Method and system for acquiring three-dimensional electron diffraction data.

Abstract

To provide a method for acquiring three dimensional electron diffraction data with reduced noise for extracting crystallographic information.SOLUTION: Crystallographic information of a crystalline sample can be determined from one or more three dimensional diffraction pattern datasets generated based on diffraction patterns collected from a plurality of crystals. Crystals for diffraction pattern acquisition may be selected based on a sample image. At a location of each selected crystal, the plurality of diffraction patterns of the crystal are acquired at different angles of incidence by tilting an electron beam, where the sample is not rotated while the electron beam is directed to the selected crystal.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This specification generally relates to methods and systems for crystallography, and more specifically, to obtaining three-dimensional electron diffraction data for extracting crystallographic information.

Background Art

[0002] The crystallographic information of crystals can be obtained based on their three-dimensional (3D) electron diffraction data, i.e., electron diffraction patterns obtained from various angles of the crystals with respect to an electron beam. In one example, the 3D diffraction data can be obtained by tilting the crystal with respect to the charged particle beam using stage tilting. The sample and / or the charged particle beam are typically shifted during the data acquisition process to compensate for any sample shift caused by the stage tilting. Such a data acquisition workflow can be time-consuming due to the need for correction and relatively slow stage tilting. Additionally, long exposures can cause radiation damage to the crystals. In particular, when it is necessary to analyze a large number of crystals, it is necessary to develop an efficient and fast workflow for obtaining 3D diffraction data. This can apply when analyzing a powder consisting of nanocrystals. Conventionally, such powders can be analyzed by powder X-ray diffraction, but the spectra obtained by this method are ensemble averages, and since the peaks of the spectra overlap, it can be difficult to extract crystallographic information from the X-ray spectra.

Summary of the Invention

[0003] In one embodiment, the method includes acquiring a sample image of a sample, selecting multiple crystals in the sample image, determining the coordinates of the multiple selected crystals, directing an electron beam to each of the multiple selected crystals, adjusting the electron beam at the location of each selected crystal to acquire multiple diffraction patterns of the selected crystal at different angles of incidence, and directing the electron beam so that the selected crystals are not rotated by the sample holder while the electron beam is directed at them, and extracting crystallographic information from the multiple diffraction patterns. By adjusting the angle of incidence through tilting the electron beam to each selected crystal, a 3D electron diffraction dataset can be acquired quickly and efficiently.

[0004] It should be understood that the above summary is provided in a simplified form to introduce the selection of concepts that will be further described in the modes for carrying out the invention. This summary is not intended to identify any major or essential features of the claimed subject matter, and the scope of the claimed subject matter is uniquely defined by the claims that follow the modes for carrying out the invention. Furthermore, the claimed subject matter is not limited to implementation examples that solve any of the defects mentioned above or in any part of this disclosure. [Brief explanation of the drawing]

[0005] [Figure 1] This shows an exemplary transmission electron microscope (TEM) system operating in one mode. [Figure 2] Figure 1 shows the TEM system operating in a different mode. [Figure 3] This shows an exemplary bifocal microscope system in the XZ plane. [Figure 4] Figure 3 shows a bifocal microscope system in the YZ plane. [Figure 5] In the XZ plane, Figure 3 shows the tilting of the diffraction beam of the bifocal microscope system. [Figure 6A] This is a zoomed-in view of a certain region in Figure 3. [Figure 6B]This is a zoomed-in view of a certain region in Figure 5. [Figure 7] An exemplary method for acquiring three-dimensional diffraction data is shown. [Figure 8A] An illustrative overview image is shown. [Figure 8B] A sample image of part of Figure 8A is shown. [Figure 9A] This is a sample image of an exemplary selection region. [Figure 9B] This is another sample image containing the imaged sample region from Figure 9A. [Figure 10] Another exemplary method for acquiring three-dimensional diffraction data is shown. [Figure 11] This is an example of a diffraction heatmap.

[0006] Similar reference numbers refer to the corresponding parts across several figures in the drawing. [Modes for carrying out the invention]

[0007] The following description concerns systems and methods for obtaining crystallographic information of a sample by acquiring 3D diffraction data, such as three-dimensional (3D) electron diffraction (ED) data. A sample may contain impurities and / or a collection of crystals with different structures. Multiple 3DED datasets may be formed from acquired 3DED data, each dataset (or diffraction gradient series) containing 3DED patterns of the crystal acquired at various angles relative to the electron beam. The crystal structure may be obtained by analyzing a subset of the 3DED datasets. Statistically relevant crystallographic information of a sample may be obtained by analyzing all 3DED datasets. ED patterns are acquired in transmission mode, i.e., scattered electrons are acquired from the side of the sample opposite the electron source. ED patterns may be acquired by a transmission electron microscope (TEM) system shown in Figures 1 and 2, a bifocal microscope system shown in Figures 3 and 4, or a scanning transmission electron microscope (STEM) system. When a TEM system is used, the microscope may be switched between imaging mode for acquiring sample images and diffraction mode for acquiring ED patterns. Switching modes in a TEM system may require adjusting one or more lenses in the optical column. Figure 1 shows a TEM system in low-magnification (LM) imaging mode. Figure 2 shows a TEM system in selected-region (SA) imaging and SA diffraction modes.

[0008] A bifocal microscope system was disclosed by Henstra et al. in U.S. Patent Application No. 16 / 834,963, filed March 30, 2020, and by Buijsse et al. in U.S. Patent Application No. 16 / 835,218, filed March 30, 2020. Both patent applications are incorporated herein by reference in their entirety for all purposes. A bifocal microscope system splits an electron beam generated from an electron source into an imaging beam and a diffraction beam using a bifocal beamformer. The bifocal beamformer alters the focal characteristics of either or both the imaging beam and the diffraction beam. For example, the two beams are focused on different focal planes perpendicular to the optical axis of the microscope. Furthermore, the bifocal beamformer can break the cylindrical symmetry of either one of the two beams. The imaging beam is used to acquire a sample image, and the diffraction beam is used to acquire an ED pattern. A bifocal system can acquire both a sample image and an ED pattern of the same crystal simultaneously. To reduce radiation exposure, either the sample image or the ED pattern can be acquired separately by blocking or blanking the imaging beam or the diffraction beam. Compared to TEM systems, bifocal microscope systems can switch between sample imaging and diffraction acquisition in less time and with fewer errors.

[0009] To solve the crystal structure, a complete 3DED dataset, i.e., ED patterns covering a sufficiently large gradient range of the crystal, is required. In “Serial electron crystallography for structure determination and phase analysis of nanocrystalline materials”, J.Appl.Cryst.51, 1262-1273, Smeets et al. disclose a method for identifying crystal positions in imaging mode and acquiring one diffraction pattern for each crystal selected at each goniometer position. The set of diffraction patterns is then used to identify the crystal structure. However, since only one diffraction pattern is acquired per crystal, prior information such as lattice constants and space group is needed to clearly index the crystal and solve the crystal structure. In “High-throughput continuous rotation electron diffraction data acquisition via software automation”, J.Appl.Cryst.51, 1652-1661, Cichocka et al. disclose determining the crystal structure based on continuous rotation electron diffraction, where diffraction patterns are acquired while the crystal rotates. However, Cichocka's method requires tracking the crystal during its rotation and frequently switching between in-focus and defocus diffraction modes. The applicant recognizes the need to obtain ED patterns of a large number of crystals to extract statistically relevant information such as the phase distribution, polymorphism, and chirality of the sample. As a result, neither Smeets' method nor Cichocka's method is suitable for efficiently extracting statistically relevant crystallographic information.

[0010] To address the above issues, various workflows for acquiring 3DED datasets are presented herein. In one example, a sample image of a crystal sample is acquired first. The sample image is acquired at a resolution that allows the size and shape of the crystal to be determined. Multiple crystals shown in the sample image may be selected to acquire ED data, and the position or coordinates of the selected crystals are also determined based on the sample image. Crystals may be selected based on one or more of their size, morphology, distribution, and contrast in the sample image. Multiple ED patterns are acquired for multiple selected crystals. The electron beam is shifted to illuminate each of the selected crystals and tilted within a range of beam tilt angles so that each ED pattern of the selected crystals has a different angle of incidence. The angle of incidence is defined by the angle between the incident beam and an axis perpendicular to the sample (such as the optical axis), as well as the plane of incidence (the plane containing the incident beam and the axis perpendicular to the sample). Different values ​​of the angle of incidence or different planes of incidence result in different angles of incidence. Only beam tilt is used to adjust the angle of incidence; sample tilt is not used. In other words, the sample is not rotated / tilted by the sample holder while the electron beam is directed at the selected crystal. The sample may remain stationary for the entire time the selected crystal is visited by the electron beam, from when the electron beam moves to the selected crystal until when the electron beam moves to the next selected crystal. In one example, the angle of incidence is -10 to 10 degrees relative to the optical axis. In another example, the angle of incidence is -5 to 5 degrees relative to the optical axis. In yet another example, the range of the angle of incidence is less than 20 degrees. The range of the angle of incidence is set small to reduce beam movement in the sample plane due to aberrations of the illumination optical system, thereby keeping the incident beam aligned with the crystal position. This range is also set small to keep the optical distortion of the diffraction pattern sufficiently small. In one example, the angle of incidence is adjusted by tilting the electron beam in fixed angle steps so that ED patterns can be acquired at multiple distinct angles of incidence. In another example, ED patterns can be acquired while continuously tilting the electron beam.The electron beam can be tilted in a plane or multiple planes orthogonal to the sample plane. In other examples, the beam tilt scheme may be precession at a fixed tilt angle, or precession combined with a change in tilt angle (e.g., spiral scanning). In some examples, the beam tilt scheme can incorporate corrections for optical aberrations to minimize beam displacement at the sample and to compensate for small deviations from the desired tilt increment. The electron beam can be tilted by a deflector located upstream of the sample. In a bifocal beam system, the deflector may be a bifocal beamformer, and the diffracted beam is tilted for ED pattern acquisition. By acquiring multiple ED patterns of multiple crystals via beam tilt, a 3DED dataset suitable for extracting statistical crystallographic information can be automatically collected. A limited tilt range has two additional advantages: 1) it reduces the need to adjust the eucentric stage height when visiting crystals of different z heights, and 2) finer angle sampling allows for better noise reduction when integrating peak intensities.

[0011] In another example, a crystal sample is held on a TEM grid containing multiple grid windows for TEM imaging. The TEM grid may include perforated support foil, such as lace-like carbon. Crystals located within the grid windows can be imaged or probed by an electron beam. The TEM grid can be translated in a sample plane perpendicular to the optical axis of the imaging system via a sample holder. To cover a large TEM grid area, an overview sample image covering multiple windows of the TEM grid is obtained by stitching together multiple sample images. The sample image can be obtained by translating the sample in the sample plane using a sample holder. The coordinates of selected crystals are determined based on the overview image. Furthermore, the coordinates of the center of each window on the TEM grid can be determined based on the overview image. After moving the center of the windows into the FOV of the imaging system by translating the TEM grid using a sample holder and / or shifting the electron beam using a deflector, selected crystals within each window, or within sub-regions of each window, are probed by a combined beam shift and beam tilt without moving the sample.

[0012] In another example, after acquiring a first sample image for crystal selection, the electron beam position is calibrated before acquiring multiple ED patterns. Beam position calibration can compensate for errors caused by sample movement, such as sample movement when translating the TEM grid to probe a specific grid window. Beam position calibration can also compensate for beam displacement due to mode switching. A second sample image may be acquired to calibrate the beam position. The current beam position can be determined by comparing the second sample image with the first sample image acquired for crystal selection. In one example, the first sample image is a schematic image acquired in LM imaging mode, and the second sample image is acquired in SA imaging mode. Calibrating the beam position involves registering the SA sample image along with the schematic image. After beam position calibration, the microscope may be adjusted to SA diffraction mode, and ED patterns of multiple crystals are acquired via combined beam shift and beam tilt. In another example, using a bifocal microscope system, the beam position can be calibrated using the imaging beam, for example, by comparing sample images acquired with the imaging beam before and after sample movement. Next, the ED pattern can be obtained using a diffracted beam, based on a calibrated beam position.

[0013] In yet another example, a diffraction heatmap can be generated by selecting a crystal in a sample image and then scanning the sample in diffraction mode. The scanned area may be the same as the field of view (FOV) of the sample image. The diffraction heatmap is formed based on the diffraction score of the ED pattern. The diffraction score represents the quality of the ED pattern. The position of the selected crystal may be updated based on the diffraction heatmap. Furthermore, based on the diffraction heatmap, the selected crystal can be re-evaluated to identify sub-regions within the crystal for ED pattern acquisition.

[0014] Referring to Figures 1 and 2, the 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 toward a condenser optical system 12 along the optical axis 110. The electron source 10 may generate high-energy electrons, i.e., electrons with typical energies of about 10 keV to 1,000 keV. In some embodiments, the condenser optical system 12 may include one or more condenser lenses and one or more apertures. A deflector 19 located 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 located downstream of the deflector 19 collimates the electron beam and directs the electron beam toward a sample 14. The sample 14 may be held in the sample plane 111 by a sample holder 13. In some examples, the sample is positioned on a TEM grid mounted on the sample holder. The sample holder 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 transmitted through the sample 14 sequentially pass through the post-sample objective lens 123 and the projector system 21 and are collected by a detector 25 positioned on the opposite side of the sample 14 relative to the electron source 10. The detector 25 can detect the received electrons and send the signal to the image processor 24 to form an image. The detector 25 may include an amplifier to amplify the signal before sending it to the image processor 24. In one example, the detector 25 may be a CCD camera or a CMOS camera. In some embodiments, different detectors may be used for diffraction pattern acquisition and sample image acquisition.

[0015] Figure 1 shows the TEM system 100 operating in low-magnification (LM) imaging mode. The dashed line 22 shows the beam path of scattered electrons from a point in the sample to the detector 25 in LM imaging mode, and the post-sample objective lens 123 is either off or operated at a low excitation voltage to acquire a sample image with a large FOV and low resolution. A beam stopper 17 can be used to block strong non-scattered beams. The projection system 21 operates differently for imaging modes (such as LM imaging mode or SA imaging mode) and diffraction modes (such as SA diffraction mode).

[0016] Figure 2 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 post-sample objective lens 123 onto the detector 25. A beam stopper 17 is inserted into the optical axis 110 to block the non-scattered 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 onto the SA plane 44, and the projector system 21 images the SA plane 44 onto the detector 25. The beam stopper 17 is retracted from the optical axis 110. Sample images acquired in SA imaging mode may have a smaller FOV and higher magnification compared to sample images acquired in LM imaging mode. In one example, an SA aperture may be inserted into the beam path. The SA aperture may be located within the SA plane 44. Alternatively, an aperture within 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 centered on the detector during beam tilt and the image remains centered on the detector during beam shift. The image deflector 45 may be positioned between the back focal plane 43 and the SA plane 44.

[0017] The controller 30 can control the operation of the TEM system 100 either manually in response to an operator's command or automatically according to computer-readable instructions stored in a non-transitory memory (or computer-readable medium) 32. The controller 30 includes a processor and may be configured to execute computer-readable instructions to control various components of the TEM system 100 in order to implement any of the methods described herein. For example, the controller may adjust the TEM system to operate in any one of the LM imaging mode, SA imaging mode, and SA diffraction mode by adjusting one or more of the SA aperture 18, the excitation of the objective lens 123, the beam stopper 17, and the projector system 21. The controller 30 can adjust the beam position on the sample and / or the beam incident angle by adjusting the deflector 19. The controller 30 may be further coupled to a display 31 to display signals detected by the notification and / or detector 25. The controller 30 can receive user input from a user input device 33. The user input device 33 may include a keyboard, a mouse, or a touch screen. The controller may be configured to extract crystallographic information of a crystal based on the acquired data set.

[0018] Although the TEM system is described as an example, it should be understood that sample images and diffraction patterns can be acquired using other charged particle microscope systems. As another example, the charged particle microscope system is a scanning transmission electron microscope (STEM) system. In that case, the sample image can be created in the scanning STEM mode, and the diffraction image can be acquired with a (quasi-)parallel beam. This discussion of the TEM system is provided merely as an example of one suitable imaging modality.

[0019] Figures 3-5 illustrate an exemplary embodiment of the two-focus microscope system 200. The two-focus microscope system 200 includes a two-focus beam former for splitting an electron beam generated by an electron source into an imaging beam for acquiring a sample image and a diffraction beam for acquiring an ED pattern. The two-focus beam former can change the focal characteristics of either one of the imaging beam and the diffraction beam. FIG. 3 shows the two-focus microscope system 200 in the XZ plane, and FIG. 4 shows the two-focus microscope system 200 in the YZ plane. The imaging beam and the diffraction beam can simultaneously irradiate the sample so that a sample image and an ED pattern are simultaneously acquired by a detector. This can be achieved by selecting an appropriate defocus value for the projector 215, in which case diffraction information can be taken while the projector system is in the image mode. Alternatively, either the ED pattern or the sample image can be acquired individually by blocking or blanking the imaging beam or the diffraction beam. Compared with the TEM system 100, the two-focus microscope system 200 does not require lens adjustment between sample image acquisition and ED pattern acquisition, thus avoiding errors caused by mode switching and shortening the overall 3DED data set acquisition time.

[0020] In the bifocal microscope system 200, the electron beam 220 generated by the electron source 201 along the optical axis 221 is focused by the lens system 202 and enters the accelerator 203. The electron beam exiting the accelerator 203 passes sequentially through the first capacitor 204 and the second capacitor 205 and enters the bifocal beamformer 206 as a focused beam. The bifocal beamformer 206 splits the electron beam into an imaging beam 223 traveling along different directions and a diffracted beam 222. In this specification, a quadrupole electromagnetic field is generated on the imaging beam by the bifocal beamformer, thereby altering the focal characteristics of the imaging beam. To alter the focal characteristics of at least one charged particle beam, the bifocal beamformer can apply at least a quadrupole lens effect to at least one of the charged particle beams, focusing, astigmatizing, and / or otherwise altering the corresponding focal characteristics of the beams. For example, a bifocal beamformer expands the imaging beam in the XZ plane (as shown in Figure 3) and focuses it in the YZ plane (as shown in Figure 4). The diffracted beam passes through the aperture of the bifocal beamformer 206 without changing its focal characteristics. The diffracted beam is focused onto a multipole device 207, which may be a stigmeter, located downstream of the bifocal beamformer. The multipole device 207 corrects the quadrupole lensing effect induced on the imaging beam by the bifocal beamformer, making the imaging beam cylindrically symmetric. In some examples, the multipole device is omitted, and the imaging beam remains cylindrically asymmetric before irradiating the sample. After leaving the multipole device 207, the imaging beam is focused onto a beam selection plane 209 by a third capacitor 208 located downstream of the multipole device 207. In one example, the imaging beam or diffracted beam may be selected by the aperture of a third focusing lens located on the beam selection plane 209. Alternatively, the aperture may be located at another position downstream of the bifocal beamformer.Downstream of the third condenser 208, one or both of the imaging beam and / or diffraction beam can be directed onto the sample 212 by sequentially passing through the minicondenser 211 and the pre-sample objective lens 213. The sample is held in the sample plane by the sample holder 216. Scattered electrons from the sample 212 are collected by the detector 217 after passing through the post-sample objective lens 214 and projector 215, which are sequentially positioned along the optical axis 221 downstream of the sample 212. A zoomed-in view of the beam path within region 210 is shown in Figure 6A.

[0021] Figure 5 shows the bifocal system 200 with the diffracted beam shifted and tilted in the XZ plane. Compared to Figure 3, the diffracted beam is tilted in the X direction by the bifocal beamformer 206, while the beam path of the imaging beam remains the same. In Figure 3, the diffracted beam 222 is focused on the optical axis 221 at the position of the multipole device 207. In Figure 5, the focus of the diffracted beam 222 at the multipole device 207 is shifted along the X axis. As a result, different locations of the sample, still within the FOV of the sample image, are probed for ED pattern acquisition. The beam path within region 210 is shown in Figure 6B.

[0022] Figures 6A and 6B show zoomed-in views of region 210 in Figures 3 and 5, respectively. In Figures 6A and 6B, the beam path of the imaging beam 223 is the same. The imaging beam 223 is focused on the optical axis 221 between the pre-sample objective lens 213 and the sample 212. By adjusting the bifocal beamformer, the diffracted beam 222 is tilted with respect to the optical axis 221 in Figure 6B compared to Figure 6A. In both Figures 6A and 6B, the diffracted beam 222 is focused on the focused diffraction pattern plane 218 between the sample 212 and the post-sample objective lens 214. The diffracted beam is quasi-parallel in the sample plane. The diffracted beam may have a focus angle of less than 5 mrad. The intersection point between the diffracted beam 222 and the sample 212 is shifted to the right along the X direction from Figure 6A to Figure 6B. By adjusting the bifocal beamformer, different sample regions within the FOV of the sample image acquired by the imaging beam can be probed with the diffraction beam. In this way, different crystals can be selected for diffraction analysis.

[0023] In one example, a second deflector (such as the image deflector 45 in Figures 1 and 2) may be positioned between the sample and the detector to shift and tilt electrons that have passed through the sample back to the optical axis.

[0024] In another example, a third beam deflector 225 may be positioned downstream of the third condenser 208 to shift and tilt the diffracted beam. Instead of a bifocal beamformer, the third beam deflector may be used to select a crystal for diffraction probing. Note that the second beam deflector 225 also affects the imaging beam.

[0025] In yet another example, the imaging beam and the diffracting beam can be swapped. For example, the diffracting beam may travel along the optical axis 221, while the imaging beam may travel through an aperture offset from the optical axis. In yet another example, the focal characteristics of the diffracting beam are modified via a bifocal beamformer, while the focal characteristics of the imaging beam remain the same. In yet another example, the focal characteristics of both the diffracting beam and the imaging beam are modified by the bifocal beamformer, but the focal characteristics of the diffracting beam and the imaging beam are different.

[0026] The controller 250 can control the operation of the bifocal microscope system 200 either manually in response to operator commands or automatically according to computer-readable commands stored in non-temporary memory (or computer-readable medium) 251. The controller 250 may include a processor 252 which can be configured to execute computer-readable commands to control various components of the system 200 in order to implement any of the methods described herein. The controller 250 can adjust the energy of the charged particle beam irradiated toward the sample by adjusting the high voltage level of the charged particle source 201. The controller 250 can adjust the sample position and / or orientation by adjusting the sample holder 216. The controller 250 receives data acquired from the detector 217 and generates a sample image and / or ED pattern based on the acquired data. The controller 250 may further be coupled to a display 253 to display notifications and / or images of the sample. The controller 250 may receive user input from a user input device 254. The user input device 254 may include a keyboard, mouse, or touchscreen. The controller may be configured to extract crystallographic information of the crystal based on the acquired ED dataset.

[0027] The controller 250 can adjust the beam characteristics of the imaging beam and / or diffracted beam in the sample plane by adjusting the bifocal beamformer 206. For example, adjusting the beam inclination angle of the diffracted beam in the sample plane may include adjusting the degree of deflection of the diffracted beam in the bifocal beamformer, which can be adjusted by adjusting the dipole intensity of the bifocal beamformer. Adjusting the optical characteristics of the charged particle beam in the sample plane (such as the irradiation area, the ratio of the diameters of the two beams, and the mutual inclination angle between the two beams) includes adjusting the excitation of one or more condenser lenses. Furthermore, the system may include additional condenser lenses to provide flexibility. In some embodiments, instead of positioning the bifocal beamformer downstream of the accelerator, the bifocal beamformer may be positioned upstream of the accelerator and sample, and the multipole element may be positioned between the bifocal beamformer and the sample.

[0028] The controller 250 can adjust the bifocal beamformer 206 and one or more lenses in the optical column to switch between bifocal multibeam imaging mode and normal TEM and / or scanning electron microscopy (STEM). In normal TEM and STEM modes, only one charged particle beam is formed by the optical column.

[0029] Figure 7 shows a method for obtaining a 3DED dataset to determine the crystal structure. Multiple crystals of a sample are positioned within the sample plane of a microscope system, such as the microscope systems shown in Figures 1-5. In one example, the crystals are randomly distributed on a TEM grid mounted on a sample holder. Crystals within a region of interest (ROI) are selected based on a sample image for ED pattern acquisition. The selected crystals are probed with parallel or quasi-parallel electron beams by shifting the electron beam within the sample plane. At the location of each selected crystal, the electron beam is tilted to acquire multiple ED patterns at different incidence angles. The ED patterns of the selected crystals can be merged to form a complete 3DED dataset for solving the 3D molecular structure. More importantly, multiple 3DED datasets of different crystals in the sample can be compared to extract statistically relevant crystallographic information.

[0030] In step 702, a sample image showing the ROI of the sample is acquired. The resolution of the sample image is high enough to resolve the crystals. For example, each crystal may be represented by multiple pixels in the sample image. The sample image can be used for crystal selection. Furthermore, the positions of the crystals and the TEM grid window can be established based on the sample image.

[0031] To cover a large sample area (or large ROI), the sample image may be an overview image formed by stitching together multiple sample images. Figure 8A shows an exemplary overview image 801 generated from multiple sample images. Each sample image covers a tile indexed from 0 to 15. The sample image may be acquired by scanning the sample in a spiral scan pattern by shifting the sample in the sample plane according to the sequence of tile indices. The sample image in this example is acquired in LM imaging mode at a magnification of 400x.

[0032] Returning to Figure 7, at 704, a subset of crystals in the sample image is selected for 3DED data acquisition. Crystals may be selected based on one or more of the following: size, morphology, shape, image contrast, and crystal distribution. In one example, crystals with a diameter within a given diameter range may be selected. The diameter range may be determined based on estimated crystal size to ensure that multiple crystals and / or large clusters of debris on the TEM grid are not probed for the ED pattern. In another example, crystals with a distance greater than a given minimum distance from adjacent crystals are selected. The minimum distance may be determined based on the size and incident angle range of the diffraction beam in the sample plane to ensure that only one crystal is probed for the ED pattern. The coordinates of the selected crystals are determined based on the sample image. Furthermore, the position of the TEM grid window (e.g., the coordinates of the center of each hole) may be determined based on the sample image. The coordinates may also be pixel numbers in the sample image. Crystals may be selected and positioned using computer vision and / or AI-assisted image processing methods.

[0033] In some examples, step 704 may include determining whether the particles shown in the sample image are crystalline based on whether diffraction peaks can be observed within the ED pattern when the particles are probed in diffraction mode. Particles that cannot produce an ED pattern are excluded from the subset of crystals.

[0034] In 706, if the area for probing crystals for ED collection is smaller than the ROI, a portion of the ROI is optionally translated into the diffraction probing region, and as a result, multiple selected crystals within that portion can be probed without sample movement or tilting by the sample holder. The diffraction probing region can be determined based on the beam deflection range for shifting the beam in the sample plane. For example, the sample holder can shift the TEM grid window into the diffraction probing region based on the coordinates of the center of the grid window. Figure 8B shows that by shifting the TEM grid with the sample holder, the optical axis is shifted from the center 805 of tile 12 to the center 802 of grid window 803 in the direction indicated by arrow 804. However, inaccuracies in sample movement can cause the optical axis to be misaligned with the center of the grid window, requiring calibration of the electron beam position.

[0035] In Figure 7, at 708, the electron beam position can be optionally calibrated. Through calibration, beam position errors caused by lens adjustments (such as lens adjustments during mode switching) and / or sample movement can be corrected. For example, beam position calibration can correct beam displacement caused by mode switching from LM imaging mode to SA imaging mode or SA diffraction mode in 702, and / or stage inaccuracies when moving a specific TEM grid window within the FOV in 706.

[0036] Calibrating the beam position may involve determining the beam displacement by comparing a second sample image acquired at the current beam position with a sample image of the ROI from 702. The second sample image may have higher resolution and a smaller FOV compared to the sample image at 702. For example, after sample movement at 706, a 10Kx magnified SA sample image, such as SA sample image Figure 9A, is acquired in SA imaging mode, as shown in Figure 2. The beam displacement is determined by registering the SA image together with the sample image Figure 9B acquired in step 702. The circled region 901 in Figure 9B is imaged in SA image Figure 9A. The circled region 901 is displaced from the center 903 of sample image Figure 9B, indicating that there is a beam displacement 904 from the beam position in step 702 to the current beam. In one example, the electron beam may be shifted back to an expected position, such as the center 903 of a grid hole. In another example, the coordinates of a selected crystal may be updated based on the beam displacement.

[0037] The beam displacement along the X and Y axes of the sample image can be expressed as [Δx, Δy], where Δx and Δy are projections of the beam displacement 904 onto the X and Y axes, respectively. The beam shift to correct for the beam displacement can be calculated as follows:

number

[0038] At 710, the electron beam is directed to one of the selected crystals within the diffraction probing region for ED pattern acquisition by shifting the electron beam within the sample plane. The position of the selected crystal may be the coordinates determined from 704 or the coordinates updated from 708.

[0039] In 712, multiple ED patterns of a crystal are acquired by tilting the electron beam so that each ED pattern is acquired at a different angle of incidence. Different angles of incidence result in different values ​​of the angle of incidence or different planes of incidence (i.e., planes of incidence). In one example, the electron beam is tilted in one or more planes perpendicular to the sample plane. In another example, the electron beam is tilted helically with respect to an axis such as the optical axis. In yet another example, the beam is tilted by precession at a fixed tilt angle or by a combination of precession and a change in tilt angle (e.g., spiral scan). The electron beam can be tilted with respect to a beam pivot point. The beam pivot point may be located between the pre-sample objective lens and the post-sample objective lens. In one example, the beam pivot point is between the sample plane and the post-sample objective lens. In another example, the beam pivot point is on the sample plane. The beam pivot point may be on the optical axis of the TEM or STEM system. The electron beam is tilted within a beam tilt range such that the beam shift caused by lens aberrations in front of the sample on the sample plane is small compared to the beam size. In other words, the same selected crystal is irradiated by the electron beam despite the beam shift caused by the beam tilt. The incident angle can be in the range of less than 20 degrees. In one example, the incident angle is -10 to 10 degrees. In another example, the incident angle is -5 to 5 degrees. In some examples, the beam tilt scheme can incorporate corrections for optical aberrations to minimize beam displacement in the sample and to compensate for small deviations from the desired tilt increment.

[0040] In one example, ED patterns can be collected at discrete incidence angles; that is, one ED pattern is acquired after tilting the beam at a step-sized angle. In another example, ED patterns can be collected while the beam is continuously tilted. Thus, each ED pattern covers a range of incidence angles, and this range depends on the beam tilt velocity and data acquisition velocity for each ED pattern.

[0041] In another example, step 714 may check the quality of the acquired diffraction patterns and decide whether to include them in the 3DED dataset. If the acquired ED patterns are not typical of the crystal, method 700 may stop collecting ED patterns at the current location and proceed to 714.

[0042] In step 714, method 700 optionally checks whether ED patterns have been collected for all selected crystals within the diffraction probing region. If the answer is NO, method 700 proceeds to 716 to obtain the ED patterns for the next selected crystals. If ED patterns have been collected for all selected crystals within the current diffraction probing region, method 700 proceeds to 718 to check if any portion of the ROI has not been probed. If any portion of the ROI remains to be probed for an ED pattern, method 700 proceeds to 720 to obtain an ED pattern from another portion of the ROI. For example, if there is a remaining TEM grid window, the next TEM grid window is moved into the diffraction probing region for ED pattern acquisition. If the entire ROI has been probed, method 700 proceeds to 722. In some examples, if the diffraction probing region is not smaller than the ROI, method 700 may skip steps 714 and 718 and proceed directly from 712 to 722.

[0043] In step 722, method 700 checks whether ED pattern acquisition is complete. If ED pattern acquisition is complete, method 700 proceeds to step 724. If the answer is NO, the ED pattern of the next crystal is acquired. In one example, the sample may optionally be rotated using a sample holder around a sample rotation axis in the sample plane, and the ED pattern of the selected crystal on the rotated sample is acquired using beam tilt. The sample rotation angle may be greater than the maximum beam tilt angle. For example, the sample rotation angle may be 30 to 60 degrees. When probing a selected crystal away from the rotation axis of the sample holder (such as a selected crystal in the TEM grid window), the sample holder may also be adjusted in the z direction along the optical axis to move the crystal toward the sample plane.

[0044] In step 724, ED patterns from all selected crystals are sorted, indexed, and classified based on various parameters (phase, crystal symmetry, pattern SNR, etc.). ED patterns of the same class are merged into a 3DED dataset, and the 3D structure of the molecule can be determined using conventional electron crystallography software packages. More importantly, sample statistics can be extracted from multiple 3DED datasets. These statistics may include sample purity, sample mixing ratio, polymorphism of a particular crystal, phase distribution, and sample chirality. In some embodiments, step 724 includes, for example, centering and strain correction of the ED patterns due to beam tilt before merging the ED patterns.

[0045] In this way, ED patterns of more crystals can be obtained efficiently and quickly. Multiple 3D ED datasets can be obtained to extract statistically relevant crystallographic information. Compared to tilting the crystal using a sample holder, Method 700 does not require setting the crystal to eucentric height or complex sample tracking. Compared to obtaining one ED pattern per crystal, tilting the electron beam allows for the acquisition of ED patterns sampled at precise and fine angles, enabling the probe of 3D sections of reciprocal lattice space, thereby eliminating the need for prior information on lattice constants to determine the crystal structure.

[0046] In another embodiment, Method 700 may be performed using a bifocal microscope system shown in Figures 3-5. Sample images of the ROI may be acquired using an imaging beam, and the ED patterns of each selected crystal may be acquired using a diffraction beam. Switching between sample imaging and ED pattern acquisition does not require adjusting the lenses in the optical column, thus reducing the frequency of beam position calibration.

[0047] Figure 10 shows another exemplary method 1000 for obtaining a 3DED dataset to determine the structure of a crystal sample. Unlike method 700 shown in Figure 7, method 1000 generates a diffraction heatmap by first selecting crystals based on sample images, then scanning the sample and acquiring ED patterns at each scan location. In one example, the sample is scanned with an electron beam using a TEM system in diffraction mode (such as the SA diffraction mode shown in Figure 2). In another example, the sample is scanned with a diffraction beam from a bifocal microscope system. At each scan location, a diffraction score representing the quality of the ED pattern is generated. The diffraction heatmap is generated using the diffraction scores. Crystals may be re-selected based on the diffraction heatmap. Furthermore, the coordinates of the selected crystals may be updated based on the diffraction heatmap.

[0048] In steps 1002 and 1004, similar to steps 702 and 704 in Figure 7, the sample image is acquired by including the sample's ROI and setting the microscope to imaging mode. The sample image may be stitched together from multiple images acquired by translating the sample stage.

[0049] In step 1006, if the diffraction probing region of the microscope is smaller than the region of the ROI, a portion of the ROI is optionally moved into the diffraction probing region. In some examples, after sample movement in step 1006, the beam position may be calibrated as shown in step 708 of Figure 7.

[0050] At 1008, a diffraction heatmap is generated. A diffraction heatmap can be generated by scanning a diffraction probing region with an electron beam (or a diffracting beam from a bifocal microscope) and / or by moving the sample position within the sample plane in a sample holder. The scan step size can be determined by the beam size in the sample plane. For example, the step size can increase with increasing beam size. The step size can also be determined based on a range of incident angles. At each scan position, an ED pattern is acquired and scored. The diffraction score represents the quality of the ED pattern. For example, the diffraction score increases with increasing intensity of the ED pattern. The pixel values ​​in the diffraction heatmap correspond to the diffraction score of the ED pattern at the corresponding scan position. Figure 11 shows an exemplary diffraction heatmap. Pixels with higher intensity (brighter) correspond to higher diffraction scores.

[0051] In step 1010, the crystals selected in step 1004 are updated based on the heatmap. For example, crystals corresponding to pixels with diffraction scores lower than the threshold diffraction score are excluded from the subset of selected crystals. Furthermore, the beam position may be corrected based on the heatmap. Since the locations of high diffraction scores in the heatmap correlate with the crystal positions, the beam position may be corrected, for example, by comparing the coordinates of the selected crystals determined in step 1004 with the heatmap. In addition, the diffraction heatmap can be used to optimize diffraction probing. For example, in the case of large crystals, sub-regions within the crystal with high diffraction scores may be selected, and multiple ED patterns may be obtained for each sub-region of the crystal.

[0052] In 1012, the electron beam is directed towards one of the selected crystals by beam shift, and multiple ED patterns of the crystal are acquired in 1014 by beam tilt. In one example, the coordinates of the selected crystal are the coordinates determined in 1004. In another example, the coordinates of the selected crystal are the updated coordinates determined in 1010. In yet another example, the coordinates of the selected crystal are determined based on a diffraction heatmap. The coordinates of the selected crystal may be a position in the diffraction heatmap that has a high diffraction score and is separated from other high diffraction scores at a distance greater than the threshold distance.

[0053] If the diffraction probing region is smaller than the region of the ROI, at 1016, method 1000 checks whether all crystals within the diffraction probing region have been probed. If the answer is NO, the ED pattern of the next selected crystal is obtained at 1018. Otherwise, method 1000 moves to 1020 to check whether any portion of the ROI has not been probed for ED pattern acquisition. If the entire ROI has been probed, method 1000 moves to 1024. If any portion of the ROI remains to be probed, method 1000 moves to 1022 to acquire the ED pattern from another portion of the ROI.

[0054] At step 1024, method 2000 checks whether ED pattern collection is complete. If the answer is NO, the ED pattern of the next selected crystal is obtained at step 1018. If the answer is YES, at step 1024, the ED patterns from all selected crystals are merged to form a complete 3DED dataset, and the crystal structure is determined based on the complete 3DED dataset.

[0055] In another example, a diffraction heatmap of the entire ROI may be generated after or before acquiring a sample image, and crystals may be further selected based on the diffraction heatmap at 1004. Furthermore, the location of the crystals may also be determined based on the diffraction heatmap. In one example, the diffraction heatmap may be acquired by operating the microscope in a diffraction mode such as the SA diffraction mode shown in Figure 2.

[0056] In one embodiment, method 1000 may be performed using a bifocal microscope system shown in Figures 3-5. Sample images of the ROI may be acquired using an imaging beam. Diffraction heatmaps and ED patterns of each selected crystal may be acquired using a diffraction beam.

[0057] The technical benefits of acquiring sample images are selecting crystals for ED pattern acquisition and determining the position or coordinates of the selected crystals. The technical benefit of directing the electron beam to each of the selected crystals for ED pattern acquisition via beam shift is that beam shift is faster and less prone to errors compared to sample movement. The technical benefit of tilting the electron beam at each selected crystal position to acquire multiple ED patterns is an improvement in data acquisition speed, which makes it possible to collect diffraction gradient series from many (hundreds to thousands) crystals. Therefore, by analyzing the diffraction gradient series, statistically relevant crystallographic information can be obtained from many crystals.

Claims

1. It is a method, Obtain a sample image of the sample, Selecting multiple crystals from the aforementioned sample image, Determining the coordinates of the selected plurality of crystals, Directing an electron beam towards each of the selected plurality of crystals, adjusting the electron beam so as to acquire multiple diffraction patterns of the selected crystal at different incident angles at the location of each selected crystal, and ensuring that the selected crystals are not rotated by the sample holder while the electron beam is directed towards them. A method comprising extracting crystallographic information from the aforementioned plurality of diffraction patterns.

2. The method according to claim 1, wherein obtaining a plurality of diffraction patterns of the selected crystal at different incident angles by tilting the electron beam includes obtaining the plurality of diffraction patterns while continuously tilting the electron beam.

3. The method according to claim 1, wherein adjusting the electron beam to acquire multiple diffraction patterns of the selected crystal at different incident angles includes acquiring multiple diffraction patterns of the selected crystal at incident angles of -10 to 10 degrees with respect to the optical axis.

4. The method according to claim 1, wherein adjusting the electron beam to acquire multiple diffraction patterns of the selected crystal at different incident angles includes acquiring multiple diffraction patterns of the selected crystal at incident angles in the range of less than 20 degrees.

5. The method according to any one of claims 1 to 4, wherein selecting a plurality of crystals in the sample image includes selecting the plurality of crystals based on one or more of the size, morphology, distribution, and contrast of the crystals in the sample image.

6. The method according to claim 1, further comprising calibrating the beam position relative to the sample before directing the electron beam towards each of the selected plurality of crystals.

7. The process involves acquiring multiple diffraction patterns at multiple scan positions within the field of view of the aforementioned sample image, For each of the aforementioned plurality of diffraction patterns, a diffraction score is generated. Based on the diffraction score, a diffraction heatmap is generated, The method according to claim 1, further comprising determining the coordinates of one or more of the selected crystals based on the diffraction heatmap.

8. It is a system, An electron source for generating an electron beam along the optical axis, A sample holder for holding the sample and adjusting the sample position, A deflector for shifting the electron beam to irradiate different positions on the sample and tilting the electron beam to adjust the incident angle, and a detector for detecting electrons that have passed through the sample, A controller including non-temporary memory for storing computer-readable instructions, wherein by executing the computer-readable instructions, the controller Obtain a sample image of the aforementioned sample, Selecting multiple crystals from the aforementioned sample image, Determining the coordinates of the selected plurality of crystals, The electron beam is directed towards each of the selected plurality of crystals, and the electron beam is adjusted so as to acquire multiple diffraction patterns of the selected crystal at different incident angles at the location of each selected crystal, and the selected crystals are not rotated by the sample holder while the electron beam is directed towards them. The process involves merging multiple diffraction patterns of the selected crystals into a diffraction dataset, A system comprising a controller configured to extract crystallographic information from the diffraction dataset, and

9. The system according to claim 8, further comprising a second deflector positioned between the sample and the detector, for shifting and tilting the electrons that have passed through the sample back to the optical axis.

10. The system according to claim 8 or 9, wherein the sample is positioned on a transmission electron microscope grid (TEM grid), and the sample holder holds the sample by being attached to the TEM grid.

11. The controller further comprises a capacitor positioned between the sample and the detector, After obtaining the aforementioned sample image, the capacitor is adjusted, A second sample image is acquired using the adjusted capacitor, The system according to claim 8 or 9, further configured to calibrate the beam position by comparing the second sample image with the sample image, and to direct the electron beam to one of the selected crystals, wherein the electron beam is directed to one of the selected crystals based on the calibrated beam position.

12. The system according to claim 8 or 9, wherein the deflector is a bifocal beamformer, and the bifocal beamformer splits the electron beam into a diffraction beam for acquiring the plurality of diffraction patterns and an imaging beam for acquiring the sample image, and the imaging beam and the diffraction beam have different focal characteristics.

13. The system according to claim 12, wherein the bifocal beamformer shifts the diffracting beam in the sample plane, tilts the diffracting beam to adjust the angle of incidence, directs the electron beam to one of the selected crystals, and acquires a plurality of diffraction patterns of the selected crystal at different angles of incidence, wherein by shifting the diffracting beam, the diffracting beam is directed to one of the selected crystals within the field of view of the sample image, and by tilting the diffracting beam, the plurality of diffraction patterns of the selected crystal are acquired.

14. The system according to claim 12, further comprising a third deflector positioned downstream of the bifocal beamformer, wherein obtaining a plurality of diffraction patterns of the selected crystal at different incidence angles includes obtaining the plurality of diffraction patterns by tilting the diffracted beam with the third deflector.

15. The system according to claim 12, further comprising: an accelerator positioned upstream of the bifocal beamformer between the bifocal beamformer and the sample; and a multipole positioned between the bifocal beamformer and the sample.

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