Preparation method of in-situ high-flux transmission electron microscope sample

Abstract

The invention belongs to the technical field of electron microscopy, and discloses a preparation method of an in-situ high-flux transmission electron microscope sample, and the preparation method comprises the following steps: (1) preparing a microsphere probe; and (2) driving the probe by using the automatic three-axis module to realize liquid transfer of the sample to be detected, and neatly arranging liquid drops of the sample to be detected on the in-situ TEM chip to complete preparation of the in-situ high-flux transmission electron microscope sample. The preparation method has the innovation points that the preparation principle is novel, the equipment automation degree is high, and the structural design is completely original. Micron-sized liquid drops can be carried by the probe and are neatly arranged on the in-situ TEM chip through the three-axis module which moves accurately. The intelligent cross-scale liquid transfer platform is utilized to transfer a large number of raw materials with different components to the in-situ TEM chip, so that the requirement of high-throughput sample observation in an in-situ experiment is met, and the problem of low observation efficiency of the in-situ experiment is solved.

Description

Technical Field

[0001] The 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] At the end of the 19th century, scientist E. Abbe proposed the Abbe imaging principle and found that the resolution limit of the microscope is related to factors such as the refractive index and the wavelength of the light source. In the early 20th century, cathode rays were discovered and de Broglie proposed the concept of matter waves. Scientists realized that electrons as light sources have shorter wavelengths and higher energy, and using them as light sources will achieve higher spatial resolution. Therefore, the transmission electron microscope (TEM) came into being. As an important means of observing the microscopic structure of matter, TEM has important application value in materials science, biomedicine and other fields. However, since the thickness of the sample that the electron beam can penetrate is limited, generally believed to be less than 20 microns, TEM sample preparation has become an important part of obtaining high-quality microscopic photos of matter.

[0003] At present, most of the TEM sample preparation at home and abroad is a single grid holding only one sample, and the way to increase the sample observation throughput is to install multiple sample holders on the sample rod. However, due to the limitation of the TEM pole shoe, it is difficult to increase the throughput of this sample rod, and the cost is very high. It is of great significance to explore a low-cost and high-throughput TEM sample preparation method.

[0004] There are many types of TEM grids, such as carbon grids, microgrids, silicon chips, etc. In the powder sample preparation process, the sample is generally dispersed in a volatile dispersant to form a uniform dispersion, and then a small amount of dispersion is dripped on the grid. After the dispersant evaporates, the powder sample remains on the surface of the grid. If more different samples can be dripped onto the same grid, the observation flux of the same grid is 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. Generally, only one sample change process can be observed in one experiment, and the efficiency is low. A sample preparation method that can improve the observation flux is needed. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for preparing an in-situ high-throughput transmission electron microscope sample. The present invention designs a probe with a polystyrene ball at the end for micron-level liquid transfer. The hydrophilic properties of the polystyrene ball enable it to carry very small droplets. The TEM sample is transferred from the raw liquid pool to the in-situ TEM chip and arranged neatly and evenly by using an automated three-axis module platform that can be precisely controlled to carry a special probe.

[0006] The technical solution of the present invention is as follows:

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

[0008] (1) Making 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 on the micrometer probe stage and move it into the field of view of the optical microscope;

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

[0014] ③Take 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 Allow the mixed solution A to stand for 24 hours until it is completely solidified to obtain 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 cleaning the probe;

[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 to the top of 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 hole 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 the top of the in-situ TEM chip with a horizontal interval of 5 microns from the 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 hole to clean the probe up and down, and 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 control file is generated using a python script based on the three sets of positions above. The transfer of one liquid, i.e., one script cycle, is divided into six steps:

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

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

[0032] c. The probe tip descends into the liquid in hole 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 TEM chip in situ;

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

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

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

[0038] The present invention independently builds an automatic pipetting platform and an automatic three-axis module to form a high-throughput cross-scale liquid transfer device. The automatic pipetting platform is used to synthesize the precursor solution with high throughput to form a raw material pool in a 96-well plate; the probe is driven by a module with high positioning accuracy to extract micron-sized droplets from the raw material liquid pool and transfer them to an in-situ TEM chip. Since the positioning accuracy of the module is high and the size of the liquid that can be carried by the probe tip is small, nanodroplets of various components can be arranged on an in-situ TEM chip. The preparation of automated high-throughput TEM samples without human intervention is achieved through efficient, simple and precise mechanical movement.

[0039] The reason why the present invention can achieve a higher throughput is due to the special probe and the three-axis module with precise movement. The problem of the traditional pipette forming droplets with large size and difficult to control size is solved by transferring liquid through the probe tip. The droplet size is reduced so that it can be precisely controlled by the module and arranged 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. The micron-sized droplets can be carried by the probe and neatly arranged on the in-situ TEM chip through a precisely moving three-axis module. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Optical microscope photo 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 is described in detail below in conjunction with the accompanying drawings and embodiments.

[0045] The epoxy resin AB glue, polystyrene beads, in-situ TEM chip and probe used in the present invention are all commercially available analytically pure products, and the deionized water is homemade in the laboratory; the glass instruments and equipment used are commonly used instruments and equipment in the laboratory.

[0046] Figure 1 The optical microscope photos of the probe prepared in the present invention; a is the probe used for liquid transfer, which is a purchased ordinary commercial probe; b is the in-situ TEM chip photo and the optical microscope magnification; c is the in-situ TEM chip simulation picture and the real photo of the hole in the in-situ TEM chip under an electron microscope.

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

[0048] The method for making the microsphere probe of the present invention is as follows:

[0049] ①Clamp the probe on the micrometer probe stage and move it into the field of view of the optical microscope;

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

[0051] ③Take a glass slide and use a thin glass rod to draw mixed solution A on one end of the slide;

[0052] ④ Take the polystyrene ball powder and sprinkle it on the other end of the slide;

[0053] 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;

[0054] ⑥ Allow the mixed solution A to stand for 24 hours until it is completely solidified to obtain the microsphere probe.

[0055] Embodiment 1:

[0056] A method for preparing 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) Prepare a target product distribution map according to the well positions of the 96-well plate, and use a Python script to generate the control file for the automatic pipetting platform.

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

[0060] (4) Import the control file into the automatic liquid transfer 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 as 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 above three sets of positions using a python script, where 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. Take out the sample from the ethanol pool and move it to well A1 of the 96-well plate;

[0067] c. The probe tip descends into the liquid in hole 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 into the ethanol bath for cleaning.

[0071] After the first set of runs, a loop command was used to allow the probe to pick up the material from the 96-well plate at a specific step length and place nanodroplets at 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] Embodiment 2:

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

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

[0077] (2) Prepare a target product distribution map according to the well positions of the 96-well plate, and use a Python script to generate the control file for 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 liquid transfer 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 as 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, use the "jogging mode" to 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. Take out the sample from the ethanol pool and move it to well A1 of the 96-well plate;

[0086] c. The probe tip descends into the liquid in hole 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 into the ethanol bath for cleaning.

[0090] After the first set of runs, a loop command was used to allow the probe to pick up the material from the 96-well plate at a specific step length and place nanodroplets at 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 by a common 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 by using a plasma cleaner is omitted. The result is different from that of Example 1 in 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 arrange the droplets neatly on the in-situ TEM chip.

[0097] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an in-situ high-throughput transmission electron microscope sample, characterized in that: The preparation method comprises the following steps: (1) Making microsphere probes; (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.

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: ①Clamp the probe on the micrometer probe stage and move it into the field of view of the optical microscope; ② Mix epoxy resin AB glue to obtain a uniform mixed liquid A; ③Take a glass slide and use a thin glass rod to draw mixed solution A on one end of the slide; ④ Take the polystyrene ball powder and sprinkle it on the other end of the slide; 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; ⑥ Allow the mixed solution A to stand for 24 hours until it is completely solidified to obtain the microsphere probe.

4. The preparation method according to claim 1, characterized in that: 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.

5. The preparation method according to claim 1, characterized in that: In step (2), the specific method is: ① Place the liquids A, B, C, ... to be transferred into a 96-well plate, and place ethanol in the last well for cleaning the probe; ② Adjust the height so that the microsphere probe can just touch the liquid in the 96-well plate; ③ Place the hydrophilized in-situ TEM chip next to the 96-well plate on the sample stage; ④ Move the Z axis so that the microsphere probe contacts liquid A, then move it to the top of 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 hole to clean the probe up and down, and obtain droplet A on the in-situ TEM chip; ⑤ Move the Z axis so that the microsphere probe contacts liquid B, then move it to the top of the in-situ TEM chip with a horizontal interval of 5 microns from the 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 hole to clean the probe up and down, and droplets A and B are obtained on the in-situ TEM chip; ⑥ Repeat steps ④ and ⑤ to obtain droplets A, B, C, ... neatly arranged on the in-situ TEM chip.

6. The preparation method according to claim 5, characterized in that: 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.

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