultrathin copper mesh support film for transmission electron microscopy and its preparation method.

[0006] To achieve the objectives of this invention, the following technical solution is adopted: First, this invention provides a method for preparing an ultrathin copper mesh support film for transmission electron microscopy, comprising the following steps: S1. Solution preparation: Prepare the film-forming material Pioloform... ® Powder is dissolved in dichloroethane to prepare a film-forming solution with a mass-volume concentration of 1% to 2%, and stored away from light. S2. Pretreatment of glass slides and copper mesh: Clean and dry the glass slides and copper mesh for later use; S3. Centrifugation to form a film: Fix the pretreated glass slide in a centrifuge, add 200-250 μl of the film-forming solution to its surface, and then start the centrifuge at 7000 rpm for 4-6 seconds to form a uniform thin film on the surface of the glass slide. S4. Underwater peeling: The glass slide covered with the film in step S3 is vertically immersed in ultrapure water at 55-60°C. The film is peeled off from the glass slide and floats flat on the water surface by utilizing the surface tension and temperature of the water. S5. Transfer and drying: Place the pretreated copper mesh on the floating film at intervals, then carefully attach it from one end of the water film with sealing film and lift it up as a whole. Finally, perform drying treatment to obtain a copper mesh covered with a support film.

[0007] In the above preparation method, the solvent dichloroethane is Pioloform. ® A good solvent for powders, with a moderate evaporation rate, which ensures sufficient fluidity for spreading and film formation during centrifugation, and also allows for rapid drying and setting after film formation.

[0008] In the centrifugation film-forming step, the strong centrifugal force compels the added solution to spread uniformly and forcefully towards the tail of the slide (in the centrifugal direction) within seconds, and the solvent rapidly evaporates to form a film. By precisely controlling the rotation speed and time, the thickness and uniformity of the film can be controlled with high repeatability. A volume of 200-250 μl is sufficient to cover the width of a standard slide and ensures the formation of a film with a sufficient area. This ensures the preparation of films with sufficiently uniform thickness and extremely high repeatability.

[0009] In the underwater peeling step, warm water at 55-60℃ provides the energy for the peeling process. The heat weakens the weak adhesion between the film and the glass slide, while simultaneously increasing the flexibility of the polymer film, making it easier to detach completely from the slide. The surface tension of the water smoothly supports the peeled ultrathin film, keeping it flat and unrolled, preventing it from sinking to the bottom or curling and wrinkling. Vertical immersion allows the film to gradually contact and peel off from one end of the water. The "lubrication" and "support" of the water surface ensures a smooth transfer to the surface, avoiding the stress caused by sudden full immersion that could lead to tearing or wrinkling. This step allows the uniform film to be transferred intact, flat, and stress-free from the rigid substrate (glass slide) to the flexible medium (water surface), preparing it for the next step of retrieval onto the copper mesh.

[0010] During the transfer and drying steps, the sealing film has moderate hydrophobicity and flexibility. Starting from one end and adhering it to the water surface, it can be applied like a screen protector to a mobile phone, from one end to the other, smoothly and layer by layer lifting the floating film (along with the copper mesh on it) and firmly adhering it to the sealing film.

[0011] Preferably, the centrifugation device described in step S3 is modified from an ordinary centrifuge. The specific modification method is as follows: remove the centrifuge rotor, directly fix the TCT cell preparation clamp used to hold the glass slides onto the rotor connector, and add an anti-splash structure to the tail of the preparation clamp.

[0012] Preferably, the anti-splash structure is a small plastic sheet attached to the tail of the slide clamp. This structure acts as a "baffle" during high-speed centrifugation, preventing the slides from being thrown out under centrifugal force, which could potentially cause centrifuge malfunction.

[0013] Preferably, the small plastic piece is the right size for the tail of the slide holder, and its height is consistent with the height of the opposite side after it is attached. The weight should not be too heavy to avoid imbalance at both ends.

[0014] Preferably, in step S2, the pretreatment of the copper mesh specifically involves: placing the copper mesh in 70% alcohol and subjecting it to ultrasonic vibration for 10-15 minutes, then removing it and air-drying it; the pretreatment of the glass slide specifically involves: rinsing it with ultrapure water and then immersing it in 70% alcohol for later use. A moderately clean, hydrophilic surface allows the film to adhere tightly during centrifugation, preventing premature slippage. However, this adhesion should not be too strong, so that it can be easily peeled off in subsequent steps (through scratching and water immersion). 70% alcohol effectively removes oil and dirt without leaving stubborn water stains on the surface.

[0015] Preferably, in step S3, after centrifugation and before underwater peeling, tweezers or a blade are used to scratch the edges of the film on the slide surface to facilitate complete peeling. Before peeling, scratching the edges of the film with tweezers essentially pre-sets a "starting point" and boundary for the peeling process. This guides the peeling path of the film, ensuring it detaches neatly from the slide from the scratches in a predetermined manner, avoiding uncontrolled tearing during the peeling process. This significantly improves the success rate and integrity of the underwater peeling step, making it easier to obtain large-area films with neat edges.

[0016] Preferably, in step S5, the drying process is to use a lamp or low-temperature baking for rapid drying, or to place the food in a petri dish for natural air drying.

[0017] Finally, the present invention provides an ultrathin sliced ​​copper mesh support film for transmission electron microscopy prepared by the above method.

[0018] The beneficial effects of this invention are as follows: High film quality and uniformity: The centrifugal method is used for film formation. Centrifugal force forces the solution to spread evenly, effectively overcoming the problem of thicker edges and thinner centers caused by surface tension or gravity in traditional methods. This results in films with sufficiently uniform thickness that are also resistant to bombardment and do not easily break under normal electron microscope bombardment. High repeatability and success rate: By precisely controlling parameters such as the type of organic solvent, solution concentration, volume, centrifugation speed, and time, the preparation process is standardized, greatly reducing the uncertainty of human operation and significantly improving intra-batch and inter-batch repeatability.

[0019] Simple and quick to operate, highly efficient: the centrifugal film formation process takes only a few seconds, far exceeding the waiting time for the solvent to evaporate naturally, and the sealing film can be retrieved easily, smoothly and quickly, thus improving the preparation efficiency.

[0020] Low cost and easy to promote: The centrifuge device provided can be easily modified from a common centrifuge commonly found in laboratories. There is no need to purchase expensive special equipment, the modification cost is extremely low, and it is easy to promote and use in various laboratories.

[0021] The copper mesh support film prepared using the method described in this application can meet the needs of various types of support meshes (50-200 mesh, single-pitch, wide-pitch, etc.), satisfying the requirements of different samples and different electron microscopy techniques (such as ultrathin sectioning and negative staining). The thickness of the film can be adjusted, and it can be made on demand. Thicker films are more resistant to bombardment and have lower costs. Attached Figure Description

[0022] Figure 1 Centrifuge device fitted with TCT cell slide clamp; Figure 2 The centrifuge device with a slide and anti-splash structure added; Figure 3 This is an electron microscope image of the copper mesh support film in Example 1; Figure 4 This is an electron microscope image of the copper mesh support film in Example 2; Figure 5 The image shown is an electron microscope image of the copper mesh support film in Example 3. The black corner in the lower right corner is a problem that occurs with all electron microscopes used at 2000x magnification and is unrelated to the film. Figure 6 Electron micrograph of the copper mesh support film in Comparative Example 1; Figure 7 Electron micrograph of the copper mesh support film in Comparative Example 2; Figure 8 Electron micrograph of the copper mesh support film in Comparative Example 3; Figure 9 Electron micrograph of the copper mesh support film in Comparative Example 4; Figure 10 Electron micrograph of commercially available Fanghua film copper mesh support membrane at 2500x magnification; Figure 11 Electron micrographs of commercially available Fanghua film copper mesh support film, under the same temperature and humidity conditions as other comparative examples and embodiments, showing that the film broke in less than one minute when observed at a magnification of 2500x. Figure 12 For comparison example 5, the sample is located within the red box in the image; Figure 13 The image is a comparison of six photos. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0024] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0026] The following are some of the sources of raw materials and reagents in the examples: Dichloroethane: Sigma-Aldrich, 100ml; analytical grade; Pioloform ® Powder: 10g, Manufacturer: TED PELLA, INC.; Sealing film: Easy-seal, size 10cm*38m; Alcohol: Anhydrous ethanol, analytical grade, Sun brand, 2500ml; Copper mesh: All purchased from Zhongjing Keyi; 150 mesh with film, bare mesh is 2*1 single-pitch copper mesh. Glass slides: Wheel Brand, 72 slides per pack; TCT cell preparation clip: purchased from Jiayiyuan Medical Devices Store (Taobao).

[0027] Example 1: Preparation of an ultrathin copper mesh support film for transmission electron microscopy S1. Solution preparation: Accurately weigh 0.15g Pioloform ® Powder was dissolved in 15 ml of dichloroethane solution and magnetically stirred until completely dissolved to prepare a 1% (w / v) film-forming solution. It was then wrapped in aluminum foil and stored in the dark.

[0028] S2. Pretreatment of glass slides and copper mesh: Glass slide treatment: Take a new glass slide, rinse the surface with ultrapure water, and then immerse it in a beaker containing 70% alcohol. Before use, remove it and wipe it dry with lint-free paper. Copper mesh treatment: Place several copper meshes in a small weighing bottle, pour in 70% alcohol until the copper meshes are completely submerged, and place the weighing bottle in an ultrasonic cleaner for ultrasonic vibration for 15 minutes. Afterward, remove it with tweezers and place it on filter paper to air dry for later use.

[0029] S3. Centrifugation for Film Formation: Take a pretreated glass slide and mount it onto the TCT cell preparation clip of the modified centrifuge apparatus. Accurately pipette 220 μl of 1% film-forming solution and add it to the center of the glass slide. Quickly close the centrifuge lid, start the centrifuge, set the speed to 7000 rpm, and the centrifugation time to 5 seconds. After centrifugation, remove the glass slide; a clear, uniform film without interference fringes will be visible on its surface. Carefully use a needle to scribble along the edge of the glass slide to separate the film from the edge of the slide.

[0030] S4. Underwater peeling: Fill a petri dish with ultrapure water and heat it to 56°C in a water bath. Slowly immerse the etched slide in the water at a near-vertical angle. The film on the surface will peel off completely from the slide under the action of surface tension and temperature, and float flat on the water surface (takes 36 seconds).

[0031] S5. Transfer and Drying: Using tweezers, gently place the pre-treated copper meshes one by one, spaced apart, on the floating film; cut a small piece of flat sealing film, and from one end of the film, horizontally close to the water surface, carefully lift the film with the copper meshes using the surface tension of the water, and attach it to the sealing film; place the sealing film (with the film side facing up) in a petri dish, cover it, and let it air dry naturally. After it is completely dry, the finished ultrathin slice copper mesh support film can be obtained.

[0032] The centrifugation device in this embodiment is a modified Dalong D1008 centrifuge: First, the original rotor of the centrifuge is removed; then, the base of the TCT cell slide clamp, commonly used in pathology, is directly installed and fixed onto the main shaft rotor connector of the centrifuge. A small plastic sheet is glued to the tail of the TCT cell slide clamp as a splash-proof structure. The device with the TCT cell slide clamp installed is shown in [link to documentation]. Figure 1 The device with the slide and anti-splash structure is shown in [reference]. Figure 2 .

[0033] Example 2 The difference between this embodiment and Embodiment 1 is that the concentration of the film-forming solution is 2%, the ultrasonic time in the glass slide and copper mesh pretreatment step is 10 min, the amount of film-forming solution in the centrifugal film-forming step is 250 μl, the centrifugation time is 6 s, and the water temperature in the underwater peeling step is 60℃ (takes 35 s). Example 3 The difference between this embodiment and Embodiment 1 is that the film-forming solution is 1.5%, the amount of film-forming solution in the centrifugal film-forming step is 200 μl, the centrifugation time is 4 s, and the water temperature in the underwater stripping step is 55℃ (takes 37 s).

[0034] Comparative Example 1 The difference from Example 1 is that the concentration of the film-forming solution is 3%.

[0035] Comparative Example 2 The difference from Example 1 is that the centrifugation time is 3 seconds.

[0036] Comparative Example 3 The difference from Example 1 is that the organic solvent used in the film-forming solution is chloroform.

[0037] Comparative Example 4 The difference from Example 1 is that in the film transfer and drying steps, filter paper is used instead of sealing film for film scooping.

[0038] Comparative Example 5 The difference from Example 1 is that the water temperature in the underwater stripping step was room temperature (23°C), which took 3 minutes and 41 seconds, and the membrane was wrinkled and adhered. See the photograph for details. Figure 12 (The sample is located within the red box in the figure).

[0039] Comparative Example 6 The difference from Example 1 is that the water temperature in the underwater stripping step is 40°C, and the time taken at this temperature is 1 minute and 24 seconds. The membrane is better than at room temperature, but it is still easy to get wet and wrinkle at the tail end.

[0040] Comparative Example 7 The difference from Example 1 is that the water temperature in the underwater peeling step is 70°C. When the water temperature reaches this temperature, the sealing film used for film retrieval will shrink due to the heat, as shown in the photo. Figure 13 .

[0041] Ultrathin sliced ​​copper mesh support films prepared in Examples 1-3 and Comparative Examples 1-4, as well as commercially available aromatic copper mesh films, were used. Their quality was observed using a transmission electron microscope (TEM). The TEM was a 120kV JEM-1400Flash from Nippon Electron, operated at room temperature. The copper mesh with the film was directly placed on the sample holder for imaging. Both the examples and comparative examples used magnifications of 2000x and 2500x. 2000x allows observation of a larger area of ​​the film, while 2500x provides clearer visualization of minute imperfections. In practical applications, TEM observation sometimes requires low magnification for overall observation and sometimes high magnification for detail observation. To demonstrate the universality of this patent application, this experiment used two magnifications.

[0042] Electron micrographs of the copper mesh support films prepared in Examples 1-3 are shown below. Figure 3-5 The results showed that the copper mesh support film prepared by the method of the present invention was uniform in thickness and moderate in thickness. No unevenness or other minor defects were observed after the electron microscope was magnified to 2500 times. It was also resistant to bombardment and would not break even after prolonged bombardment by the electron microscope.

[0043] The electron microscope image of the copper mesh support film in Comparative Example 1 is shown below. Figure 6Under a 2000x electron microscope, the film showed obvious granular texture and was relatively thick. This indicates that the concentration of the film-forming solution has a significant impact on the quality of the film. The applicant further experimented with other concentrations and found that above 2%, the film was too thick and the powder had granular precipitation; below 1%, the film was too clear and difficult to form. At a concentration of 0.5%, film formation was impossible.

[0044] The electron microscope image of the copper mesh support film in Comparative Example 2 is shown below. Figure 7 Under a 2500x electron microscope, uneven thickness can be observed, with some areas dark and some bright, and streaks are visible. This indicates that centrifugation time has a significant impact on the uniformity of film formation. At a speed of 7000 pm, a speed of less than 4 seconds results in uneven film formation.

[0045] The electron microscope image of the copper mesh support film in Comparative Example 3 is shown below. Figure 8 Under a 2500x electron microscope, the membrane appears to be relatively thick overall, with significant unevenness in thickness, and contains obvious pores and impurities. The lower right corner appears to contain membrane debris. Example 1, compared to Comparative Example 3, demonstrates that using dichloromethane as the membrane solution solvent can significantly improve membrane quality.

[0046] Comparative Example 4: Electron micrograph of the copper mesh support film (see Figure 4). Figure 9 Under a 2500x electron microscope, needle-like impurities were visible in the center, most likely fibers from the filter paper. Water stains were also visible in the upper right corner and slightly to the right of center. Although the membrane thickness was uniform, the use of filter paper to retrieve the membrane resulted in impurities or water stains, which would affect observation. This indicates that using a sealing film to retrieve the membrane can prevent membrane contamination and water stains. On the other hand, retrieving the membrane with filter paper not only contaminates the membrane, but the filter paper itself also becomes wet. While the surface dries easily, the filter paper under the copper mesh remains wet. If it is placed in the electron microscope before it is completely dry, it is prone to discharge, affecting observation.

[0047] Commercially available Fanghua film copper mesh, when observed under an electron microscope at 2500x magnification (see...), Figure 10 The film showed obvious unevenness, and within one minute of observation, it was ruptured due to electron beam bombardment (see...). Figure 11 Furthermore, there were unidentified impurities, while no cracking was observed in Examples 1-3.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an ultrathin copper mesh support film for transmission electron microscopy, characterized in that, Includes the following steps: S1. Solution preparation: Prepare the film-forming material Pioloform. ® Powder is dissolved in dichloroethane to prepare a film-forming solution with a mass-volume concentration of 1% to 2%, and stored away from light. S2. Pretreatment of glass slides and copper mesh: Clean and dry the glass slides and copper mesh for later use; S3. Centrifugation to form a film: Fix the pretreated glass slide in a centrifuge, add 200-250 μl of the film-forming solution to its surface, and then start the centrifuge at 7000 rpm for 4-6 seconds to form a uniform thin film on the surface of the glass slide. S4. Underwater peeling: The glass slide covered with the film in step S3 is vertically immersed in ultrapure water at 55-60°C. The film is peeled off from the glass slide and floats flat on the water surface by utilizing the surface tension and temperature of the water. S5. Transfer and drying: Place the pretreated copper mesh on the floating film at intervals, then carefully attach it from one end of the water film with sealing film and lift it up as a whole. Finally, perform drying treatment to obtain a copper mesh covered with a support film.

2. The preparation method according to claim 1, characterized in that, The centrifugation device mentioned in step S3 is modified from an ordinary centrifuge. The specific modification method is as follows: remove the centrifuge rotor, fix the TCT cell preparation clamp used to hold the glass slide directly to the rotor connector, and add an anti-splash structure to the tail of the preparation clamp.

3. The preparation method according to claim 2, characterized in that, The anti-splash structure is a small plastic sheet that is attached to the tail of the film clamp.

4. The preparation method according to claim 1, characterized in that, In step S2, the pretreatment of the copper mesh is specifically as follows: the copper mesh is placed in 70% alcohol and subjected to ultrasonic vibration for 10-15 minutes, then removed and dried; the pretreatment of the glass slide is specifically as follows: after rinsing with ultrapure water, it is soaked in 70% alcohol for later use.

5. The preparation method according to claim 1, characterized in that, In step S3, after centrifugation and before underwater peeling, a tool is used to scratch the periphery of the film on the surface of the glass slide.

6. The preparation method according to claim 1, characterized in that, In step S5, the drying process is to use a lamp or low-temperature baking for rapid drying, or to place the food in a petri dish for natural air drying.

7. The ultrathin copper mesh support film for transmission electron microscopy prepared by the method according to any one of claims 1-6.

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