A nanoparticle sample retrieval method for TEM

Abstract

The present invention relates to a method for retrieving nanoparticle samples for TEM, and a method for retrieving nanoscale target samples using micron-scale markers for positioning. The method of the present invention is universally applicable to various brands and models of TEM instruments and coordinate support film grids. No special pattern needs to be designed. Only inert markers need to be added to the grid already loaded with the target sample before testing to achieve the positioning and retrieval of nanoscale targets. It is universal for various brands and models of coordinate grids and TEM equipment; the target position is retrieved based on the coordinate grid and the micron-scale markers, and the retrieval accuracy is high, the efficiency is high, the cost is low, the operation is simple, and the practicality is strong. The mesh size of the selected coordinate grid matches the maximum field of view of most commercial transmission electron microscopes on the market, that is, the periodic grid of the coordinate grid is smaller than the maximum field of view of the TEM, and at least one complete period can be displayed in the TEM field of view, so that the large grid where the target is located can be accurately locked.

Description

Technical Field

[0001] The present invention relates to a method for retrieving a specific microscopic area on a nanoparticle sample which is generally applicable to various types of TEMs. Background Art

[0002] Transmission electron microscopes (TEMs) are one of the most commonly used instruments in microanalysis, playing an irreplaceable role in analyzing nanoparticle morphology and compositional structure. TEM equipment is expensive and complex to operate, making machine time extremely valuable in universities and research institutions. TEMs are categorized by their illumination source: tungsten or lanthanum hexaboride filaments, or field emission filaments. Tungsten or lanthanum hexaboride filaments are inexpensive, have short lifespans, and require significantly lower vacuum levels than field emission TEMs. Pumping down to a vacuum takes only one-third to one-fifth the time of field emission TEMs, resulting in high efficiency. Even with multiple samples loaded simultaneously, the required vacuum can be quickly achieved, making them ideal for initial sample screening. However, the large tip size and low brightness of tungsten or lanthanum hexaboride filaments make them unsuitable for high-resolution imaging. They are typically used for initial screening or imaging biological samples, and mainstream models on the market generally lack numerous accessories, resulting in limited analytical capabilities. Due to the limited space within the transmission electron microscope's pole piece, after placing multiple samples, there's insufficient room for sample rotation and detector placement. This limits selected area electron diffraction and energy spectrum analysis capabilities, resulting in insufficient analytical capabilities for models suitable for sample screening. Field emission transmission electron microscopes, with their thinner filament tips, higher brightness, and higher resolution, coupled with higher accelerating voltages and larger beam currents, are more suitable as comprehensive analysis platforms. However, field emission electron microscopes are limited by their high vacuum requirements, resulting in lengthy pumping times and impacting test efficiency.

[0003] To improve the efficiency of transmission electron microscopes, a strategy of combining different models with different magnifications is generally adopted. First, a low-magnification transmission electron microscope with fast vacuuming and the ability to load multiple samples at once is used for initial sample screening. Samples worthy of further research are then loaded into models with higher magnifications and more analytical functions for analysis and testing. In practice, since the observation target is at the nanoscale, once the grid is removed from the instrument, it is difficult to find its original position for subsequent observations. In this case, when the grid is transferred to another instrument for observation, a considerable amount of time is still required to find the target area, resulting in low work efficiency and wasted machine time. In this context, it is of great significance to develop a micro-area positioning and retrieval system that is universally applicable to various brands and models of TEMs.

[0004] In addition, the universal micro-area positioning system can also realize repeatable and traceable observation and analysis of micro-area targets, and can provide technical support for improving the credibility and traceability of data. It is of great significance both in instrument operation training and in improving scientific research integrity. Summary of the Invention

[0005] In view of the above situation, in order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a nanoparticle sample retrieval method for TEM, which can effectively solve the problem of positioning and retrieval of nanoscale targets.

[0006] The technical solution provided by the present invention is:

[0007] A method for retrieving nanoparticle samples for TEM, comprising the following steps:

[0008] Step 1: Add inert markers

[0009] Drop the nanoparticle target sample onto the grid, disperse the inert marker in a dispersant, drop it onto the grid, and place the grid in an oven or vacuum oven to evaporate the dispersant;

[0010] Step 2: First Observation

[0011] After the grid containing the inert marker and the target sample is mounted on the sample holder of the transmission electron microscope, it is loaded into the transmission electron microscope and observed after the vacuum is drawn.

[0012] Step 3: Positioning

[0013] Find the target sample and, in the low mag mode of the transmission electron microscope, confirm the coordinate grid of the target sample and take a photo to record it, which is recorded as Picture A;

[0014] Place the inert marker and the location to be marked on the target sample in the same field of view, measure the distance from the characteristic point at the target location to the inert marker, mark it in the image, and record it as image C;

[0015] In the low mag mode of the transmission electron microscope, record the distance between the inert marker and the grid lines of the coordinate grid. Place at least one edge of the coordinate grid and the inert marker in the same field of view. Measure the distance from the two end points of the inert marker to the nearest coordinate grid line, mark it in the image, and record it as image B.

[0016] Step 4: Retrieve

[0017] Place the grid into the TEM specimen holder. First, find the coordinate grid where the target sample is located in low mag mode. Adjust the Z focus so that the grid is stable in wobble mode.

[0018] Rotate the image A taken in step 3 by an angle α so that it coincides with the coordinate grid direction in the current field of view. Rotate the image B taken in step 3 by an angle α synchronously with image A. Find the inert marker based on the positional relationship between the inert marker measured in step 3 in image B and the grid lines of the coordinate grid.

[0019] The image C of the positional relationship between the inert marker and the target sample recorded in step 3 is rotated synchronously with image A by an angle α, and the target sample is retrieved based on the positional relationship and distance between the inert marker and the target sample in image B in step 3.

[0020] At present, commercial coordinate grids can solve the problem of sample retrieval to a certain extent and are suitable for the retrieval of micron-level samples. However, due to the limitations of processing technology, the mesh size of the coordinate grid is small and the period is large, making it more difficult to retrieve nano-samples.

[0021] In addition, since the TEM's goniometer stage can adjust the sample height to accommodate the focal length of the electromagnetic lens, allowing it to be clearly focused and imaged, thus clearly displayed in the field of view, under normal circumstances, the coordinate grid and the support film are at different heights. Therefore, the coordinate grid and the nanoscale sample attached to the support film cannot be clearly focused at the same time (clear focus is defined as the image of the test target in wobble mode without shaking). Therefore, using only the coordinate grid cannot accurately determine the target's in-focus position. In the non-focused case, the shape of the test target cannot be clearly displayed, making it difficult to determine which sample is the target.

[0022] Based on this, the present invention has developed a method for locating and retrieving nanoscale target samples using commercially available coordinate grids and micron-scale markers. This method is universally applicable to various brands and models of TEM instruments and coordinate support film grids. It eliminates the need for specialized pattern design and simply requires adding inert markers to the grid already loaded with the target sample before testing to achieve nanoscale target location and retrieval. Compared to existing technologies, this method offers the following advantages:

[0023] 1. The method of the present invention is universal for coordinate grids and TEM equipment of various brands and models;

[0024] 2. The method of the present invention retrieves the target position based on the coordinate grid and micron-level markers, with high retrieval accuracy, high efficiency, low cost, simple operation and strong practicality.

[0025] 3. The mesh size of the coordinate grid selected in the method of the present invention matches the maximum field of view of most commercial transmission electron microscopes on the market, that is, the periodic grid of the coordinate grid is smaller than the maximum field of view of the TEM, and at least one complete period can be displayed in the TEM field of view, so the large grid where the target is located can be accurately locked.

[0026] 4. The method of the present invention has a good error tolerance rate and can find the target position based on the image information when the distance is not completely matched. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the positioning process of Application Example 1 of the present invention; testing instrument JEOL JEM 1400PLUS; test sample: Au nanorods, marker BaTi2O4 microrods; the direction of the arrow represents the order in which the positioning process images are obtained.

[0028] Figure 2 Schematic diagram of the retrieval process of Application Example 1 of the present invention; testing instrument JEOL 2100F; rotation angle 135° counterclockwise; arrow directions represent the order in which images of the retrieval process are obtained.

[0029] Figure 3 Schematic diagram of the positioning process of Application Example 2 of the present invention; testing instrument JEOL JEM 1400PLUS; test sample: Au nanorods, marker BaTi2O4 microrods; the direction of the arrow represents the order in which the positioning process images are obtained.

[0030] Figure 4 Schematic diagram of the retrieval process of Application Example 2 of the present invention; the testing instrument is a Thermal Fisher Talos120EC; all images are mirror images of the sample projected onto a fluorescent screen, with Low Mag up to 2300x and Mag starting at 1250x. The Mag image is rotated 180° based on the Low Mag image; the directions of the arrows indicate the order in which the images of the retrieval process are obtained.

[0031] Description of the accompanying drawings:

[0032] (1) 80* field of view size: 328um*220um; copper mesh period is 100um, 200 mesh hole diameter is 75um, which is equivalent to a square side length of 132um. 80* field of view can cover all of it.

[0033] (2) The meaning of Axy, Bxy, and Cxy in the figure: the letters refer to the field of view within the specified magnification range, A = 80-200 times, B = 200-800 times, and C = 5000-50000; x refers to the retrieval order within the above field of view, that is, the number of retrievals, and 0 represents the positioning process; y refers to the number of photos taken within the corresponding photo magnification range during the positioning or retrieval. DETAILED DESCRIPTION

[0034] The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings and examples.

[0035] A method for retrieving nanoparticle samples for TEM, comprising the following steps:

[0036] Step 1: Add inert markers

[0037] Drop the nanoparticle target sample onto the grid, disperse the inert marker in the dispersant, drop it onto the grid, and place the grid in an oven or vacuum oven (35-200°C) to evaporate the dispersant;

[0038] The inert marker is a rod-shaped object with a length of micrometer level and a width or diameter of nanometer level.

[0039] The inert marker has a length of 1-50 μm and a width or diameter of 30-200 nm.

[0040] The inert markers are micro-nanorods made of BaTi2O4, ZnO, TiO2, Au, Pt, or Pd. When selecting an inert marker, it must be clearly different in composition from the target nanoparticle sample, with the primary constituent elements of the two clearly distinguishable in the energy spectrum (distinguishing in the energy spectrum means that the energy difference between the main peaks of the energy spectrum of the primary constituent elements of the two must be no less than 500 eV). In practice, an elemental composition analysis can be performed on a sample containing only the target sample, and then a material with a non-overlapping elemental composition can be selected as the inert marker.

[0041] The dispersant is deionized water, anhydrous ethanol, acetone, N,N-dimethylformamide (DMF), chloroform or dimethyl sulfoxide (DMSO), and the dispersant does not chemically react with the inert marker and the nanoparticle target sample, and the inert marker and the nanoparticle target sample cannot be dissolved in the dispersant; the molar ratio of the inert marker to the dispersant is 10 -7 ~10 -9 between.

[0042] The grid is a coordinate support film grid with a mesh size of 200-250 meshes; the grid is made of copper, molybdenum, nickel, gold or stainless steel.

[0043] Before dropping the target nanoparticle sample onto the carrier mesh, the target sample needs to be dispersed in a dispersant, then dropped onto the carrier mesh, and the carrier mesh is placed in an oven or vacuum oven (35-200°C) to evaporate the dispersant. The target sample is then dispersed in the dispersant and then dropped onto the carrier mesh for drying. This is the prior art.

[0044] Step 2: First Observation

[0045] After the grid containing the inert marker and the target sample is mounted on the sample holder of the transmission electron microscope, it is loaded into the transmission electron microscope and observed after the vacuum is drawn.

[0046] Step 3: Positioning

[0047] Find the target sample based on the pre-estimated morphology and composition, take photos or perform other analyses (such as selected area diffraction or energy spectrum analysis) as required, and in the transmission electron microscope low mag mode, confirm the coordinate grid of the target sample and take photos to record it, which is recorded as image A;

[0048] Place the inert marker and the location to be marked on the target sample in the same field of view, measure the distance from the characteristic point at the target location to the inert marker (extend the longest side of the inert marker-micro-nanorod closest to the characteristic point into a straight line, and record the distance from the characteristic point to this extended line as the distance from the characteristic point to the specific micron-rod marker), mark it in the image, and record it as Image C;

[0049] The characteristic point of the target position is the vertex, endpoint or geometric center of the target position closest to the inert marker.

[0050] In the low mag mode of the transmission electron microscope, record the distance between the inert marker and the grid lines of the coordinate grid. Place at least one edge of the coordinate grid and the inert marker in the same field of view. Measure the distance from at least one endpoint of the inert marker to the nearest coordinate grid line, mark it in the image, and record it as image B.

[0051] Step 4: Retrieve

[0052] Place the grid into the TEM specimen holder. First, find the coordinate grid where the target sample is located in low mag mode. Adjust the Z focus so that the grid is stable in wobble mode.

[0053] Rotate the image A taken in step 3 by an angle α (the low mag mode of some models is a mirror image of the projection. If it is a mirror image, you need to flip image A before rotating it, and then rotate it again) so that it coincides with the coordinate grid direction in the current field of view. Rotate image B taken in step 3 by an angle α synchronously with image A (if it is a mirror image, you need to flip image B before flipping it, and then rotate it again). Find the inert marker based on the positional relationship between the inert marker measured in step 3 and the grid lines of the coordinate grid;

[0054] Rotate the image C of the positional relationship between the inert marker and the target sample recorded in step 3 synchronously with image A by an angle α (if it is a mirror image, flip image C first, then rotate it after flipping), and retrieve the target sample based on the positional relationship and distance between the inert marker and the target sample in image B in step 3.

[0055] When retrieving, the magnification ratio remains the same as in Picture ABC. Whether the photo is flipped depends on the camera model used. Pictures that need to be flipped in low mag need to remain flipped in Mag mode.

[0056] Since the support film grid used in the experiment is a metal grid with a thickness of less than 200um, some grids are soft in texture and easily deformed during loading and unloading with tweezers. In order to maximize the flatness of the metal grid and the integrity of the support film, a vacuum sampler is used to load, unload and move the grid.

[0057] The present invention has achieved good technical effects through practical application. The following are application examples:

[0058] Application Example 1:

[0059] The JEOL 1400plus TEM was used for observation and the JEOL 2100F TEM was used for retrieval: 1. First observation, such as Figure 1 As shown:

[0060] 1. Select a 200-250 mesh grid and add the sample to be tested: Au nanorods dispersed in deionized water, with a concentration of 10 - 9 g / L;

[0061] 2. Dry the sample in an oven at 100°C. After the sample is completely dry, add the marker (BaTi2O4 microrods dispersed in deionized water, concentration: 10 -8 -10 -9 The sample was then dried in an oven at 100°C and tested on a JEOL JEM 1400Plus instrument.

[0062] 3. Find the sample to be tested, take a photo or perform other analysis as required (photos are recorded as C-0-3).

[0063] 4. After photographing the target position, reduce the magnification until both the micron-sized rod and the target can be seen in the field of view. Aim at the end of the marker microrod closest to the target nanorod and adjust the Z focus so that it is in focus (no shaking in Wobble mode). Take a photo and record it as C-0-1. Measure and record the distance between the endpoint closest to the target and the marker microrod (when there is only one microrod nearby, in addition to recording the distance D1 from the measurement target to the microrod, also record the distance D2 from the foot of the perpendicular segment to the part of the microrod that is in focus. This measurement D1 = 0.26um, D2 = 0.36um. If there are multiple microrods nearby, select the two closest to the target and measure the distance between the center of the target and the two microrods respectively, and record the photos.).

[0064] 5. Continue to reduce the magnification and take photos in LOW MAG mode. Take at least two photos, one to determine the relative position of the marked microrod in the large grid, recorded as B-0-1, and the other to determine the large grid where the target is located on the coordinate copper mesh, recorded as A-0-1.

[0065] 2. Second recovery: Instrument JEOL 2100F;

[0066] Place the coordinate copper grid face up on the sample holder.

[0067] 1. In LOW MAG mode, determine the rotation angle of the copper mesh placed this time compared to the copper mesh placed last time. Rotate the first LOW MAG photo A-0-1 counterclockwise by the corresponding angle α1 (α1 = 135°) to match the direction of the current placement.

[0068] 2. Find the large grid on the copper mesh where the target is located according to Figure A-0-1 after the rotation angle.

[0069] 3. Rotate photo B-10 counterclockwise by an angle of α1 and find the marked micrometer rod corresponding to the rotated photo B-0-1 in the current field of view.

[0070] 4. Switch the instrument to Mag mode and use the instrument's Z focus function to adjust the micrometer rod in step 3 so that it does not wobble while the wobble is on. Determine the foot of the perpendicular segment from the target point to the micrometer rod based on the distance D2 marked on the micrometer rod in Figure C-0-1. Then, retrieve the test target based on the length of the perpendicular segment in Figure C-0-1.

[0071] In this embodiment, the test target is a gold nanorod. The TEM photo of the recovered test target is as follows: Figure 2As shown in Figure C-1-1 and Figure C-1-2, their shape, size, and contrast are consistent with the shapes of the surrounding microrod markers. Compared with Figure C-0-2 and Figure C-0-3, Figure C-1-1 and Figure C-1-2 are rotated 135° counterclockwise.

[0072] Application Example 2:

[0073] 1. First observation, such as Figure 3 As shown:

[0074] 1. Select a 200-250 mesh grid and add the sample to be tested: Au nanorods dispersed in deionized water, with a concentration of 10 -9 g / L.

[0075] 2. Dry the sample in an oven at 100°C. After the sample is completely dry, add the marker (BaTi2O4 microrods dispersed in deionized water, concentration: 10 -8 -10 -9 The samples were then dried in an oven at 100°C and tested on a JEOL JEM 1400Plus instrument.

[0076] 3. Find the sample to be tested, take a photo or perform other analysis as required (photos are recorded as C-0-3).

[0077] 4. After photographing the target location, reduce the magnification until both the micron-sized rod and the target can be seen in the field of view. Aim at the end of the marker microrod closest to the target nanorod, adjust the Z focus so that it is in focus (no shaking in Wobble mode), and take a photo, recorded as C-0-1. Measure and record the distance between the endpoint closest to the target and the marker microrod and the marker microrod (when there is only one microrod, in addition to recording the distance D1 from the measurement target to the microrod, also record the distance D2 from the perpendicular segment to the in-focus section of the microrod. This measurement D1 = 0.26um, D2 = 0.36um; if there are multiple microrods nearby, select two of them and measure the distance between the center of the target and each microrod, taking photos and recording them).

[0078] 5. Continue to reduce the magnification and take photos in LOW MAG mode. Take at least two photos, one to determine the relative position of the marked microrod in the large grid, recorded as B-1, and the other to determine the large grid where the target is located on the coordinate copper mesh, recorded as A-0-1.

[0079] Second, retrieve and test instrument Thermal Fisher Talos 120EC;

[0080] Place the coordinate copper grid face up on the sample holder.

[0081] 1. In LOW MAG mode, obtain an overall image of the copper mesh, as shown in Figure A-2-1; mirror image A-0-1 (flip along the right side of the image - horizontal flip), and then rotate the rotated image counterclockwise by α2 (α2 = 52°) to make it consistent with the direction of the image in the field of view at this time.

[0082] 2. Find the large grid on the copper mesh where the target is located according to Figure A-2-1 after horizontal flipping and rotation angle α2.

[0083] 3. Flip photo B-0-1 horizontally and rotate it counterclockwise by an angle of α2. Find the marked micrometer rod corresponding to the rotated photo B-0-1 in the current field of view.

[0084] 4. Zoom in further and enter Mag mode. The image will rotate 180° clockwise, as shown in Figure B-2-2. Flip photo C-0-1 horizontally and then rotate it 128° clockwise (180° - 52° = 128°). Find the location of the target sample in the flipped and rotated image.

[0085] 5. Use the instrument's Z focus function to adjust the micrometer rod in step 4 above so that it does not shake when the wobble is on. Determine the foot of the perpendicular segment from the target point to the micrometer rod based on the distance D2 marked on the micrometer rod in the flipped and rotated Figure C-0-1. Then, find the test target D1 based on the length of the perpendicular segment in the flipped and rotated Figure C-0-1.

[0086] In this application example, the test target is a gold nanorod. The TEM photo of the recovered test target is as follows: Figure 4 As shown in Figure C-2-1 and Figure C-2-2, their shape, size, and contrast are consistent with the shapes of the surrounding microrod markers. Compared with Figure C-0-2 and Figure C-0-3, Figure C-2-1 and Figure C-2-2 are horizontally flipped and rotated 128° clockwise.

Claims

1. A method for retrieval of nanoparticle samples for TEM, characterized in that: The following steps are involved: Step 1: Add inert markers The nanoparticle target sample is dropped onto the grid, the inert marker is dispersed in a dispersant, and the inert marker is dropped onto the grid, and the grid is placed in an oven or a vacuum oven to evaporate the dispersant; Step 2: First Observation After the grid containing the inert marker and the target sample is mounted on the sample holder of the transmission electron microscope, it is loaded into the transmission electron microscope and observed after the vacuum is drawn. Step 3: Positioning Find the target sample and, in the low mag mode of the transmission electron microscope, confirm the coordinate grid of the target sample and take a photo to record it, which is recorded as picture A; Place the inert marker and the location to be marked on the target sample in the same field of view, measure the distance from the characteristic point at the target location to the inert marker, mark it in the image, and record it as image C; In the low mag mode of the transmission electron microscope, record the distance between the inert marker and the grid lines of the coordinate grid. Place at least one edge of the coordinate grid and the inert marker in the same field of view. Measure the distance from the two end points of the inert marker to the nearest coordinate grid line, mark it in the image, and record it as image B. Step 4: Retrieve Place the grid into the TEM specimen holder. First, find the coordinate grid where the target sample is located in low mag mode. Adjust the Z focus so that the grid is stable in wobble mode. Rotate the image A taken in step 3 by an angle α so that it coincides with the coordinate grid in the current field of view. Rotate the image B taken in step 3 by an angle α synchronously with image A. Find the inert marker based on the positional relationship between the inert marker measured in step 3 in image B and the grid lines of the coordinate grid. Rotate the image C of the positional relationship between the inert marker and the target sample recorded in step 3 synchronously with image A by an angle α, and retrieve the target sample based on the positional relationship and distance between the inert marker and the target sample in image B in step 3; The inert marker is a rod-shaped object with a length of micrometer level and a width or diameter of nanometer level; The inert marker is a micro-nano rod made of BaTi2O4, ZnO, TiO2, Au, Pt or Pd material; The grid is a coordinate support film grid, and the mesh number of the grid is 200-250 meshes.

2. The method for retrieval of nanoparticle samples for TEM according to claim 1, characterized in that: The inert marker has a length of 1-50 μm and a width or diameter of 30-200 nm.

3. The method for retrieval of nanoparticle samples for TEM according to claim 2, characterized in that: The dispersant is deionized water, anhydrous ethanol, acetone, N,N-dimethylformamide, chloroform or dimethyl sulfoxide. The dispersant does not chemically react with the inert marker and the nanoparticle target sample, and the inert marker and the nanoparticle target sample cannot be dissolved in the dispersant. The molar ratio of the inert marker to the dispersant is 10 -7 ~10 -9 between.

4. The method for retrieval of nanoparticle samples for TEM according to claim 1, characterized in that: The material of the grid is copper, molybdenum, nickel, gold or stainless steel.

5. The method for retrieval of nanoparticle samples for TEM according to claim 1, characterized in that: The characteristic point of the target position in step 2 is the vertex, endpoint or geometric center of the target position closest to the inert marker.

Images