Method and system for determining crystal structure

Abstract

To provide a method and a system for determining a crystal structure.SOLUTION: A molecular structure of a crystal may be solved based on at least two diffraction tilt series acquired from a sample. The two diffraction tilt series include multiple diffraction patterns of at least one crystal of the sample acquired at different electron doses. In some examples, the two diffraction tilt series are acquired at different magnifications.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] This specification generally relates to methods and systems for crystal structure analysis, and more specifically, to determining a three-dimensional electron potential map of the molecules constituting a crystal based on a tilt series of diffraction patterns of the crystal across a portion of the crystal's 3D reciprocal lattice space.

Background Art

[0002] The molecular structure of a crystal sample can be obtained by analyzing an electron diffraction tilt series of the crystal, i.e., electron diffraction patterns acquired from various angles of the crystal. The electron diffraction tilt series can be acquired in a transmission mode where electrons scattered from the crystal are acquired from the opposite side of the electron source. The angle between the crystal and the electron beam can be adjusted by tilting the electron beam using a deflector or by rotating the sample using a sample stage. The large dynamic range of the intensity and spatial distribution of the diffraction spots in the diffraction pattern can make it difficult to accurately collect the tilt series of diffraction.

Summary of the Invention

[0003] In one embodiment, the method includes obtaining a first tilt series of diffraction of a sample, wherein each diffraction pattern in the first tilt series has a first electron dose and a first magnification; obtaining a second tilt series of diffraction of the sample, wherein each diffraction pattern in the second tilt series has a second electron dose and a second magnification; and solving for the molecular structure of the crystal based on the first tilt series of diffraction and the second tilt series of diffraction.

[0004] In another embodiment, the method includes acquiring one or more first diffraction patterns of a crystal with a first electron dose; acquiring one or more second diffraction patterns of a crystal with a second electron dose; acquiring a high-resolution dataset based on the first diffraction patterns; acquiring a low-resolution dataset based on the second diffraction patterns; generating a merged dataset by combining the high-resolution dataset and the low-resolution dataset in reciprocal space; and solving the molecular structure of the crystal based on the merged dataset. In this way, high-quality diffraction patterns can be obtained for accurate crystal molecular structure analysis.

[0005] It should be understood that the above summary is provided in a simplified manner to introduce a selection of concepts that will be further described in the modes for carrying out the invention. It is not intended to identify the main or essential features of the claimed subject matter, and its scope is uniquely defined by the claims that follow the modes for carrying out the invention. Furthermore, the claimed subject matter is not limited to implementations that solve any of the defects mentioned above or in any part of this disclosure. [Brief explanation of the drawing]

[0006] [Figure 1] An example of a transmission electron microscope system is shown.

[0007] [Figure 2] Selected field electron diffraction pattern.

[0008] [Figure 3] This paper describes a method for determining the crystal structure based on diffraction gradient series.

[0009] [Figure 4A] This shows the change in diffraction spots in response to adjustments in electron dose and magnification. [Figure 4B] This shows the change in diffraction spots in response to adjustments in electron dose and magnification.

[0010] [Figure 5] This paper presents another method for determining the crystal structure based on diffraction gradient series.

[0011] Similar reference numbers refer to corresponding parts across several figures in the drawing. [Modes for carrying out the invention]

[0012] Electron diffraction (ED) is a technique for determining the molecular (or atomic) structure of large (biomolecules). In this technique, a relatively large quantity of the molecule to be structured is generated and then processed to form a large crystal. The unit cells of this crystal are formed by these (biomolecules). Due to the repeating structure of the crystal, each interatomic distance within the molecule is repeated many times (the same number of times as the number of unit cells). These repeating distances act as a lattice, so the diffracted portion of the electron beam has a specific direction, and its magnitude corresponds to this interatomic distance according to Bragg's law of diffraction. The direction of scattering indicates the direction of this interatomic distance within the (biomolecule). The intensity (or probability) of this scattering is proportional to the square of the number of electrons in the atom (the atom corresponding to this distance) and approximately proportional to the fourth power of the scattering angle. The complete set of diffracted beam forms a diffraction pattern within the back focal plane of the first imaging lens of the electron microscope. This diffraction pattern is imaged onto the camera / detector by appropriately adjusting the electron-optical lens between the first imaging lens and the microscope's camera / detector.

[0013] The center of a diffraction pattern is formed by the undiffracted beam. Typically, the intensity of this central spot is much higher than that of the adjacent diffraction spots. The camera's dynamic range is limited between the smallest signal a pixel can detect (e.g., one primary electron) and the largest signal a pixel can detect before it saturates (e.g., 10,000 primary electrons). This dynamic range of the camera is generally not large enough to cover the very large intensity difference (up to 10⁸ times) between the central spot and the weak diffraction spots. Therefore, to record the weak spots with sufficient accuracy, the central spot must be blocked to prevent the camera from oversaturating. Such blocking can be done by a so-called "beam stopper," which is a small needle that can be inserted into the electron beam and shaped to precisely block the central portion of the diffraction pattern.

[0014] Diffraction patterns show a 2D projection of the 3D distribution of interatomic distances within a (biomolecule). Reconstructing the complete 3D structure of a molecule generally requires combining many of these 2D projections, each collected under different projection directions (or different angles of incidence). Such a collection can be obtained in several ways, for example, by rotating the sample between different image collections. Alternatively, many crystals, each with a different orientation, can be dispersed across the sample, and images of each of these crystals can be collected.

[0015] In particular, when each of a multipolar crystal is irradiated simultaneously in its own orientation, it is desirable to record only the diffraction pattern of a single crystal. This can be achieved by appropriately applying a selective aperture in the intermediate (usually first) image plane of the sample. Such an aperture is called a limited-field aperture, and the diffraction pattern is called a limited-field diffraction pattern. Often, the term "limited-field (electron) diffraction (pattern)" is used even when a limited-field aperture is not actually used.

[0016] ED (Electron Diode) is used to decompose the molecular (or atomic) structure of a (biomolecule) of interest. Strictly speaking, ED decomposes a 3D map of potentials within a molecule, rather than atomic positions, because electrons in the beam are scattered not on atoms, but on potentials formed by electrons bonded to atoms within the molecule. However, since this potential map is (almost) identical to the atomic structure, ED is commonly referred to as a method for decomposing molecular structure.

[0017] The term "crystal structure" can be constrained to refer only to the structure and symmetry (e.g., hexagonal, cubic, or orthorhombic) in which unit cells are assembled within a crystal. However, the term is often also used to refer to the structure within the crystal, i.e., the molecular (or atomic) structure of the (bio)molecule of interest. In this literature, the term "crystal structure" is used to refer to both the packing of unit cells and the atomic structure within the unit cells. The term "molecular structure of a crystal" is used to refer to the molecular or atomic structure of the unit cells (i.e., the (bio)molecule of interest) that make up the crystal.

[0018] The following description concerns electron diffraction (ED) tilt series, i.e., systems and methods for determining the structure of a crystal based on 3D ED patterns acquired from various angles of the crystal. For example, the ED pattern of a crystal can be acquired by adjusting the incident angle of the electron beam by rotating the crystal using a sample stage and / or by tilting the electron beam. In some examples, multiple crystals in a crystal sample may be probed for the ED pattern. For crystal structure analysis of the structure, it is important to accurately acquire the intensity and spatial distribution of diffraction spots within the ED pattern. However, the dynamic range of the diffraction spot intensity may exceed the dynamic range of the detector / camera. In the case of selected area (SA) ED patterns, as shown in Figure 2, some lower-resolution diffraction spots are blocked by the beam stopper and therefore not captured by the detector. These lower-resolution diffraction spots may be important for determining the structure of crystals with large unit cells, such as unit cells larger than 10 nm.

[0019] To address the above issues, a method and system for determining molecular structure based on 3D ED diffraction patterns are presented. The 3D ED pattern may include multiple ED gradient series acquired at different electron doses and / or different magnifications. The electron dose is e / Å. 2 It may have units of . 3D ED diffraction patterns can be acquired using a transmission electron microscopy (TEM) system such as the TEM system shown in Figure 1. In one example, two diffraction gradient series may be constructed from the 3D ED pattern: a first diffraction gradient series containing multiple ED patterns acquired at a first higher electron dose, and a second diffraction gradient series containing multiple ED patterns acquired at a second lower electron dose. The first electron dose is just below the critical dose that causes radiation damage to the finer details of molecules in the crystal.

[0020] The first and second diffraction gradient series may be acquired from one or more crystals in the sample. The electron dose may be adjusted while probing the same crystal or after probing multiple crystals. In one example, the first and second diffraction gradient series are acquired from a single crystal. The first diffraction gradient series is acquired by directing the electron beam to the crystal and rotating the crystal using a sample stage and / or tilting the electron beam using a deflector. After acquiring the first diffraction gradient series, the electron dose is adjusted from the first to the second electron dose, and the second diffraction gradient series is acquired from the same crystal by rotating the crystal using a sample stage and / or tilting the electron beam using a deflector. In another example, the sample contains multiple crystals. As in the previous example, multiple ED patterns of the first crystal are acquired at the first and second electron doses. The electron beam is then directed to the second crystal via beam shift or movement of the sample stage. Multiple ED patterns of the second crystal are acquired at the first and second electron doses. The first and second diffraction gradient series can be constructed from the ED patterns of the first and second crystals based on electron dose. In yet another example, the first diffraction gradient series is acquired by directing an electron beam to each of several crystals. At each crystal position, one or more ED patterns are acquired at the first electron dose. Next, the second diffraction gradient series is acquired by directing an electron beam to each of several crystals, and one or more ED patterns are acquired at each crystal position at the second electron dose. In some examples, all ED patterns of at least one crystal are acquired at only one electron dose. Thus, the two diffraction gradient series may contain ED patterns generated from different crystal subsets.

[0021] In this way, different dose ED patterns are acquired for each crystal, with higher dose ED patterns acquired before lower dose ED patterns. The first electron dose is selected to ensure visibility of higher resolution diffraction spots within the higher dose ED pattern. In one example, the first electron dose is 80% to 100% of the critical dose. In another example, the first dose is 50% to 100% of the critical dose. The critical dose is the dose at which radiation damages the finer details of molecules within the crystal, significantly reducing the intensity of high-resolution spots within the diffraction pattern. The critical dose depends on the type of crystal. After acquiring higher dose ED patterns, radiation damage may destroy the finer details (or structures) corresponding to higher resolution diffraction spots. Lower resolution diffraction spots, which are more resistant to radiation damage, may be captured in lower dose ED patterns. Furthermore, because the electron dose decreases, it is possible to accurately record the intensity of lower resolution diffraction spots within lower dose ED patterns without saturating the detector. In this way, diffraction spots with a wide dynamic range can be recorded. The electron dose can be adjusted by adjusting one or more of the exposure time per frame and the dose rate. The dose rate can be adjusted by adjusting the settings of the illumination optical system.

[0022] In some examples, the ED pattern of the first diffraction gradient series is acquired at a lower magnification using a shorter camera length, and the ED pattern of the second diffraction gradient series is acquired at a higher magnification using a longer camera length. Here, "camera length" refers to the magnification at the camera / detector from the back focal plane of the first imaging lens multiplied by the focal length of this first imaging lens. By adjusting the magnification, lower resolution diffraction spots closer to the center of the ED pattern in the first diffraction gradient series are broadened and can be easily distinguished in the second diffraction gradient series. Furthermore, lower resolution diffraction spots are enlarged and may occupy more pixels in the second diffraction gradient series, thereby further avoiding detector saturation.

[0023] The molecular structure of the crystal can be obtained based on the first diffraction tilt series and the second diffraction tilt series. The molecular structure can be revealed as a 3D electron potential map of the molecule. In one example, a high-resolution data set is obtained based on the first diffraction tilt series, and a low-resolution data set is obtained based on the second diffraction tilt series. The high-resolution data set and the low-resolution data set are merged in reciprocal lattice space. The molecular structure of the crystal is determined based on the merged data set. The high-resolution data set and the low-resolution data set each contain a sorted list of reflections generated based on their respective diffraction tilt series and known structural characteristics of the crystal (such as symmetry and shape). Combining the high-resolution data set and the low-resolution data set in reciprocal lattice space is advantageous compared to directly merging the diffraction tilt series by adding the ED patterns obtained at the same incident angle. This is because complex image processing procedures such as image correction and registration for merging ED patterns from different tilt series can be avoided. Also, the ED patterns from different diffraction tilt series can correspond to different incident angles.

[0024] Referring to FIG. 1, a transmission electron microscopy (TEM) system 100 is shown in different operating modes. The TEM system 100 includes an electron source 10 that emits an electron beam 11 along an optical axis 110 towards a condenser optical system 12. The electron source 10 can generate high-energy electrons, i.e., electrons having a typical energy of about 10 keV to 1,000 keV. In some embodiments, the condenser optical system 12 can include one or more condenser lenses and one or more apertures. A deflector 19 positioned downstream of the condenser optical system 12 shifts and / or tilts the electron beam with respect to the optical axis 110. A pre-sample objective lens 16 positioned downstream of the deflector 19 collimates the electron beam and directs the electron beam towards a sample 14. The sample 14 can be held by a sample stage 13 within a sample plane 111. In some examples, the sample is positioned on a TEM grid attached to the sample stage. The sample stage 13 can adjust the sample position by tilting the sample with respect to the optical axis and / or translating the sample within the sample plane. Scattered electrons that pass through the sample 14 sequentially pass through a post-sample objective lens 123 and a projection system 21 and are collected by a detector 25 positioned on the opposite side of the sample 14 with respect to the electron source 10. The projection system 21 performs different operations in an imaging mode and a diffraction mode. The detector 25 can detect the received electrons and send a signal to an image processor 24 to form an image. The detector 25 can include an amplifier for amplifying the signal before sending the signal to the image processor 24. In one example, the detector 25 can be a CCD camera or a CMOS camera. In some embodiments, different detectors can be used for obtaining a diffraction pattern and obtaining a sample image.

[0025] Figure 1 shows the TEM system 100 operating in SA imaging mode and SA diffraction mode. The dashed line 41 shows the beam path of scattered electrons from sample 14 to detector 25 in SA diffraction mode. In SA diffraction mode, the projector system 21 images the back focal plane 43 of the back objective lens 123 of the sample to detector 25. A beam stopper 17 is inserted into the optical axis 110 to block the unscattered beam. The dashed line 42 shows the beam path of scattered electrons from sample 14 to detector 25 in SA imaging mode. In SA imaging mode, the sample plane 111 is imaged into the SA plane 44, and the projector system 21 images the SA plane 44 to detector 25. The beam stopper 17 is retracted from the optical axis 110. In one example, an SA aperture may be inserted into the beam path. The SA aperture may be positioned within the SA plane 44. Alternatively, an SA aperture in the condenser optics 12 may function as a beam limiting aperture. In another example, an image deflector may be positioned between the sample and the detector to shift and tilt electrons transmitted through the sample back to the optical axis so that the ED pattern remains at the center of the detector during beam tilt and the image remains at the center of the detector during beam shift. The image deflector 45 may be positioned between the back focal plane 43 and the SA plane 44. In some embodiments, the TEM system does not include a beam stopper, and the detector receives an unscattered beam.

[0026] The controller 30 can control the operation of the TEM system 100 manually in response to operator commands, or automatically according to computer-readable commands stored in non-temporary memory (or computer-readable medium) 32. The controller 30 may include a processor and be configured to execute computer-readable commands to control various components of the TEM system 100 in order to carry out any of the methods described herein. For example, the controller may adjust the TEM system to operate in different modes by adjusting one or more of the aperture 18, the intensity of the objective lens 123, the beam stopper 17, and the projector system 21. The controller 30 may adjust the beam position and / or beam incidence angle relative to the sample by adjusting the deflector 19. The controller 30 may adjust the electron dose of each ED pattern by adjusting one or more of the illumination optical system settings, the exposure time for each frame acquired by the detector, and the angular velocity of beam tilt / sample rotation. The controller 30 may adjust the magnification by adjusting the projector system 21. The controller 30 may be further coupled to the display 31 to display notifications and / or signals detected by the detector 25. The controller 30 may receive user input from a user input device 33. The user input device 33 may include a keyboard, mouse, or touchscreen. The controller may be configured to solve the molecular structure of a crystal based on a 3D ED diffraction pattern.

[0027] While a TEM system is described as an example, it should be understood that sample images and diffraction patterns can be obtained using other charged particle microscopy systems. Another example of a charged particle microscopy system is a scanning transmission electron microscope (STEM) system. Sample images can be created in scanning STEM mode, and diffraction images can be obtained using (quasi-)parallel beams. This discussion of the TEM system is provided merely as an example of one suitable imaging modality.

[0028] Figure 2 shows an example of an ED pattern acquired in SA diffraction mode at an electron energy of 300 kV. Crystal lattice planes with relatively large separation (coarse details / structure) resulted in lower resolution diffraction spots located closer to the center of the diffraction pattern, while crystal lattice planes with narrower spacing (finer details / structure) resulted in higher resolution diffraction spots located further from the center of the diffraction pattern. The shadow 204 at the center of the diffraction pattern is a shadow projected by a beamstopper (e.g., beamstopper 17 in Figure 1). Low-resolution diffraction spots 201, 202, and 203 saturate even when higher-resolution diffraction spots (diffraction spots 205, 206, etc.) are weaker. Furthermore, in some examples, low-resolution diffraction spots may be blocked by the beamstopper shadow 204 and not recorded in the ED pattern.

[0029] Figure 3 shows an exemplary method 300 for determining a crystal structure based on diffraction gradient series of different electron doses. The sample may contain one or more crystals. After identifying the crystals for ED pattern acquisition, a first set of multiple ED patterns is acquired at a first electron dose at each location of the selected crystal. Then, a second set of multiple ED patterns is acquired for the same selected crystal. While acquiring each of the first and second sets of multiple ED patterns, the incident angle is adjusted by the tilt of the beam and / or the rotation of the sample. The first and second diffraction gradient series are constructed from the ED patterns of the selected crystals based on the corresponding electron doses. The crystal structure is then determined based on the two diffraction gradient series.

[0030] In 302, the parameters of the microscope system are set. The parameters may include one or more of the following: beam current, first and second electron doses, first and second magnifications, and tilt parameters. The setting of the first and second electron doses may include setting the electron dose rate and the detector frame time. The first electron dose may be determined based on the radiation damage threshold of the crystal. The first electron dose is set high so that after acquiring the ED pattern, structures that produce higher resolution spots are destroyed or nearly destroyed. The setting of the tilt parameter may include setting the beam tilt and / or the angular step size and / or angular velocity of the sample rotation. The first and second magnifications may be determined based on prior knowledge of the beam stopper size and / or the unit cell size. For example, the second magnification is selected so that the diffraction spots necessary to solve the 3D potential map are not blocked by the beam stopper. In one example, the second magnification is 5 times the first magnification.

[0031] In step 304, one or more sample images of the region of interest (ROI) of the sample are acquired. The sample images are acquired in imaging mode. The sample images may have a resolution that allows for the determination of the crystal size and shape. If the area of ​​the ROI is larger than the field of view of a single sample image, multiple sample images may be stitched together to cover the ROI.

[0032] In step 306, one or more crystals within the ROI are selected. The crystals may be selected based on one or more of the size, distribution, morphology, and image contrast of the crystals in the sample image acquired in step 304. In some cases, it may be possible to determine whether a particle is crystalline by probing one or more particles in the sample image in diffraction mode. If the diffraction pattern does not show clear diffraction spots, the particle may not be crystalline. Non-diffractive particles are excluded from the selected crystals. Furthermore, the position or coordinates of the selected crystals may also be determined based on the sample image.

[0033] In 308, the electron beam is directed to one of the selected crystals via beam shift and / or sample stage shift in the sample plane. The electron beam may be directed to the crystal based on the coordinates of the selected crystal. The electron beam or sample stage may be guided, alternatively or additionally, by real-time sample imaging.

[0034] In 310, the microscope system is adjusted to a first higher electron dose. The magnification can also be adjusted to a lower first magnification. Multiple first ED patterns are acquired at different incidence angles using different probing schemes. In one example, the ED pattern is acquired while continuously or incrementally rotating the sample through a sample stage. The crystal is positioned at the eucentric center of the sample stage. In another example, the ED pattern is acquired by tilting the electron beam through a beam deflector. In yet another example, the ED pattern is acquired by combining beam tilt and sample rotation. For example, after rotating the sample in large angular steps, the electron beam is tilted to acquire an ED pattern covering finer angular steps. While acquiring multiple first ED patterns, the sample shift may be corrected based on, for example, a sample image acquired in imaging mode.

[0035] In 312, the microscope system is adjusted to a second, lower electron dose. The magnification may be adjusted to a second, higher magnification. The second magnification may be determined based on prior knowledge of the beamstopper size, the first magnification, and the sample characteristics. Sample characteristics may include the range of unit cell sizes and possible diffraction spot distributions. Multiple second ED patterns are acquired at different incidence angles using the same probing scheme as in 310. The data acquisition times for the first and second diffraction gradient series may be the same. The dose rate for the second diffraction gradient series may be lower than that of the first diffraction gradient series. The scanning parameters for the first and second diffraction gradient series may be the same.

[0036] Figures 4A and 4B show the radial intensity distribution of ED patterns of the same crystal. The y-axis represents intensity, and the x-axis represents distance from the center of the ED pattern. Figure 4A corresponds to a first ED pattern acquired at a higher electron dose and lower magnification. Figure 4B corresponds to a second ED pattern acquired at a lower electron dose and higher magnification. At lower magnifications, higher resolution diffraction peaks 404 can be captured in Figure 4A. However, due to the high electron dose, lower resolution peaks 406 are saturated in Figure 4A. As the magnification decreases, diffraction peaks 408 move away from the center of the ED pattern. The intensity of diffraction peaks 408 also decreases with decreasing electron dose. If the ED pattern is an SA diffraction pattern acquired using a beamstopper 402, lower resolution diffraction peaks 406 cannot be captured within the first ED pattern. By increasing the magnification, in Figure 4B, lower resolution peaks 406 are no longer blocked by the beamstopper 402. Furthermore, as the electron dose decreases, the lower-resolution peak 406 in Figure 4B is not saturated. By acquiring both the first and second ED patterns of the same crystal, diffraction spots can be captured with a large dynamic range intensity and resolution distribution.

[0037] Returning to Figure 3, at 314, method 300 checks whether the ED patterns for all selected crystals have been obtained. If the answer is no, the electron beam is directed to the next selected crystal at 308. Otherwise, method 300 proceeds to 316.

[0038] In 316, first and second diffraction gradient series are constructed based on a first and second set of ED patterns from a selected crystal. For example, the first diffraction gradient series includes all first set of ED patterns acquired at a first dose, and the second diffraction gradient series includes all second set of ED patterns acquired at a second dose. High-resolution and low-resolution datasets are generated based on the first and second diffraction gradient series. For each ED pattern in the gradient series, it can be determined whether (some of) these patterns should be considered useful data or outliers by comparing the correlation of unit cell parameters and / or intensity. For example, ED patterns from damaged crystals and / or ED patterns acquired from adjacent gradient angles that differ too greatly from those obtained from adjacent gradient angles are removed from the diffraction gradient series. Dataset acquisition may involve 3D integrating the intensity of each diffraction spot within a diffraction gradient series, scaling the integrated intensity based on electron dose, applying corrections (e.g., Lorentz factor, absorption correction), and generating a sorted list of reflections based on the integrated intensity and known structural properties of the crystal (such as symmetry and shape). For example, each reflection may have an index (h, k, l), intensity, and standard deviation of intensity.

[0039] In 318, the crystal structure is determined based on high-resolution and low-resolution datasets. In one example, in 320, a merged dataset is obtained based on the high-resolution and low-resolution datasets. The high-resolution and low-resolution datasets can be scaled, after correction, to minimize the intensity difference between identical reflections (i.e., reflections with the same index) from different measurements (e.g., different gradient series), and all their symmetry-associated equivalents.

[0040] In 322, the crystal structure can be determined based on the merged dataset using a conventional crystal structure analysis software package. In 324, the crystal structure is improved using the merged dataset to optimize atomic positions while fitting other parameters, for example, to represent disorder and twinning, using a conventional crystal structure analysis software package.

[0041] Figure 5 shows another exemplary method 500 for determining crystal structure based on diffraction gradient series acquired at different electron doses. Multiple selected crystals within an ROI are scanned multiple times at different electron doses. In each scan, one or more ED patterns are acquired from each crystal at the same electron dose. Unlike method 300, where the electron dose is adjusted while probing one crystal before moving to the next, method 500 acquires ED patterns of multiple crystals at a first electron dose, and then acquires ED patterns of multiple crystals at a second electron dose.

[0042] In 502, system parameters are set. Similar to 302 of Method 300, the system parameters may include one or more of the beam current, first and second electron doses, first and second magnifications, and gradient parameters.

[0043] In 504 and 506, sample images are acquired, similar to 304 and 306 of method 300, and one or more crystals are selected based on the sample images.

[0044] In 508, the microscope system is set up to acquire an ED pattern at a first higher electron dose. The microscope system may also be adjusted to a first lower magnification. The electron beam is directed to one or more selected crystals via beam shift and / or translation of the sample stage in the sample plane, and one or more ED patterns of the crystals are acquired.

[0045] In 510, the microscope system is set up to acquire an ED pattern at a second, lower electron dose. The microscope system may also be adjusted to a second, higher magnification. The electron beam is directed to one or more selected crystals via beam shift and / or translation of the sample stage in the sample plane, and one or more ED patterns of the crystals are acquired. The crystals probed in 508 and 510 may belong to different subsets of the selected crystals.

[0046] In step 512, first and second slope series are constructed. The first slope series may be constructed based on the ED pattern acquired in step 508, and the second slope series may be constructed based on the ED pattern acquired in step 510. Furthermore, high-resolution and low-resolution datasets are generated based on the first and second slope series, respectively.

[0047] In 514, the crystal structure is determined based on high-resolution and low-resolution datasets, similar to 318 of method 300.

[0048] In this way, diffraction spots with a wide range of intensity and resolution can be captured. Based on multi-dose and / or multi-magnification diffraction gradient series, complex crystal structures can be accurately determined.

[0049] The technical benefit of acquiring ED patterns of the same crystal at different doses is that the dynamic range of the captured signal intensity can be greater than the dynamic range of the detector. The technical benefit of acquiring ED patterns of the same crystal at different magnifications is that diffraction spots with higher and lower spatial resolutions can be obtained. Furthermore, diffraction spots blocked by beam stoppers can be captured. The technical benefit of solving high-resolution and low-resolution datasets in reciprocal space is that complex imaging procedures for directly merging higher and lower-resolution ED patterns can be avoided. [Explanation of symbols]

[0050] 100 Transmission Electron Microscopy (TEM) Systems 24 Image Processors 30 controllers 31 displays 32 memory 33 User Input Devices

Claims

1. It is a method, To acquire a first diffraction gradient series of a sample, wherein each diffraction pattern within the first diffraction gradient series has a first electron dose and a first magnification. To obtain a second diffraction gradient series of the sample, wherein each diffraction pattern in the second diffraction gradient series has a second electron dose and a second magnification. This includes solving the molecular structure of the crystal based on the first diffraction gradient series and the second diffraction gradient series, A method wherein the first electron dose is higher than the second electron dose, and the first magnification is lower than the second magnification.

2. The method according to claim 1, wherein a beam stopper is used during the acquisition of the first diffraction gradient series and the second diffraction gradient series, and the method further comprises adjusting the first magnification to the second magnification based on the size of the beam stopper.

3. The method according to claim 1, further comprising adjusting the first magnification to the second magnification based on the unit cell size.

4. The method according to any one of claims 1 to 3, wherein solving the molecular structure of a crystal based on the first diffraction gradient series and the second diffraction gradient series comprises obtaining a higher resolution dataset based on the first diffraction gradient series, obtaining a lower resolution dataset based on the second diffraction gradient series, obtaining a merged dataset by combining the higher resolution dataset with the lower resolution dataset in reciprocal space, and solving the molecular structure based on the merged dataset.

5. The method according to any one of claims 1 to 4, wherein obtaining the first diffraction gradient series includes obtaining a plurality of diffraction patterns of the first crystal at a first electron dose by adjusting the angle between the first crystal of the sample and the electron beam, and obtaining the second diffraction gradient series includes obtaining a plurality of diffraction patterns of the first crystal at a second electron dose by adjusting the angle between the first crystal and the electron beam.

6. The method according to claim 5, wherein the second diffraction gradient series is obtained after obtaining the first diffraction gradient series.

7. The method according to claim 5, wherein the angle between the first crystal and the electron beam is adjusted by tilting the electron beam and / or by rotating a sample stage for holding the sample.

8. The method according to claim 5, wherein obtaining the first diffraction gradient series further comprises obtaining a plurality of diffraction patterns of the second crystal at the first electron dose by adjusting the angle between the second crystal of the sample and the electron beam.

9. The method according to claim 1, wherein the sample comprises a plurality of crystals, and obtaining the first diffraction gradient series comprises directing an electron beam to one or more of the plurality of crystals and obtaining one or more diffraction patterns for each of the plurality of crystals, and obtaining the second diffraction gradient series comprises directing the electron beam to one or more of the plurality of crystals and obtaining one or more diffraction patterns for each of the plurality of crystals.

10. The method according to claim 1, wherein the data acquisition time for the first diffraction gradient series is the same as the data acquisition time for the second diffraction gradient series.

11. It is a system, An electron source for generating an electron beam along the optical axis, A sample stage for holding the sample and adjusting the sample position, A detector for detecting electrons that have passed through the aforementioned sample, A controller including non-temporary memory for storing computer-readable instructions, wherein the controller executes the computer-readable instructions, To obtain a first diffraction gradient series of the sample, wherein each diffraction pattern in the first diffraction gradient series has a first electron dose and a first magnification. To obtain a second diffraction gradient series of the sample, wherein each diffraction pattern in the second diffraction gradient series has a second electron dose and a second magnification. The system is configured to determine the molecular structure of the sample crystal based on the first diffraction gradient series and the second diffraction gradient series, A system in which the first electron dose is higher than the second electron dose, and the first magnification is lower than the second magnification.

12. The system according to claim 11, wherein the first diffraction gradient series and the second diffraction gradient series are obtained by rotating the sample stage with respect to the electron beam at the same rotational speed.

Images