A method and apparatus for collecting three-dimensional electron diffraction data of liquid crystal structures

Abstract

The application provides a liquid-phase crystal structure three-dimensional electron diffraction data acquisition method and device, and belongs to the field of crystal structure analysis. The method provided by the application comprises the following steps: immersing a nanocrystal to be collected in a solvent; adding a first volume of the solvent on the front surface of a first continuous carbon support net; covering a second continuous carbon support net on the first continuous carbon support net, and the edges and front surface of the first continuous carbon support net and the second continuous carbon support net form a sealed double continuous carbon support net liquid pool; loading the double continuous carbon support net liquid pool on a sample rod; positioning the nanocrystal to be collected in the double continuous carbon support net liquid pool by using an electron microscope, rotating the sample rod, and collecting three-dimensional electron diffraction data of the nanocrystal to be collected. The liquid-phase crystal structure three-dimensional electron diffraction data acquisition method and device provided by the application can realize high-quality three-dimensional electron diffraction data acquisition of nanocrystals in a liquid-phase environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crystal structure analysis, and in particular to a liquid-phase crystal structure three-dimensional electron diffraction data acquisition method and device. BACKGROUND

[0002] The structure of a substance determines its properties, and accurate analysis of the crystal structure is the basis for understanding its function and performance. Single-crystal X-ray diffraction is currently the most authoritative method for determining crystal structures at atomic resolution, but it has stringent requirements for crystal size, typically requiring high-quality single crystals with dimensions greater than 5 microns in all three dimensions. However, in the fields of chemistry, materials science, and life science, many newly developed functional materials (such as metal-organic frameworks, nanopharmaceutical crystals, protein microcrystals, etc.) are difficult to grow to the required size, limiting the application of single-crystal X-ray diffraction.

[0003] To meet the needs of micro- and nanocrystal structure analysis, three-dimensional electron diffraction technology has emerged. Due to the much stronger interaction between electrons and matter than X-rays, this technology can obtain high-quality diffraction data from nanometer or even sub-micron crystals, and has been successfully applied to the structure determination of various materials. However, conventional three-dimensional electron diffraction technology must be performed in a high vacuum environment, which makes it impossible to characterize many crystals that exist stably or function in a liquid environment (such as solvent-driven flexible frameworks, biological macromolecular crystals, etc.) in their original state. Although there have been attempts to use specially designed chip liquid cells and in-situ sample rods for electron diffraction experiments in liquid phase, these methods still have obvious limitations: chip liquid cells usually result in a liquid layer that is too thick, severely attenuating the electron beam intensity and reducing the signal-to-noise ratio; special in-situ rods are expensive, and their mechanical structure limits the tilt angle of the sample, resulting in insufficient completeness of the diffraction data collection, and so far no crystal structure analysis based on the direct method has been achieved.

[0004] Therefore, there is an urgent need for a general method that can achieve high-quality three-dimensional electron diffraction data acquisition and structure analysis of nanocrystals in a liquid environment without relying on complex special equipment. SUMMARY

[0005] In view of this, the present application provides a liquid-phase crystal structure three-dimensional electron diffraction data acquisition method and device, which can achieve high-quality three-dimensional electron diffraction data acquisition of nanocrystals in a liquid environment.

[0006] Specifically, the present application is implemented by the following technical solutions:

[0007] The first aspect of the present application provides a liquid-phase crystal structure three-dimensional electron diffraction data acquisition method, which comprises:

[0008] Soaking the nanocrystal to be collected in a solvent;

[0009] adding a first volume of the solvent containing the nanocrystals to be collected on the front surface of the first continuous carbon support net;

[0010] covering the second continuous carbon support net on the first continuous carbon support net, the edges and front surfaces of the first continuous carbon support net and the second continuous carbon support net forming a sealed double continuous carbon support net liquid pool, and the double continuous carbon support net liquid pool encapsulating the solvent; wherein the front surface of the second continuous carbon support net is opposite to the front surface of the first continuous carbon support net, and the second continuous carbon support net has the same horizontal dimension as the first continuous carbon support net;

[0011] mounting the double continuous carbon support net liquid pool on a sample rod;

[0012] positioning the nanocrystals to be collected in the double continuous carbon support net liquid pool by using an electron microscope, controlling the rotation of the sample rod, and collecting three-dimensional electron diffraction data of the nanocrystals to be collected.

[0013] The second aspect of the present application provides a liquid crystal structure three-dimensional electron diffraction data collection device, which comprises:

[0014] The double continuous carbon support net liquid pool is formed by covering the second continuous carbon support net on the first continuous carbon support net after adding the solvent containing the nanocrystals to be collected on the front surface of the first continuous carbon support net; wherein the front surface of the second continuous carbon support net is opposite to the front surface of the first continuous carbon support net, the edges and front surfaces of the second continuous carbon support net and the first continuous carbon support net form a sealed structure, and the horizontal dimension of the second continuous carbon support net is the same as that of the first continuous carbon support net, which is used to encapsulate the solvent and maintain the liquid phase environment of the nanocrystals to be collected;

[0015] The sample rod is used to mount the double continuous carbon support net liquid pool;

[0016] The electron microscope is used to position the nanocrystals to be collected after the double continuous carbon support net liquid pool is placed in the sample chamber of the electron microscope, control the rotation of the sample rod, and collect three-dimensional electron diffraction data of the nanocrystals to be collected.

[0017] The liquid phase crystal structure three-dimensional electron diffraction data collection method and device provided by the application greatly facilitates the rapid positioning and measurement of target crystals in an electron microscope, and successfully realizes high-quality three-dimensional electron diffraction data collection of nanocrystals in a liquid phase environment in a conventional transmission electron microscope. Compared with the existing chip liquid pool technology, the liquid pool made of the double continuous carbon carrier net has a thinner liquid layer, and can only cover the crystal with a trace of liquid drop. On the one hand, the trace of liquid drop ensures that the crystal is in a liquid environment, and on the other hand, the trace of liquid drop can prevent the phenomenon that the crystal moves with the liquid during the three-dimensional electron diffraction data collection process, ensuring the stability of the data collection process, and significantly reducing the scattering and energy loss of the electron beam in the liquid, thereby obtaining higher quality diffraction signals; at the same time, the liquid pool structure can accommodate crystals of different sizes, and is compatible with conventional sample rods, supports continuous tilting at a larger angle, and effectively improves the completeness of three-dimensional diffraction data; by combining the continuous rotation collection mode, a single set of three-dimensional electron diffraction data can be collected in a short time, the electron cumulative dose is minimized, the damage of liquid radiation decomposition to the crystal structure is reduced, and thus the quality of the diffraction data and the authenticity of the crystal structure are ensured; by adding a trace of solvent on the front surface of the first continuous carbon carrier net and covering it with the second continuous carbon carrier net, a sealed double continuous carbon carrier net liquid pool is constructed, which can stably maintain a local liquid phase microenvironment in the high vacuum environment of the electron microscope, and ensure that the crystal is observed in a real functional state. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The flowchart of the liquid phase crystal structure three-dimensional electron diffraction data collection method provided by the application is provided.

[0019] Figure 2 The structure diagram of MIL-53(Al) obtained by the liquid phase crystal structure three-dimensional electron diffraction data collection method provided by the application is provided.

[0020] Figure 3 The structure diagram of MIL-53(Al) obtained by the conventional three-dimensional electron diffraction data collection method provided by the application is provided.

[0021] Figure 4 The structure diagram of the liquid phase crystal structure three-dimensional electron diffraction data collection device provided by the application is provided. DETAILED DESCRIPTION

[0022] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0023] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used herein are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0024] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0025] The following specific embodiments are given to illustrate the technical solution of this application in detail.

[0026] Example 1

[0027] Figure 1 This is a flowchart of an embodiment of the three-dimensional electron diffraction data acquisition method for liquid-phase crystal structures provided in this application. Please refer to... Figure 1 The method provided in this embodiment may include:

[0028] S101. Immerse the nanocrystals to be collected in a solvent.

[0029] It should be noted that this application aims to construct a stable and realistic liquid environment for the nanocrystal to be tested and optimize its structural state to meet the requirements of subsequent three-dimensional electron diffraction data acquisition. Therefore, the nanocrystal to be collected needs to be immersed in a suitable solvent.

[0030] The purpose of soaking is to place the crystal in a target liquid environment. For different types of crystals, soaking can produce one or more of the following effects: for some crystals (such as some metal-organic frameworks, biological macromolecular crystals), soaking can improve their structural stability or crystallinity, prevent their structural collapse under vacuum, and thus obtain clearer diffraction signals; for other crystals (such as flexible porous materials), the entry of solvent molecules into the pores can induce structural phase transitions, and soaking is used to achieve the equilibrium or functional configuration in a specific solvent; for general crystals, soaking helps to maintain their intrinsic state related to the solvent. Regardless of the specific effect, the common purpose of the soaking operation is to make the crystal reach a structural equilibrium state in the solvent before analysis, so as to ensure that the final resolved structure reflects its true functional state and lays the foundation for collecting high-quality diffraction data. Among them, the specific performance of high-quality diffraction data is that the diffraction pattern collected from a single crystal in the selected area electron diffraction mode should have a high enough resolution, with sharp, discrete and continuous diffraction spots, rather than a state of dispersion, line connection or high background.

[0031] Among them, the nanocrystal to be collected is a micro- or nanoscale crystalline material that needs to be structurally analyzed, which can be obtained by chemical synthesis methods such as hydrothermal method or solvothermal method, or by commercial purchase and separation and extraction from natural products, which is not limited in the present application. As an optional embodiment, the nanocrystal can be prepared by the following hydrothermal method: dissolving metal salt (such as aluminum nitrate nonahydrate) and organic ligand (such as terephthalic acid) in solvent (such as water), reacting at high temperature (such as 220°C) for several days (such as 3 days), and then washing and drying after cooling to obtain the nanocrystal; the obtained crystal can also be subjected to subsequent treatment as needed, such as calcination at a certain temperature (such as 350°C) to obtain an activated phase. Specifically, the selected nanocrystal material at least includes any one of oxides, alloys, inorganic-organic hybrid materials, zeolites, organic crystals, metal complexes, metal-organic frameworks (MOFs), covalent organic frameworks (COFs), protein crystals, nucleic acid crystals, and drug molecule crystals. The reason for selecting these materials is that they are widely used in the fields of chemistry, materials, biology and pharmacy, and their structure, properties and functions are often highly dependent on the liquid environment they are in. Therefore, it is of great practical significance to analyze the structure of these crystals in the liquid phase.

[0032] It should be noted that the solvent herein is used to simulate the real existence or reaction environment (in liquid phase environment) of the nanocrystal to be collected, which includes polar solvents and non-polar solvents. Among them, the polar solvent at least includes any one of water, ethylene glycol, chlorobenzene, pyridine, DMF, methanol, ethanol, THF, isopropanol; the non-polar solvent at least includes any one of benzene, tetrachloromethane, cyclohexane, n-hexane. Specifically, the polar solvent is suitable for hydrophilic systems such as metal organic frameworks and protein crystals; the non-polar solvent is suitable for materials such as organic crystals and some drug molecules which are more stable in non-polar environment. By selecting these solvents, a stable dispersion medium can be provided for the nanocrystal to be collected, and necessary physical and chemical interactions can occur. Among them, the solvent can be obtained from the market and purified (such as distillation, degassing, etc.) before use as needed to remove impurities and avoid adverse effects on the crystal structure or diffraction quality.

[0033] Specifically, the nanocrystal to be collected is soaked in the solvent, and the obtained crystal suspension is uniformly dispersed and in its target liquid phase environment. The reactions or changes that may occur during this soaking process include: for porous crystalline materials (such as MOFs, zeolites), solvent molecules will diffuse into their pores, which may cause lattice expansion, contraction or conformational change; for biological macromolecular crystals, soaking ensures that they maintain a highly hydrated natural active conformation; for general crystals, soaking helps to repair surface defects and remove surface-adsorbed impurities. Therefore, soaking is performed to make the nanocrystal to be collected reach a structural equilibrium state in the solvent before analysis, so as to ensure that the structure obtained by analysis finally reflects the real functional state of the nanocrystal, rather than the structure under dry or non-equilibrium conditions.

[0034] It should also be noted that the soaking condition is set to be soaked at a temperature of 0-90°C for more than 5 minutes. The wide temperature range of 0-90°C can not only protect the heat-sensitive biological samples such as proteins and nucleic acids (which can be performed at 0-4°C), but also accelerate the diffusion and exchange process of the solvent in the pore material by increasing the temperature (such as 50-90°C). The soaking time of more than 5 minutes ensures that there is enough time for the solvent and the crystal to interact fully, ensuring that the nanocrystal to be collected reaches or approaches its equilibrium state in the liquid phase. Through such condition setting, a basis can be provided for subsequent acquisition of high-quality diffraction data and successful structure resolution.

[0035] Furthermore, the maximum size of the nanocrystals to be collected must be less than 3 μm. Firstly, given the limited penetration depth of electron beams in solid materials, excessively large crystals can cause multiple scattering of electrons or prevent penetration, thus weakening the diffraction signal intensity and introducing unnecessary background noise. Secondly, excessively large crystals can prevent the two subsequent continuous carbon support grids from adhering tightly, resulting in an excessively thick liquid layer. This not only exacerbates electron beam scattering and energy loss but may also affect the stability of the liquid pool, leading to solvent leakage or evaporation. Finally, the advantage of three-dimensional electron diffraction technology lies in its ability to obtain single-crystal diffraction data from crystals at the nanometer to submicrometer scale, thereby overcoming the limitations of traditional single-crystal X-ray diffraction on crystal size. Therefore, controlling the crystal size to below 3 μm ensures the acquisition of high-quality diffraction patterns.

[0036] S102. A first volume of the solvent is dropped onto the front side of the first continuous carbon carrier network, the solvent containing the nanocrystals to be collected.

[0037] It should be noted that the first continuous carbon grid is a sample grid commonly used in transmission electron microscopy, and its surface is covered with a complete, non-porous continuous carbon film. Since the carbon film on the surface of the continuous carbon grid provides a uniform, conductive, and impermeable substrate, the use of a continuous carbon grid can form a stable liquid cell and obtain diffraction images with a high signal-to-noise ratio.

[0038] Specifically, the droplet application must be performed on the front side of the first continuous carbon mesh (i.e., the side covered with the continuous carbon film). This is because only the front side has uniform hydrophobic / hydrophilic properties and a complete surface, ensuring the droplet's stable shape and its position in the center of the mesh, thus guaranteeing the success of subsequent coating steps. If the droplet is applied to the back side (the metal mesh side without the carbon film), the liquid will rapidly seep into the mesh gaps due to capillary action or become unevenly distributed, making effective encapsulation impossible.

[0039] It should be noted that this application chooses the dropwise addition method to transfer liquid because this method can achieve precise and controllable transfer of micro-volume liquids, for example, by using a micropipette or capillary tube.

[0040] The initial volume must be trace, typically 0.05 μL to 2 μL. If the volume is too small, a sufficient liquid environment may not be formed to completely encapsulate the nanocrystals, resulting in some crystals being exposed at the vacuum interface, thus negating the significance of liquid-phase testing. If the volume is too large, an excessively thick liquid layer will form, severely scattering the electron beam and causing energy loss, making the diffraction signal unclear or preventing the detection of effective diffraction spots. Therefore, the initial volume needs to be controlled. Through the above operations, the nanocrystals to be tested, currently in a liquid environment, are transferred to a first continuous carbon support network, preparing for the subsequent construction of a dual continuous carbon support network liquid cell.

[0041] S103, covering the second continuous carbon carrier net on the first continuous carbon carrier net, the edges and the front of the first continuous carbon carrier net and the second continuous carbon carrier net form a sealed double continuous carbon carrier net liquid pool, and the solvent is encapsulated in the double continuous carbon carrier net liquid pool; wherein, the front of the second continuous carbon carrier net is opposite to the front of the first continuous carbon carrier net, and the horizontal direction size of the second continuous carbon carrier net is completely same as that of the first continuous carbon carrier net.

[0042] It should be noted that the second continuous carbon carrier net should be consistent with the first continuous carbon carrier net in material, structure and specification, and the reason why the material and the horizontal direction size of the two are the same is that it can ensure that the edges of the two continuous carbon carrier nets can be aligned and closely fitted to the maximum extent, thereby forming effective encapsulation and preventing liquid from leaking or volatilizing from the edges. The size or shape deviation will generate a gap at the interface, which will destroy the sealing of the liquid pool.

[0043] Specifically, the opposite covering means that the front of the second continuous carbon carrier net (i.e. the side covered with continuous carbon film) is opposite to the front of the first continuous carbon carrier net, and covers it. The front-to-front covering method must be used here, because the carbon film surface of the two continuous carbon carrier nets is the most flat, smooth and consistent in chemical properties, and the van der Waals force and other close-range interaction between them can help form a relatively closed microcavity, which can stably trap the liquid droplet in it.

[0044] After covering, the edges and the front of the two continuous carbon carrier nets together form a microcavity which encapsulates the solvent inside, i.e. a double continuous carbon carrier net liquid pool. The sealing here does not mean an absolute airtight or liquid-tight state, but means that within the limited time window of the transmission electron microscopy data acquisition, the structure can effectively block the rapid volatilization of the solvent and limit the liquid flow inside the microcavity, thereby maintaining a stable local liquid phase environment for the nanocrystals to be tested which is sufficient for data acquisition.

[0045] By forming the double-continuous carbon grid liquid pool, the high-vacuum environment of the electron microscope can be prevented from instantaneously sucking dry the solvent, ensuring that the crystal is always observed in its original or target liquid phase state, while effectively preventing the solvent vapor from escaping too quickly and polluting the high-precision electron optical system in the electron microscope barrel. The cavity size of the liquid pool is determined by the gap between the two grids, and the height (i.e. thickness) is much smaller than the width (i.e. grid diameter). Among them, the relationship between the liquid volume and the cavity volume is that the micro-liquid added should be sufficient to fill the flat cavity formed after the two grids are attached, forming a thin liquid film. In the ideal state, the liquid volume is slightly smaller than or equal to the theoretical volume of the cavity, to ensure that the crystal is covered while not causing the grid to bulge or the liquid layer to be too thick due to too much liquid. The relationship between the cavity size and the crystal size is that the height (i.e. liquid layer thickness) of the cavity must be greater than the size of the crystal to be measured, to ensure that the crystal is completely immersed in the liquid phase environment; at the same time, the width of the cavity is much larger than the size of the crystal, which provides sufficient search range for finding and positioning a single dispersed crystal under the electron microscope.

[0046] S104, load the double-continuous carbon grid liquid pool on the sample rod.

[0047] It should be noted that the sample rod is a component used to hold, fix and accurately manipulate the sample grid in the transmission electron microscope. The image acquisition system involved here is composed of an electron microscope, a sample rod and a double-continuous carbon grid liquid pool. The relative position relationship is that the double-continuous carbon grid liquid pool is held at the distal end of the sample rod and inserted into the sample chamber of the electron microscope together with the sample rod, and finally accurately located between the objective lens pole pieces of the electron microscope. The purpose of loading the double-continuous carbon grid liquid pool on the sample rod is to safely and stably deliver it to the vacuum sample chamber of the electron microscope through the sample rod, and to use the mechanical transmission mechanism of the sample rod to achieve the multi-dimensional tilting and positioning of the sample during data acquisition.

[0048] It should be noted that the prepared bicontinuous carbon grid liquid cell has the same size as the standard electron microscope grid, and thus can be directly adapted to various conventional transmission electron microscope sample rods, such as single-tilt rods, double-tilt rods, TOMO rods, and liquid nitrogen rods. Specifically, the bicontinuous carbon grid liquid cell miniaturizes and encapsulates the complex liquid environment in a unit that has the same size as a standard solid sample grid. Therefore, any conventional sample rod that can hold a standard grid can directly hold and manipulate the liquid cell, so that researchers can use the existing conventional sample rods with larger tilt angles and more functions to perform liquid three-dimensional electron diffraction experiments, thereby achieving high-quality analysis of liquid crystal structures without sacrificing data integrity or purchasing expensive special equipment. Existing technologies usually rely on in-situ sample rods that are tailored for specific chip liquid cells. Such in-situ rods are not only extremely expensive, but also have a serious sacrifice in the tilt freedom of the sample due to the mechanical design to accommodate liquid flow lines, and insufficient tilt angle will directly result in incomplete collection of three-dimensional electron diffraction data, thereby failing to resolve high-quality crystal structures by direct method.

[0049] S105, positioning the nanocrystal to be collected in the bicontinuous carbon grid liquid cell using an electron microscope, controlling the rotation of the sample rod, and collecting three-dimensional electron diffraction data of the nanocrystal to be collected.

[0050] It should be noted that the collection of three-dimensional electron diffraction data of the nanocrystal to be collected includes:

[0051] (1) In the low magnification imaging mode of the electron microscope, the nanocrystal to be collected wrapped by liquid in the bicontinuous carbon grid liquid cell is positioned.

[0052] In the low magnification imaging mode of the electron microscope, the bicontinuous carbon grid liquid cell is systematically scanned by observing the bright field image or dark field image of the sample. Specifically, the operator finds and records the accurate position of one or more single nanocrystals to be collected wrapped by liquid, with clear edges and good diffraction contrast, by image contrast difference. The wrapping of liquid will make the crystal edge contrast slightly lower than that in vacuum, but its regular geometric shape and bright diffraction contrast can still be clearly identified.

[0053] (2) Switch to selected area diffraction mode and adjust the camera length.

[0054] The selected area diffraction mode is a working state of the electron microscope, in which the intermediate mirror current is adjusted so that the back focal plane of the objective lens is imaged on the screen, thereby displaying the diffraction spots formed by the superposition of the diffraction waves generated by the crystal. Switching from the imaging mode to the selected area diffraction mode is to convert the observation target from the real space morphology of the crystal to its reciprocal space diffraction information, so as to obtain crystal structure data.

[0055] The camera length determines the magnification and angular resolution of the diffraction pattern. The purpose of adjusting the camera length is to achieve the best balance between the effective separation of diffraction points and the high resolution of the diffraction pattern. On the one hand, a long enough camera length can make the adjacent diffraction points sufficiently dispersed to avoid overlapping, which is convenient for accurate identification and integration. On the other hand, too long a camera length will sacrifice the angular resolution, resulting in the loss of high-frequency (high-resolution) diffraction information. Therefore, in actual operation, the camera length needs to be adjusted to a preset value that can ensure clear separation of diffraction points and obtain the highest possible resolution according to the diffraction ability of the crystal. The value is usually set in a suitable range according to the expected diffraction ability of the crystal and the required resolution, and preferably can be 100-600 mm. Through such design, it is helpful to obtain high-quality diffraction data while managing the electron beam dose and reducing the radiation damage to sensitive samples (crystals and liquids).

[0056] (3) The sample rod on which the double-continuous carbon-carrier mesh liquid cell is mounted is continuously tilted, and the continuous diffraction image acquisition of the nanocrystals to be collected is carried out during the tilting.

[0057] It should be noted that a single diffraction pattern is only a two-dimensional section of the reciprocal space of the crystal. In order to reconstruct the three-dimensional reciprocal space, it is necessary to obtain two-dimensional diffraction patterns under different orientations by tilting the crystal. Using continuous tilting instead of step-by-step tilting can make the data acquisition faster, the total electron dose lower, and the data points continuous, which is more conducive to subsequent data processing and accurate integration. Specifically, by controlling the electron microscope software or an external controller, the sample rod is driven to rotate continuously and uniformly around its axis. At the same time of continuous tilting of the sample rod, the electron detector is started to automatically and synchronously trigger the acquisition of diffraction images at fixed time or angular intervals. In this way, a sequence containing multiple continuous diffraction patterns covering a certain range of tilt angles is obtained.

[0058] In addition, the collection process can be performed through the following detailed steps to ensure that high-quality data is obtained: in the low-magnification imaging mode, the position of at least one liquid-encapsulated nanocrystal to be collected is found; the selected area diffraction mode is switched to, and the camera length is adjusted to a preset value; in the selected area diffraction mode, a selected area diaphragm is inserted to select a single liquid-encapsulated nanocrystal to be collected; the sample rod is controlled to continuously tilt, and during the tilting, a series of diffraction patterns and the sample tilt angles corresponding to each diffraction pattern are recorded. The selected area diaphragm is an adjustable aperture diaphragm in an electron microscope for selecting a specific area in the objective lens image plane. Inserting the diaphragm can ensure that the recorded diffraction signals only come from the selected single crystal, so that pure single-crystal diffraction data is obtained. The single crystal encapsulated by liquid is selected to collect a complete single-crystal diffraction data set that can be used for three-dimensional reconstruction while maintaining the liquid environment of the single crystal. In addition, the tilt angle range of the sample rod is 40°-140°.

[0059] It should be noted that after the diffraction image is collected, the crystal structure can also be analyzed. Specifically, the diffraction pattern is indexed, intensity integrated, corrected, and data merged using professional crystallographic data processing software, so that the three-dimensional reciprocal lattice and structure factor amplitude of the crystal are restored. Then, the phase information of the crystal is solved from the structure factor by using a direct method or a classic Patterson method to determine the initial structure model. Finally, the atomic coordinates, occupancy, and thermal vibration parameters are optimized by least squares refinement, and the accurate atomic-level crystal structure of the nanocrystal to be collected in the liquid environment is finally obtained, including its cell parameters, space group, and atomic arrangement.

[0060] The method provided in this embodiment successfully realizes the collection of high-quality three-dimensional electron diffraction data of nanocrystals in a liquid environment in a conventional transmission electron microscope by using a double-continuous carbon carrier net to construct a sealed liquid pool and combining a continuous rotation data collection mode. Compared with a traditional chip liquid pool, the liquid layer formed by the method is thinner, which effectively improves the diffraction signal quality; the compatibility with a conventional sample rod ensures that the sample can be continuously tilted in a large angle range, so that more complete three-dimensional diffraction data is obtained; and the continuous rotation collection mode greatly reduces the electron cumulative dose and reduces the influence of radiation damage on the crystal structure. The method can effectively capture the real structure state of the crystal in the liquid environment, and provides a reliable technical means for studying solvent effect and phase transition behavior.

[0061] For better illustration, this embodiment provides a specific liquid crystal structure three-dimensional electron diffraction data collection and analysis method, which takes the structure analysis of metal organic framework material MIL-53(Al) in water as an example, and specifically includes the following steps:

[0062] S1, preparing MIL-53(Al) crystals:

[0063] 0.45 g of aluminum nitrate nonahydrate and 0.33 g of terephthalic acid were added to 20 mL of pure water and reacted at 220 °C for 3 days. After the reaction, the sample was cooled, washed with DMF and pure water three times in turn, and then dried at 80 °C overnight to obtain a white powder of MIL-53(Al) synthesis phase. The synthesis phase was calcined at 350 °C for 3 days to obtain the activated phase of MIL-53(Al).

[0064] S2, liquid-phase environment preparation:

[0065] The activated MIL-53(Al) crystals were quickly immersed in pure water and soaked at room temperature for more than 6 hours to allow water molecules to diffuse into the crystal pores and induce a structural transformation.

[0066] S3, construction of a double-continuous carbon-loaded network liquid pool:

[0067] A small drop of the aqueous solution containing MIL-53(Al) crystals obtained in S2 was added to the front of the first continuous carbon-loaded network, and then the front of the second continuous carbon-loaded network was placed on top of the first one to form a double-continuous carbon-loaded network liquid pool encapsulating a liquid.

[0068] S4, sample loading:

[0069] The liquid pool prepared in S3 was mounted on a conventional transmission electron microscope sample rod and sent to the device sample chamber.

[0070] S5, positioning and preparation:

[0071] In the low-magnification imaging mode of the electron microscope, the position of the MIL-53(Al) nanocrystals encapsulated by the liquid was found and recorded; then the selected area diffraction mode was switched to, and the camera length was adjusted to reduce the electron dose and reduce the radiation damage to the crystals.

[0072] S6, collection of three-dimensional electron diffraction data:

[0073] In the selected area diffraction mode, a selected area diaphragm was inserted to select a single MIL-53(Al) nanocrystal encapsulated by the liquid, the sample rod was continuously rotated, and a series of diffraction patterns and the corresponding sample tilt angles were recorded simultaneously during the tilting process.

[0074] S7, crystal structure analysis:

[0075] The collected diffraction patterns were analyzed, and the crystal structure of MIL-53(Al) in the hydrated state was obtained based on the three-dimensional electron diffraction data obtained in S6.

[0076] It should be noted that the reduction of the diffraction data in this embodiment uses the same mature method as in single crystal X-ray diffraction or neutron single crystal diffraction. The method used for structure analysis and refinement is consistent with the general mature method in single crystal X-ray diffraction, neutron single crystal diffraction, powder X-ray diffraction or neutron powder diffraction.

[0077] Further, the MIL-53(Al) soaked in pure water for more than 6 hours was collected by the liquid crystal structure three-dimensional electron diffraction data collection method described in the present application, and the structure obtained by analysis is as shown in Figure 2 Figure 2 The structure diagram of MIL-53(Al) obtained by the liquid crystal structure three-dimensional electron diffraction data collection method provided in the present application). As a control, the MIL-53(Al) soaked in pure water for the same time was collected by the conventional three-dimensional electron diffraction data collection method, and the structure obtained by analysis is as shown in Figure 3 Figure 3 The structure diagram of MIL-53(Al) obtained by the conventional three-dimensional electron diffraction data collection method provided in the present application). The structure data collected and analyzed by the two data collection methods is shown in Table 1. Table 1 is a comparison of the key parameters of the three-dimensional electron diffraction data reduction and structure refinement of MIL-53(Al) in vacuum and hydrated state obtained by analysis in this embodiment:

[0078] Table 1 Comparison of structure analysis parameters of MIL-53(Al) in vacuum and liquid cell

[0079]

[0080] From Table 1, it can be seen that the structure obtained by the conventional method after soaking MIL-53(Al) is a large pore phase, indicating that the water molecules in the pores of MIL-53(Al) are removed by the high vacuum environment of the transmission electron microscope; and the structure obtained by the liquid crystal structure collection method described in the present application is a narrow pore phase, indicating that the double continuous carbon carrier network liquid cell can well maintain the water molecules in the pores of MIL-53(Al) from being removed by vacuum. Moreover, the narrow pore structure of MIL-53(Al) obtained by the double continuous carbon carrier network liquid cell method is also the first time to analyze a single crystal by a direct method at room temperature, which reflects the outstanding advantages of the double carbon film liquid cell in the analysis of liquid crystal structure.

[0081] ​​To verify the universality of the liquid phase crystal structure three-dimensional electron diffraction data collection method described in the application, we also collected and analyzed the three-dimensional electron diffraction data of zeolite material ZSM-5 under liquid phase: ZSM-5 was soaked in pure water for more than 5 minutes, and a double-continuous carbon-loaded net liquid pool was prepared by the method described in the application, and then the sample rod was sent into the transmission electron microscope sample chamber, and then the three-dimensional electron diffraction data was collected, and the structure parameters obtained by direct method analysis are shown in Table 2.

[0082] Table 2 Structure parameters of ZSM-5 in liquid pool

[0083]

[0084] Referring to Table 2, the three-dimensional electron diffraction data of zeolite ZSM-5 under pure water environment was successfully collected and analyzed using the method described in the application, and reasonable cell parameters, space group and refinement index were obtained, which shows that the method is suitable for different types of crystal materials (such as zeolite), and further verifies the universality and reliability of the method in analyzing the structure of nanocrystals under liquid phase environment.

[0085] Example two

[0086] Corresponding to the foregoing embodiment of the liquid phase crystal structure three-dimensional electron diffraction data collection method, the application also provides an embodiment of a liquid phase crystal structure three-dimensional electron diffraction data collection device.

[0087] Figure 4 The structure diagram of the second embodiment of the liquid phase crystal structure three-dimensional electron diffraction data collection device provided by the application is shown in FIG. 2. Referring to FIG. 2, Figure 4 The device provided in this embodiment comprises:

[0088] The double-continuous carbon-loaded net liquid pool is formed by dropping the solvent containing the nanocrystal to be collected on the front surface of the first continuous carbon-loaded net, and then covering the second continuous carbon-loaded net opposite to the first continuous carbon-loaded net; wherein the front surface of the second continuous carbon-loaded net is opposite to the front surface of the first continuous carbon-loaded net, the second continuous carbon-loaded net and the first continuous carbon-loaded net form a sealed structure at the edges and front surfaces, and the horizontal direction size is completely the same, for packaging the solvent and maintaining the liquid phase environment of the nanocrystal to be collected;

[0089] The sample rod is used to carry the double-continuous carbon-loaded net liquid pool;

[0090] The electron microscope is used to position the nanocrystal to be collected after the double-continuous carbon-loaded net liquid pool is placed in the sample chamber, control the rotation of the sample rod, and collect the three-dimensional electron diffraction data of the nanocrystal to be collected.

[0091] Specifically, please refer to the foregoing description, which will not be repeated here.

[0092] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for acquiring three-dimensional electron diffraction data of a liquid-phase crystal structure, characterized in that, The method comprises: immersing the nanocrystals to be collected in a solvent; dropping a first volume of the solvent containing the nanocrystals to be collected on the front surface of a first continuous carbon support net; covering a second continuous carbon support net on the first continuous carbon support net, the edges and front surfaces of the first continuous carbon support net and the second continuous carbon support net forming a sealed double continuous carbon support net liquid pool, and the double continuous carbon support net liquid pool encapsulating the solvent; wherein the front surface of the second continuous carbon support net is opposite to the front surface of the first continuous carbon support net, and the horizontal dimension of the second continuous carbon support net is the same as that of the first continuous carbon support net; mounting the double continuous carbon support net liquid pool on a sample rod; positioning the nanocrystals to be collected in the double continuous carbon support net liquid pool by using an electron microscope, controlling the rotation of the sample rod, and collecting three-dimensional electron diffraction data of the nanocrystals to be collected; The collection of three-dimensional electron diffraction data of the nanocrystals to be collected comprises: positioning the nanocrystals to be collected wrapped by liquid in the double continuous carbon support net liquid pool in a low-power imaging mode of the electron microscope; switching to a selected area diffraction mode and adjusting the camera length; continuously tilting the sample rod mounted with the double continuous carbon support net liquid pool and continuously collecting diffraction images of the nanocrystals to be collected during the tilting; The collection of three-dimensional electron diffraction data of the nanocrystals to be collected specifically comprises: finding the position of recording at least one nanocrystal to be collected wrapped by liquid in the low-power imaging mode; switching to a selected area diffraction mode and adjusting the camera length to a preset value; selecting a single nanocrystal to be collected wrapped by liquid by inserting a selected area diaphragm in the selected area diffraction mode; controlling the continuous tilting of the sample rod and recording a series of diffraction images and the sample tilt angles corresponding to each diffraction image during the tilting.

2. The method of claim 1, wherein, The solvent comprises polar solvents and non-polar solvents, and the polar solvents at least include any one of water, ethylene glycol, chlorobenzene, pyridine, DMF, methanol, ethanol, THF, and isopropanol; and the non-polar solvents at least include any one of benzene, tetrachloromethane, cyclohexane, and n-hexane.

3. The method of claim 1, wherein, The immersion of the nanocrystals to be collected in the solvent comprises: immersing the nanocrystals to be collected in the solvent at a temperature of 0-90°C for a time greater than 5 minutes.

4. The method of claim 1, wherein, The nanocrystals to be collected at least include any one of oxides, alloys, inorganic-organic hybrid materials, zeolites, organic crystals, metal complexes, metal-organic frameworks, covalent-organic frameworks, protein crystals, nucleic acid crystals, and drug molecule crystals; and the maximum size of the nanocrystals to be collected is less than 3 mm.

5. The method of claim 1, wherein, The rotation angle of the sample rod is 40°-140°.

6. The method of claim 1, wherein, The camera length is adjusted to 100-600 mm.

7. The method of claim 1, wherein, The first volume is 0.05-2 μL.

8. A liquid-phase crystal structure three-dimensional electron diffraction data collection device, which is prepared based on the method according to any one of claims 1-7, and comprises: A double-continuous carbon-supported mesh liquid cell is formed by dropping a second continuous carbon-supported mesh on the front side of a first continuous carbon-supported mesh after dropping a solvent containing nanocrystals to be collected on the front side of the first continuous carbon-supported mesh; wherein the front side of the second continuous carbon-supported mesh is opposite to the front side of the first continuous carbon-supported mesh, the second continuous carbon-supported mesh and the first continuous carbon-supported mesh form a sealed structure with the edges and the front sides, and the horizontal dimensions are completely the same, for packaging the solvent and maintaining the liquid phase environment of the nanocrystals to be collected; A sample rod for carrying the double-continuous carbon-supported mesh liquid cell; An electron microscope for positioning the nanocrystals to be collected, controlling the rotation of the sample rod, and collecting three-dimensional electron diffraction data of the nanocrystals to be collected after the double-continuous carbon-supported mesh liquid cell is placed in the sample chamber of the electron microscope.

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