A method for preparing an in-situ high-throughput transmission electron microscope sample

Abstract

The application belongs to the field of electron microscopy, and discloses a preparation method of in-situ high-throughput transmission electron microscope samples, which comprises the following steps: (1) making a microsphere probe; (2) using an automatic three-axis module to drive the probe to realize liquid transfer of the sample to be measured, and arranging the sample droplets on the in-situ TEM chip in order, thereby completing the preparation of the in-situ high-throughput transmission electron microscope samples. The innovation of the application lies in that the preparation principle is novel, the degree of automation of the equipment is high, and the structure design is completely original. The probe can carry micron-level droplets, and the three-axis module with precise movement can arrange the droplets on the in-situ TEM chip in order. The application uses an intelligent cross-scale liquid transfer platform to transfer a large number of raw materials with different components to the in-situ TEM chip, thereby meeting the requirement of high-throughput sample observation in in-situ experiments, and solving the problem of low observation efficiency in in-situ experiments.

Description

Technical Field

[0001] The present invention relates to the technical field of electron microscopes, and in particular to a method for preparing an in-situ high-throughput transmission electron microscope sample. Background Art

[0002] In the late 19th century, scientist Erich Abbe proposed the Abbe imaging principle, which revealed that the resolution limit of a microscope is related to factors such as the refractive index and the wavelength of the light source. In the early 20th century, the discovery of cathode rays and the introduction of the concept of matter waves by de Broglie led scientists to realize that electrons, as a light source, have shorter wavelengths and higher energies, resulting in higher spatial resolution. This led to the development of the transmission electron microscope (TEM). As a crucial tool for observing the microstructure of matter, TEM has significant applications in materials science, biomedicine, and other fields. However, the electron beam's ability to penetrate a sample is limited, generally considered to be less than 20 microns. Therefore, sample preparation for TEMs is crucial for obtaining high-quality micrographs.

[0003] Currently, most TEM sample preparation methods, both domestically and internationally, use a single grid to hold only one sample. Improving sample throughput often involves attaching multiple sample holders to the sample holder. However, this type of holder is limited by the TEM pole piece, making it difficult to significantly increase throughput. Furthermore, it is very expensive to manufacture. Therefore, developing a low-cost, high-throughput TEM sample preparation method is of great significance.

[0004] There are many types of TEM grids, such as carbon grids, microgrids, silicon chips, etc. The powder sample preparation process generally involves dispersing the sample in a volatile dispersant to form a uniform dispersion, and then taking a small amount of the dispersion and dropping it on the grid. After the dispersant evaporates, the powder sample remains on the surface of the grid. If more different samples can be dropped onto the same grid, the observation flux of the same grid will be effectively improved. With the deepening of research in the microscopic field, in-situ experimental observations in transmission electron microscopes are becoming increasingly important. Taking in-situ heating chips as an example, the chips produced by mainstream TEM chip companies such as protochip and dense are mostly silicon chips with silicon nitride window panes. The window pane aperture is small, and generally only the change process of one sample can be observed in one experiment, which is inefficient. A sample preparation method that can improve the observation flux is needed. Summary of the Invention

[0005] To address the aforementioned issues with the existing technology, the present invention provides a method for preparing in situ high-throughput transmission electron microscopy samples. This method employs a probe with a polystyrene sphere at its tip for micrometer-scale liquid transfer. The hydrophilic nature of the polystyrene sphere enables it to carry very small droplets. A precisely controlled automated three-axis modular platform, carrying the specially designed probe, enables transfer of TEM samples from a raw liquid pool to an in-situ TEM chip, achieving uniform and orderly arrangement.

[0006] The technical solutions of the present invention are as follows:

[0007] A first object of the present invention is to provide a method for preparing an in-situ high-throughput transmission electron microscopy sample, the method comprising the following steps:

[0008] (1) Preparation of microsphere probes;

[0009] (2) The automated three-axis module is used to drive the probe to transfer the sample liquid to be tested, and the sample droplets to be tested are neatly arranged on the in-situ TEM chip to complete the preparation of in-situ high-throughput transmission electron microscopy samples.

[0010] In one embodiment of the present invention, in step (1), the microsphere probe is prepared by bonding polystyrene beads with AB glue; the average particle size of the polystyrene beads is 15 μm.

[0011] In one embodiment of the present invention, in step (1), the method for preparing the microsphere probe is:

[0012] ①Clamp the probe onto the micrometer probe stage and move it into the field of view of the optical microscope;

[0013] ②Mix epoxy resin AB glue to obtain a uniform mixed liquid A;

[0014] ③ Take a glass slide and use a thin glass rod to draw mixed solution A on one end of the slide;

[0015] ④ Take the polystyrene ball powder and sprinkle it on the other end of the slide;

[0016] ⑤ Use the probe station to move the probe, gently dip the mixed solution A on the slide, and immediately move it above the polystyrene ball to stick one on the needle tip;

[0017] ⑥ The mixed solution A was allowed to stand for 24 hours until it was completely solidified, thereby obtaining the microsphere probe.

[0018] In one embodiment of the present invention, in step (2), the automated three-axis module includes an X-axis for left-right linear motion, a Y-axis for front-back linear motion, and a Z-axis for up-down linear motion; the microsphere probe is locked on the Z-axis.

[0019] In one embodiment of the present invention, in step (2), the specific method is:

[0020] ① Place the liquids A, B, C, ... to be transferred into a 96-well plate, and place ethanol in the last well for probe cleaning;

[0021] ② Adjust the height so that the microsphere probe can just touch the liquid in the 96-well plate;

[0022] ③ Place the hydrophilized in-situ TEM chip next to the 96-well plate on the sample stage;

[0023] ④ Move the Z axis so that the microsphere probe contacts liquid A, then move it above the in-situ TEM chip. Control the Z axis downward so that the droplet at the lower end of the microsphere probe just contacts the surface of the in-situ TEM chip. Then lift the Z axis and move it to the ethanol well to clean the probe up and down, and obtain droplet A on the in-situ TEM chip.

[0024] ⑤ Move the Z axis so that the microsphere probe contacts liquid B, then move it to a position 5 μm above the in-situ TEM chip and droplet A. Control the Z axis downward so that the droplet at the lower end of the probe just contacts the surface of the in-situ TEM chip. Then lift the Z axis and move it to the ethanol well to clean the probe up and down. Droplets A and B are obtained on the in-situ TEM chip.

[0025] ⑥ Repeat steps ④ and ⑤ to obtain droplets A, B, C, ... neatly arranged on the in-situ TEM chip.

[0026] In one embodiment of the present invention, in step (2) ①, a target product distribution map is compiled according to the well positions of a 96-well plate, and a control file for an automatic pipetting platform is generated using a python script; the automatic pipetting platform configuration file is simulated in a simulator to check whether the parameters are correct; the control file is imported into the automatic pipetting platform and run to obtain a raw material pool in the 96-well plate.

[0027] In one embodiment of the present invention, in step (2) ③, the method for in-situ hydrophilization treatment of the TEM chip is: using a plasma cleaner at 150W, 50% air, and 50% argon for 3 minutes.

[0028] In one embodiment of the present invention, in step (2) ④, the microsphere probe is locked on the Z axis of the automated three-axis module, the highest point is set as 0, the height is adjusted so that the microsphere probe can just touch the liquid in the first well of the 96-well plate, recorded as position (X1, Y1, Z1), the height is adjusted so that the probe can just touch the in-situ TEM chip, recorded as position (x2, y2, z2), and the height is adjusted so that the probe can be immersed in the ethanol pool, recorded as position (a, b, c);

[0029] A Python script is used to generate a control file based on the three sets of positions. The transfer of one liquid is divided into six steps in a script loop:

[0030] a. Starting from point 0, the probe is immersed in the ethanol pool;

[0031] b. Remove the sample from the ethanol pool and move it to well A1 of a 96-well plate;

[0032] c. The probe lowers its tip into the liquid in well A1;

[0033] d. Lift the probe and move it above the in-situ TEM chip;

[0034] e. Lower the needle so that the droplet remains on the in-situ TEM chip;

[0035] f. Lift the probe and place it in the ethanol bath for cleaning.

[0036] After the first set of runs, a loop command was used to enable the probe to pick up the raw materials from the 96-well plate at a specific step size and place the nanodroplets at the designed intervals on the in situ TEM chip.

[0037] The beneficial technical effects of the present invention are:

[0038] This invention independently constructs an automated pipetting platform linked to an automated three-axis module to form a high-throughput, cross-scale liquid transfer device. The automated pipetting platform is used to synthesize precursor solutions at high throughput, forming a raw material pool in a 96-well plate. A probe driven by a module with high positioning precision extracts micron-sized droplets from the raw material liquid pool and transfers them to an in-situ TEM chip. Due to the module's high positioning precision and the relatively small size of liquid that can be carried by the probe tip, a single in-situ TEM chip can accommodate nanodroplets of various components. This enables automated, high-throughput TEM sample preparation without human intervention through efficient, simple, and precise mechanical motion.

[0039] The high throughput achieved by this invention is due to a specially designed probe and a precise three-axis module. Transferring liquid through the probe tip solves the problem of traditional pipettes producing large, difficult-to-control droplets. Reducing the droplet size allows the module to precisely control and position it on the in-situ TEM chip.

[0040] The present invention uses epoxy resin AB glue as an adhesive and 15-micron polystyrene beads as a liquid dipping medium, and cooperates with an automated three-axis module to achieve high-throughput sample preparation on an in-situ TEM chip.

[0041] The preparation principle of the present invention is novel, and the equipment has a high degree of automation. Micron-sized droplets can be carried by a probe and neatly arranged on an in-situ TEM chip through a precisely moving three-axis module. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Optical microscope photograph of the probe prepared by the present invention;

[0043] Figure 2 These are photos of the equipment of the present invention, schematic diagram of the experimental process, and photos of samples. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0045] The epoxy resin AB glue, polystyrene beads, in-situ TEM chip and probe used in the application are commercially available analytical pure products, and the deionized water is laboratory self-made; the glass apparatus and equipment used are commonly used apparatus and equipment in the laboratory.

[0046] Figure 1 Optical microscope photos of the probe prepared in the application; wherein a is a liquid transfer probe used, which is a commonly purchased commercial probe; b is a photo of an in-situ TEM chip and an optical micrograph; c is a simulation diagram of an in-situ TEM chip and a real photo of a hole in the in-situ TEM chip under an electron microscope.

[0047] Figure 2 a is a device photo containing a three-axis module on the left and a high-throughput liquid transfer platform on the right; b is a schematic diagram of a cross-scale liquid transfer technology path, i.e., a 6-step cycle schematic diagram; c is an optical micrograph of liquid on an in-situ TEM chip after successful transfer.

[0048] The preparation method of the microsphere probe of the application is as follows:

[0049] ①The probe is clamped on the micrometer probe table and moved into the field of view of the optical microscope;

[0050] ②The epoxy resin AB glue is mixed to obtain a uniform mixed liquid A;

[0051] ③A glass slide is taken, and the mixed liquid A is drawn on one end of the glass slide using a fine glass rod;

[0052] ④Polystyrene bead powder is taken and sprinkled on the other end of the glass slide;

[0053] ⑤The probe table is used to move the probe, and the mixed liquid A is gently dipped on the glass slide and immediately moved above the polystyrene beads to stick one on the needle tip;

[0054] ⑥After 24 hours of static state, the mixed liquid A is completely solidified to obtain the microsphere probe.

[0055] Example 1:

[0056] A preparation method of an in-situ high-throughput transmission electron microscope sample, the preparation method comprising the following steps:

[0057] (1) Build a cross-scale high-throughput liquid transfer device.

[0058] (2) According to the hole position of the 96-well plate, write a target product distribution map, and use a python script to generate a control file of the automatic pipetting platform.

[0059] (3) Simulate the configuration file of the automatic pipetting platform in the simulator, and check whether the parameters are correct.

[0060] (4) Import the control file into the automatic pipetting platform and run it to obtain the raw material pool in the 96-well plate.

[0061] (5) Place an ethanol liquid pool for probe cleaning.

[0062] (6) The in-situ TEM chip was hydrophilized using a plasma cleaner (150 W, 50% air, 50% argon, 3 minutes) and then placed next to the 96-well plate on the sample stage.

[0063] (7) Lock the microsphere probe on the Z axis of the module, set the highest point to 0, adjust the height so that the probe can just touch the liquid in the first well of the 96-well plate, record it as position (X1, Y1, Z1), adjust the height so that the probe can just touch the in-situ TEM chip, record it as position (x2, y2, z2), adjust the height so that the probe can be immersed in the ethanol pool, record it as position (a, b, c).

[0064] (8) A control file is generated based on the three sets of positions using a Python script. The transfer of one liquid, i.e., one script cycle, is divided into six steps:

[0065] a. Starting from point 0, the probe is immersed in the ethanol pool;

[0066] b. Remove the sample from the ethanol pool and move it to well A1 of a 96-well plate;

[0067] c. The probe lowers its tip into the liquid in well A1;

[0068] d. Lift the probe and move it above the in-situ TEM chip;

[0069] e. Lower the needle so that the droplet remains on the surface of the in-situ TEM chip;

[0070] f. Lift the probe and place it in the ethanol bath for cleaning.

[0071] After the first set of runs, a loop command was used to enable the probe to pick up the raw materials from the 96-well plate at a specific step size and place the nanodroplets at the designed intervals on the in situ TEM chip.

[0072] (9) Observe the preparation process using a light microscope.

[0073] The droplets with a diameter of 2 microns obtained in this embodiment have the most reasonable arrangement and the most precise mechanical control among all the prepared materials.

[0074] Example 2:

[0075] A method for preparing an in-situ high-throughput transmission electron microscope sample, comprising the following steps:

[0076] (1) Build a cross-scale high-throughput liquid transfer device.

[0077] (2) Prepare the target product distribution map according to the well positions of the 96-well plate, and use the Python script to generate the control file of the automatic pipetting platform.

[0078] (3) Simulate the automatic pipetting platform configuration file in the simulator to check whether the parameters are correct.

[0079] (4) Import the control file into the automatic pipetting platform and run it to obtain the raw material pool in the 96-well plate.

[0080] (5) Place an ethanol liquid pool for probe cleaning.

[0081] (6) The in-situ TEM chip was hydrophilized using a plasma cleaner (150 W, 50% air, 50% argon, 3 minutes) and then placed next to the 96-well plate on the sample stage.

[0082] (7) Lock the microsphere probe on the Z axis of the module, set the highest point to 0, adjust the height so that the probe can just touch the liquid in the first well of the 96-well plate, record it as position (X1, Y1, Z1), adjust the height so that the probe can just touch the in-situ TEM chip, record it as position (x2, y2, z2), adjust the height so that the probe can be immersed in the ethanol pool, record it as position (a, b, c).

[0083] (8) Using the computer visualization interface software in “jogging mode”, manually control the cross-scale liquid transfer. Each cycle consists of six steps:

[0084] a. Starting from point 0, the probe is immersed in the ethanol pool;

[0085] b. Remove the sample from the ethanol pool and move it to well A1 of a 96-well plate;

[0086] c. The probe lowers its tip into the liquid in well A1;

[0087] d. Lift the probe and move it above the in-situ TEM chip;

[0088] e. Lower the needle so that the droplet remains on the surface of the in-situ TEM chip;

[0089] f. Lift the probe and place it in the ethanol bath for cleaning.

[0090] After the first set of runs, a loop command was used to enable the probe to pick up the raw materials from the 96-well plate at a specific step size and place the nanodroplets at the designed intervals on the in situ TEM chip.

[0091] (9) Observe the preparation process using a light microscope.

[0092] The droplets with a diameter of 1.5 microns obtained in this embodiment are the smallest droplets that can be obtained from all the prepared materials and have the lowest safety risks.

[0093] Comparative Example 1

[0094] Compared with Example 1, the only difference is that the high-throughput liquid transfer platform is replaced with an ordinary pipette. The difference from the results of Example 1 is that the liquid preparation efficiency is low, the labor cost is extremely high, and the concentration fluctuation is large.

[0095] Comparative Example 2

[0096] Compared with Example 1, the only difference is that the hydrophilization treatment of the in-situ TEM chip using a plasma cleaner is omitted. The difference from the results of Example 1 is that the wetting angle between the liquid and the in-situ TEM chip is too large, and the droplet morphology is unstable during the liquid transfer process, making it difficult to align the droplets on the in-situ TEM chip.

[0097] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing in-situ high-throughput transmission electron microscopy samples, characterized in that: The preparation method comprises the following steps: (1) Preparation of microsphere probes; (2) Using an automated three-axis module to drive the probe to transfer the sample liquid, the sample droplets are neatly arranged on the in-situ TEM chip, and the in-situ high-throughput transmission electron microscopy sample preparation is completed; In step (2), the automated three-axis module includes an X-axis for left-right linear motion, a Y-axis for front-back linear motion, and a Z-axis for up-down linear motion; the microsphere probe is locked on the Z-axis; In step (2), the specific method is: Place the liquids A, B, C, ... to be transferred into a 96-well plate, and place ethanol in the last well for probe cleaning; Adjust the height so that the microsphere probe just touches the liquid in the 96-well plate; The hydrophilized in-situ TEM chip was placed next to the 96-well plate on the sample stage; Move the Z axis so that the microsphere probe contacts liquid A, then move it above the in-situ TEM chip. Control the Z axis downward so that the droplet at the lower end of the microsphere probe just contacts the surface of the in-situ TEM chip. Then lift the Z axis and move it into the ethanol well to clean the probe up and down, and obtain droplet A on the in-situ TEM chip. Move the Z axis so that the microsphere probe contacts liquid B, then move it to a position 5 μm above the in-situ TEM chip and droplet A. Control the Z axis downward so that the droplet at the lower end of the probe just contacts the surface of the in-situ TEM chip. Then, lift the Z axis and move it to the ethanol well to clean the probe up and down. Droplets A and B are then obtained on the in-situ TEM chip. Repeat steps 、 Then, droplets A, B, C,… are neatly arranged on the in-situ TEM chip.

2. The preparation method according to claim 1, characterized in that In step (1), the microsphere probe is prepared by bonding polystyrene beads with AB glue; the average particle size of the polystyrene beads is 15 μm.

3. The preparation method according to claim 1, characterized in that In step (1), the method for preparing the microsphere probe is: Clamp the probe onto the micrometer probe stage and move it into the optical microscope field of view; Mix epoxy resin AB glue to obtain a uniform mixed liquid A; Take a glass slide and use a thin glass rod to draw mixed solution A on one end of the slide; Take the polystyrene ball powder and sprinkle it on the other end of the slide; Use the probe station to move the probe, gently dip the mixture A on the slide, and immediately move it over the polystyrene ball to stick one on the needle tip; The mixed solution A was allowed to stand for 24 hours until it was completely solidified, thereby obtaining the microsphere probe.

4. The preparation method according to claim 1, wherein Step (2) In situ hydrophilization treatment of TEM chips was performed by using a plasma cleaner at 150 W, 50% air, and 50% argon for 3 minutes.

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