Method and system for collecting three-dimensional electron diffraction data

Abstract

Methods and systems for collecting three-dimensional electron diffraction data. Crystallographic information of a crystalline sample can be determined from one or more three-dimensional diffraction pattern data sets generated based on diffraction patterns collected from multiple crystals. A crystal for diffraction pattern collection can be selected based on an image of the sample. At the location of each selected crystal, a plurality of diffraction patterns of the crystal at different angles of incidence are collected by tilting an electron beam, wherein the sample does not rotate while the electron beam is directed at the selected crystal.

Description

Technical Field

[0001] This description generally relates to methods and systems for crystallography, and more specifically, to the acquisition of three-dimensional electron diffraction data for the extraction of crystallographic information. Background Technology

[0002] Crystallographic information about a crystal can be obtained from three-dimensional (3D) electron diffraction data of a crystal, i.e., electron diffraction patterns acquired from the crystal at different angles relative to an electron beam. In one instance, 3D diffraction data can be acquired by tilting the crystal relative to a charged particle beam using a stage tilt. During data acquisition, the sample and / or charged particle beam typically shift to compensate for any sample shift introduced by the stage tilt. Such data acquisition workflows can be time-consuming due to the need for correction and the relatively slow stage tilting. Furthermore, prolonged exposure can cause radiation damage to the crystal. There is a need to develop efficient and rapid workflows for acquiring 3D diffraction data, especially when analyzing large quantities of crystals. This is often the case when analyzing powders composed of nanocrystals. Traditionally, such powders can be analyzed using X-ray powder diffraction, but the spectra obtained in this way are overall averages, and extracting crystallographic information from X-ray spectra can be challenging due to peak overlap. Summary of the Invention

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

[0004] It should be understood that the above overview is provided to introduce some concepts further described in the detailed embodiments in a simplified form. This is not intended to identify key or essential features of the claimed subject matter, the scope of which is uniquely defined by the claims following the detailed embodiments. Furthermore, the claimed subject matter is not limited to embodiments that address any shortcomings mentioned above or in any part of this disclosure. Attached Figure Description

[0005] Figure 1 An exemplary transmission electron microscope (TEM) system operating in one mode is shown.

[0006] Figure 2 It shows operation in another mode. Figure 1 The TEM system.

[0007] Figure 3 An exemplary bifocal microscope system in the XZ plane is shown.

[0008] Figure 4 It shows the YZ plane Figure 3 A bifocal microscope system.

[0009] Figure 5 It shows the effect in the XZ plane. Figure 3 The diffraction beam of the bifocal microscope system is tilted.

[0010] Figure 6A yes Figure 3 A magnified view of the central area.

[0011] Figure 6B yes Figure 5 A magnified view of the central area.

[0012] Figure 7 An exemplary method for acquiring three-dimensional diffraction data is shown.

[0013] Figure 8A An exemplary overview image is shown.

[0014] Figure 8B It shows Figure 8A A partial sample image.

[0015] Figure 9A This is an example of a sample image of a selected area.

[0016] Figure 9B Is included Figure 9A Another sample image of the sample region imaged in the middle.

[0017] Figure 10 Another exemplary method for acquiring three-dimensional diffraction data is shown.

[0018] Figure 11 This is an example diffraction thermogram.

[0019] Throughout the various views in the accompanying drawings, similar reference numerals refer to the corresponding parts. Detailed Implementation

[0020] The following description relates to systems and methods for acquiring three-dimensional (3D) diffraction data, such as 3D electron diffraction (ED) data, to obtain crystallographic information about a sample. The sample may include impurities and / or a collection of crystals with different structures. Multiple 3D ED datasets can be formed from the acquired 3D ED data, where each dataset (or diffraction tilt series) includes 3D ED patterns of the crystal acquired at various angles relative to the electron beam. The structure of the crystal can be obtained by analyzing a subset of the 3D ED datasets. Statistically relevant crystallographic information about the sample can be obtained by analyzing all 3D ED datasets. ED patterns are acquired in a transmission manner, i.e., by acquiring scattered electrons from the sample side opposite the electron source. This can be achieved through... Figure 1-2 The transmission electron microscope (TEM) system shown Figure 3-4 The bifocal microscope system or scanning transmission electron microscope (STEM) system shown is used to acquire ED patterns. If a TEM system is used, the microscope can switch between an imaging mode for acquiring an image of the sample and a diffraction mode for acquiring the ED pattern. Mode switching in a TEM system may require adjusting one or more lenses in the optical column. Figure 1 The TEM system in low magnification (LM) imaging mode is shown. Figure 2 The TEM system is shown in selected area (SA) imaging and SA diffraction mode.

[0021] Bifocal microscope systems have been disclosed in U.S. Patent Application No. 16 / 834,963, filed March 30, 2020 by Henstra et al., and in U.S. Patent Application No. 16 / 835,218, filed March 30, 2020 by Buijsse et al. The entire contents of both patent applications are incorporated herein by reference and used for all purposes. The bifocal microscope system uses a bifocal beamformer to split an electron beam generated from an electron source into an imaging beam and a diffraction beam. The bifocal beamformer alters the focusing characteristics of one or both of the imaging beam and the diffraction beam. For example, the two beams are focused on different focal planes orthogonal to the microscope's optical axis. Furthermore, the bifocal beamformer can disrupt the cylindrical symmetry of either of the two beams. The imaging beam is used to acquire an image of the sample, and the diffraction beam is used to acquire an ED pattern. The bifocal system can acquire both an image of a sample and an ED pattern of the same crystal simultaneously. To reduce radiation exposure, the image of the sample 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 a shorter time and with smaller errors.

[0022] To solve for crystal structures, a complete 3D ED dataset is needed, i.e., an ED pattern covering a sufficiently large tilt range of the crystal. In "A Series of Electron Crystallography for Structure Determination and Phase Analysis of Nanocrystalline Materials," J. Appl. Cryst. 51, 1262–1273, Smeets et al. disclosed a method for identifying crystal locations in an imaging mode and acquiring one diffraction pattern for each selected crystal at each goniometer position. The set of diffraction patterns was then used to identify the crystal structure. However, since only one diffraction pattern is obtained for each crystal, prior information such as lattice parameters and space group is required to explicitly index the crystal and solve for the crystal structure. In "High-Throughput Continuous Rotation Electron Diffraction Data Acquisition by Software Automation," J. Appl. Cryst. 51, 1652–1661, Cichocka et al. disclosed a method for determining crystal structures based on continuous rotation electron diffraction, where diffraction patterns are acquired while the crystal is rotating. However, Cichocka's method requires crystal tracking during crystal rotation and frequent switching between focused and defocused diffraction modes. The applicant recognizes that a large number of crystallographic patterns must be acquired in order to extract statistically relevant information (such as phase distribution, polymorphism, and chirality of the samples). Therefore, neither Smeets' nor Cichocka's methods are suitable for efficiently extracting statistically relevant crystallographic information.

[0023] To address the aforementioned issues, this paper describes various workflows for acquiring 3D ED datasets. In one example, a sample image of a crystalline sample is first acquired. The sample image is acquired at a resolution that allows for the determination of the crystal's size and shape. Multiple crystals displayed in the sample image can be selected for ED data acquisition, and the location or coordinates of the selected crystals are also determined based on the sample image. Crystals can be selected based on one or more of the crystal's size, morphology, distribution, and contrast in the sample image. Multiple ED patterns are acquired for the multiple selected crystals. The electron beam is moved to illuminate each selected crystal and tilted within a range of beam tilt angles, thus giving each ED pattern of the selected crystal 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 (e.g., the optical axis) and the plane of incidence (which contains the plane of incident beam and the axis perpendicular to the sample). The angle of incidence differs when the value of the angle of incidence is different or the plane of incidence is different. Only the beam is tilted, not the sample, to adjust the angle of incidence. In other words, the sample is not rotated / tilted by the sample holder when the electron beam is directed to the selected crystal. Throughout the entire time the electron beam visits a selected crystal, from the time the electron beam moves to the selected crystal to the time the electron beam moves to the next selected crystal, the sample can remain stationary. In one example, the incident angle is -10 to 10 degrees relative to the optical axis. In another example, the incident angle is -5 to 5 degrees relative to the optical axis. In yet another example, the range of the incident angle is less than 20 degrees. Setting the range of the incident angle small reduces beam movement on the sample plane due to aberrations in the illumination optics, and this also aligns the incident beam with the crystal position. The range is also set small to keep the optical distortion of the diffraction pattern sufficiently small. In one example, the incident angle is adjusted by tilting the electron beam in fixed angular steps, allowing ED patterns to be acquired at multiple discrete incident angles. In another example, ED patterns can be acquired while the electron beam is tilted continuously. The electron beam can be tilted in a plane orthogonal to the sample plane or in multiple planes orthogonal to the sample plane. In other examples, the beam tilting scheme can be precession at a fixed tilt angle, or precession combined with changes in the tilt angle (e.g., helical scanning). In some instances, beam tilting schemes can be combined with optical aberration correction to minimize beam displacement at the sample and correct for small deviations from the desired tilt increment. The electron beam can be tilted via a deflector upstream of the sample. For bifocal beam systems, the deflector can be a bifocal beamformer, where the diffracted beam is tilted for ED pattern acquisition. By acquiring multiple ED patterns from multiple crystals via beam tilting, a 3D ED dataset suitable for extracting statistical crystallographic information can be automatically collected. The limited tilt range has two additional advantages: 1) no adjustment of the eccentric stage height is required when accessing crystals at different z-heights, and 2) finer angular sampling allows for better noise reduction when integrating peak intensities.

[0024] In another example, a crystalline sample is held on a TEM grid comprising multiple grid windows for TEM imaging. The TEM grid may include a perforated support foil, such as Lyssey carbon. Crystals located within the grid windows can be imaged or probed by an electron beam. The TEM grid can be translated within a sample plane orthogonal 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 acquired by stitching together multiple sample images. Sample images can be acquired by translating the sample within the sample plane using a sample holder. The coordinates of a selected crystal 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 window into the FOV of the imaging system by translating the TEM grid with a sample holder and / or moving the electron beam with a deflector, a selected crystal in each window or a sub-region of each window can be probed by combining beam shifting and beam tilting without moving the sample.

[0025] 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 corrects for errors introduced by sample movement, such as when translating a TEM grid to probe a specific grid window. Beam position calibration also corrects for beam displacement due to mode switching. To calibrate the beam position, a second sample image can be acquired. 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 an overview 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 with the overview image. After calibrating the beam position, the microscope can be switched to SA diffraction mode, and multiple ED patterns for the crystals can be acquired via a combination of beam shift and beam tilt. In another example, using a bifocal microscope system, the beam position can be calibrated with the imaging beam, for example, by comparing the acquired sample images with the imaging beam before and after sample movement. ED patterns can then be acquired using the diffraction beam based on the calibrated beam position.

[0026] In another example, after selecting a crystal in the sample image, a diffraction thermogram can be generated by scanning the sample in a diffraction mode. The scanned area may be the same as the field of view (FOV) of the sample image. The diffraction thermogram is formed based on the diffraction fraction of the ED pattern. The diffraction fraction represents the quality of the ED pattern. The position of the selected crystal can be updated based on the diffraction thermogram. Furthermore, based on the diffraction thermogram, the selected crystal can be re-evaluated to identify sub-regions within the crystal used for ED pattern acquisition.

[0027] Turn Figure 1-2The transmission electron microscope (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 toward a condenser system 12. The electron source 10 can generate high-energy electrons, typically between about 10 keV and 1,000 keV. In some embodiments, the condenser system 12 may include one or more condenser lenses and one or more apertures. A deflector 19 positioned downstream of the condenser system 12 deflects and / or tilts the electron beam relative to the optical axis 110. A sample front objective 16 positioned downstream of the deflector 19 collimates the electron beam and guides it onto a sample 14. The sample 14 may be held in a sample plane 111 by a sample holder 13. In some instances, the sample is positioned on a TEM grid attached to the sample holder. The sample holder 13 can adjust the sample position by tilting the sample relative to the optical axis and / or translating the sample within the sample plane. Scattered electrons transmitted through sample 14 pass sequentially through sample rear objective 123 and projector system 21, and are collected by detector 25 positioned on the opposite side of sample 14 relative to electron source 10. Detector 25 detects the received electrons and sends the signal to image processor 24 to form an image. Detector 25 may include an amplifier for amplifying the signal before sending it to image processor 24. In one example, 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.

[0028] Figure 1 A TEM system 100 operating in low magnification (LM) imaging mode is shown. Dashed line 22 illustrates the beam path of scattered electrons from a point on the sample to detector 25 in LM imaging mode, where the sample post-objective 123 is closed or operated with a low excitation voltage to acquire sample images with a large FOV and low resolution. Beam blocker 17 can be used to intercept strong, unscattered beams. Projection system 21 operates differently in imaging modes (such as LM imaging mode or SA imaging mode) and diffraction modes (such as SA diffraction mode).

[0029] Figure 2A TEM system 100 operating in SA imaging mode and SA diffraction mode is shown. Dashed line 41 shows the beam path of scattered electrons from sample 14 to detector 25 in SA diffraction mode. In SA diffraction mode, projector system 21 images the back focal plane 43 of sample rear objective 123 onto detector 25. Beam blocker 17 is inserted into optical axis 110 to block unscattered beams. Dashed line 42 shows the beam path of scattered electrons from sample 14 to detector 25 in SA imaging mode. In SA imaging mode, sample plane 111 is imaged onto SA plane 44, and projector system 21 images SA plane 44 onto detector 25. Beam blocker 17 is retracted from optical axis 110. Sample images acquired in SA imaging mode can have a smaller FOV and higher magnification compared to sample images acquired in LM imaging mode. In one example, an SA aperture can be inserted into the beam path. The SA aperture can be positioned in SA plane 44. Alternatively, the aperture in the condenser system 12 can be used as a beam-limiting aperture. In another example, an image deflector can be positioned between the sample and the detector to move and tilt electrons transmitted through the sample back onto the optical axis, such that the ED pattern remains centered on the detector during beam tilting and the image remains centered on the detector during beam shifting. Image deflector 45 can be positioned between the back focal plane 43 and the SA plane 44.

[0030] The controller 30 can control the operation of the TEM system 100, either manually in response to operator commands or automatically according to computer-readable instructions stored in non-transitory memory (or computer-readable medium) 32. The controller 30 may include a processor and is configured to execute computer-readable instructions and control various components of the TEM system 100 to implement any of the methods described herein. For example, the controller can adjust the TEM system to operate in any 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 blocker 17, and the projector system 21. The controller 30 can adjust the beam position and / or beam incident angle on the sample by adjusting the deflector 19. The controller 30 may be further coupled to a 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 can be configured to extract crystallographic information of the crystal based on the acquired dataset.

[0031] Although the TEM system has been described by way of example, it should be understood that sample images and diffraction patterns can be acquired using other charged particle microscopy systems. As another example, a charged particle microscopy system is a scanning transmission electron microscope (STEM) system. In this case, sample images can be prepared in scanning STEM mode, and diffraction images can be obtained using a (quasi-)parallel beam. This discussion of the TEM system is provided only as an example of a suitable imaging method.

[0032] Figure 3-5 An exemplary embodiment of a bifocal microscope system 200 is shown. The bifocal microscope system 200 includes a bifocal beamformer for splitting an electron beam generated by an electron source into an imaging beam for acquiring an image of a sample and a diffraction beam for acquiring an ED pattern. The bifocal beamformer can modify the focal characteristics of either the imaging beam or the diffraction beam. Figure 3 A bifocal microscope system 200 in the XZ plane is shown. Figure 4 A bifocal microscope system 200 in the YZ plane is illustrated. The imaging beam and diffraction beam can simultaneously illuminate the sample, allowing the detector to acquire both a sample image and an ED pattern simultaneously. This can be achieved by selecting an appropriate defocus value for the projector 215, in which case diffraction information can be obtained when the projector system is in image mode. Alternatively, the ED pattern or sample image can be acquired separately by blocking or blanking the imaging beam or the diffraction beam. Compared to the TEM system 100, the bifocal microscope system 200 eliminates the need for lens adjustments between sample image and ED pattern acquisition, thus avoiding errors introduced by mode switching and reducing the overall 3D ED dataset acquisition time.

[0033] In a bifocal microscope system 200, an electron beam 220 generated by an electron source 201 along the optical axis 221 is focused by a lens system 202 and enters an accelerator 203. The electron beam emitted from the accelerator 203 passes sequentially through a first condenser 204, a second condenser 205, and then enters a bifocal beamformer 206 as a converging beam. The bifocal beamformer 206 splits the electron beam into an imaging beam 223 and diffracted beams 222 traveling in different directions. In this paper, the bifocal beamformer generates a quadrupole electromagnetic field on the imaging beam, which causes a change in the focusing characteristics of the imaging beam. To change the focusing characteristics of at least one charged particle beam, the bifocal beamformer can apply a quadrupole lensing effect to at least one of the charged particle beams. The quadrupole lensing effect focuses, stigmates, and / or otherwise modifies at least one of the beams, resulting in different corresponding focusing characteristics. For example, the bifocal beamformer focuses the imaging beam in the XZ plane (e.g., ...). Figure 3 (as shown in the diagram) expands and focuses the imaging beam onto the YZ plane (as shown in the diagram). Figure 4(As shown in the diagram). The diffracted beam passes through the aperture of the bifocal beamformer 206 without altering its focusing characteristics. The diffracted beam is focused onto a multipole device 207, which may be an astigmatism corrector located downstream of the bifocal beamformer. The multipole device 207 corrects for the quadrupole lensing effect of the bifocal beamformer on the imaging beam and makes the imaging beam cylindrically symmetrical. In some instances, the multipole device is omitted and the imaging beam remains cylindrically asymmetrical before illuminating the sample. After exiting the multipole device 207, the imaging beam is focused onto a beam selection plane 209 by a third condenser 208 located downstream of the multipole device 207. In one instance, either the imaging beam or the diffracted beam can be selected by the aperture of the third focusing lens located at the beam selection plane 209. Alternatively, the aperture may be located at other locations downstream of the bifocal beamformer. Downstream of the third condenser 208, one or both of the imaging beam and the diffracted beam can be guided onto the sample 212 by passing sequentially through a micro condenser 211 and a sample front objective lens 213. The sample is held in the sample plane by a sample holder 216. Scattered electrons from sample 212 are collected by detector 217 after passing through sample rear objectives 214 and projector 215, which are sequentially positioned downstream of sample 212 along optical axis 221. Figure 6A The image shows an enlarged view of the beam path in region 210.

[0034] Figure 5 The diagram illustrates a bifocal system 200 in which the diffracted beam moves and tilts within the XZ plane. (Comparison) Figure 3 The bifocal beamformer 206 tilts the diffracted beam in the X direction while keeping the beam path of the imaging beam unchanged. Figure 3 In this configuration, the diffracted beam 222 is focused onto the optical axis 221 at the position of the multipole device 207. Figure 5 In this process, the focal point of the diffracted beam 222 at the multipole device 207 shifts along the X-axis. As a result, different locations of the sample, still within the FOV of the sample image, are detected for ED pattern collection. Figure 6B The beam path in region 210 is shown in the figure.

[0035] Figure 6A and 6B They are shown respectively Figure 3 and Figure 5 A magnified view of area 210 Figures 6A-6B As shown, the beam path of imaging beam 223 is the same. Imaging beam 223 is focused on the optical axis 221 between the sample front objective lens 213 and the sample 212. Figure 6A In contrast, by adjusting the bifocal beamformer, the diffracted beam 222 relative to Figure 6B The optical axis 221 is tilted. Figure 6A and Figure 6BIn the process, the diffracted beam 222 is focused onto the focusing diffraction pattern plane 218 between the sample 212 and the sample rear objective lens 214. The diffracted beam is quasi-parallel on the sample plane. The diffracted beam can have a convergence angle of <5 mrad. The intersection point between the diffracted beam 222 and the sample 212 is along the X-direction from... Figure 6A Move to the right Figure 6B By adjusting the bifocal beamformer, different sample regions within the field of view (FOV) of the sample image acquired by the imaging beam can be probed using a diffracted beam. This allows for the selection of different crystals for diffraction analysis.

[0036] In one instance, the second deflector (such as...) Figure 1-2 The image deflector 45 in the image can be positioned between the sample and the detector to move and tilt electrons transmitted through the sample back to the optical axis.

[0037] In another example, a third beam deflector 225 can be positioned downstream of the third condenser 208 to move and tilt the diffracted beam. Instead of a bifocal beamformer, the third beam deflector can be used to select the crystal for diffraction detection. Note that the second beam deflector 225 also affects the imaging beam.

[0038] In another example, the imaging beam and the diffracted beam can be interchanged. For instance, the diffracted beam can travel along optical axis 221, and the imaging beam travels through an aperture off-axis. In yet another example, the focusing characteristics of the diffracted beam are altered by a bifocal beamformer, while the focusing characteristics of the imaging beam remain unchanged. In yet another example, the focusing characteristics of both the diffracted beam and the imaging beam are altered by a bifocal beamformer, and the focusing characteristics of the diffracted beam and the imaging beam are different.

[0039] 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 instructions stored in non-transitory memory (or computer-readable medium) 251. The controller 250 may include a processor 252 and is configured to execute computer-readable instructions and control various components of the system 200 to implement any of the methods described herein. The controller 250 can adjust the energy of the charged particle beam irradiating 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 sample images and / or ED patterns based on the acquired data. The controller 250 may be further 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, and / or touchscreen. The controller may be configured to extract crystallographic information of the crystal based on the acquired ED dataset.

[0040] The controller 250 can adjust the beam characteristics of the imaging beam and / or diffracted beam at the sample plane by adjusting the bifocal beamformer 206. For example, adjusting the beam tilt angle of the diffracted beam at the sample plane may include adjusting the degree of deflection of the diffracted beam at the bifocal beamformer, wherein the degree of deflection can be adjusted by adjusting the dipole intensity of the bifocal beamformer. Adjusting the optical characteristics of the charged particle beam at the sample plane (such as the irradiation area, the ratio of the diameters of the two beams, and the mutual tilt 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 the sample, and the multipole element is positioned between the bifocal beamformer and the sample.

[0041] The controller 250 can adjust the bifocal beamformer 206 and one or more lenses in the optical column to switch between bifocal multi-beam imaging mode and normal TEM and / or scanning transmission electron microscopy (STEM). In normal TEM and STEM modes, the optical column forms only a single beam of charged particles.

[0042] Figure 7 A method 700 for acquiring 3D ED datasets to determine crystal structures is illustrated. Multiple crystals of the sample are positioned within the sample plane of the microscope system, such as... Figure 1-5 The microscope system is described. In one example, crystals are randomly distributed on a TEM grid attached to a sample holder. Crystals within a region of interest (ROI) are selected based on sample images used for ED pattern acquisition. The selected crystals are probed with parallel or quasi-parallel electron beams by moving the electron beam within the sample plane. At the location of each selected crystal, the electron beam is tilted to acquire multiple ED patterns with different incident angles. The ED patterns of the selected crystals can be merged to form a complete 3D ED dataset for solving 3D molecular structures. More importantly, multiple 3D ED datasets of different crystals in the sample can be compared to extract statistically relevant crystallographic information.

[0043] At 702, a sample image displaying the ROI of the sample is acquired. The sample image has a sufficiently high resolution to resolve the crystals. For example, each crystal can 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 TEM grid windows can be determined based on the sample image.

[0044] To cover a large sample area (or a large ROI), a sample image can be an overview image formed by stitching together multiple sample images. Figure 8AAn exemplary overview image 801 generated from multiple sample images is shown. Each sample image covers tiles with indices from 0 to 15. Sample images can be acquired by moving the sample within the sample plane and scanning the sample in a helical scan manner according to the image index. In this example, the sample images were acquired in LM imaging mode at a magnification of 400x.

[0045] Back Figure 7 In step 704, a subset of crystals within the sample image is selected for 3D ED data acquisition. Crystals can be selected based on one or more of the following: crystal size, morphology, shape, image contrast, and distribution. In one example, crystals with diameters within a predetermined diameter range can be selected. The diameter range can be determined based on estimated crystal sizes, thus avoiding detection of ED patterns by large clusters of polycrystals and / or fragments on the TEM grid. In another example, crystals at a distance greater than a predetermined minimum distance from neighboring crystals are selected. The minimum distance can be determined based on the size of the diffraction beam and the range of incident angles on the sample plane to ensure that only the ED pattern of one crystal is detected. The coordinates of the selected crystals are determined based on the sample image. Furthermore, the position of the TEM grid windows (such as the coordinates of the center of each aperture) can be determined based on the sample image. The coordinates can be the number of pixels in the sample image. Computer vision and / or AI-assisted imaging processing methods can be used to select and locate the crystals.

[0046] In some instances, step 704 may include determining whether a particle shown in the sample image is a crystal based on whether diffraction peaks can be observed in the ED pattern when the particle is probed in diffraction mode. Any particle that cannot produce an ED pattern is excluded from the crystal subset.

[0047] In step 706, if the area used to detect crystals collected by the ED is smaller than the ROI, a portion of the ROI can be optionally translated into the diffraction detection region, allowing multiple selected crystals within that portion to be detected without moving or tilting the sample by the sample holder. The diffraction detection region can be determined based on the beam deflection range used to move the beam within the sample plane. For example, the sample holder can move the TEM grid window to the diffraction detection region based on the coordinates of the grid window's center. Figure 8B The diagram illustrates how the optical axis moves from the center 805 of patch 12 to the center 802 of grid window 803 in the direction indicated by arrow 804 by moving the TEM grid with a sample holder. However, inaccurate sample movement may cause the optical axis to misalign with the center of the grid window, necessitating calibration of the electron beam position.

[0048] exist Figure 7708 can optionally calibrate the electron beam position. Calibration corrects beam position errors introduced by lens adjustments (such as lens adjustments during mode switching) and / or sample movement. 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 at 702 and / or stage inaccuracies when moving a specific TEM grid window into the FOV at 706.

[0049] Calibrating the beam position can include determining beam displacement by comparing a second sample image acquired at the current beam position with a sample image from the ROI at 702. The second sample image can have higher resolution and a smaller FOV compared to the sample image at 702. For example, after sample movement at 706, an SA sample image with a magnification of 10Kx is acquired in SA imaging mode, such as an SA sample image. Figure 9A ,like Figure 2 As shown in the figure. By comparing the SA image with the sample image acquired in step 702. Figure 9B Registration is used to determine beam displacement. Figure 9B The circular region 901 in the image is imaged in the SA image. Figure 9A In the middle. The center of circle region 901 is offset from the sample image. Figure 9B The center 903 indicates a beam displacement 904 from the beam position in step 702 to the current beam. In one instance, the electron beam can be moved back to its intended position, such as the center 903 of the gate aperture. In another instance, the coordinates of the selected crystal can be updated based on the beam displacement.

[0050] The beam displacement along the X and Y axes of the sample image can be expressed as [Δx, Δy], where Δx and Δy are the projections of the beam displacement 904 onto the X and Y axes, respectively. The beam shift used to correct the beam displacement can be calculated as: Equation 1 Where BS_x and BS_y are beam shifts used to correct beam displacement; a and b are known parameters representing the relationship between beam displacement and beam shift (such as rotation and scaling). Beam shifts BS_x and BS_y can be the current settings of the deflector coils. When the beam is moved to each selected crystal at 710, beam shifts BS_x and BS_y can be applied to the deflector.

[0051] At 710, the electron beam is guided to one of the selected crystals in the diffraction detection region for ED pattern collection by moving the electron beam within the sample plane. The position of the selected crystal can be the coordinates determined from 704, or the coordinates updated from 708.

[0052] In 712, multiple ED patterns of the crystal are acquired by tilting the electron beam, such that each ED pattern is acquired at different incident angles. The incident angles differ when the value of the incident angle or the plane of incidence (or incident plane) is different. In one example, the electron beam is tilted in one or more planes orthogonal to the sample plane. In another example, the electron beam is helically tilted relative to an axis such as the optical axis. In yet another example, the beam precesses at a fixed tilt angle, or a combination of precession and varying tilt angles (e.g., helical scanning). The electron beam can be tilted relative to a beam pivot point. The beam pivot point can be located between the front and rear sample objectives. In one example, the beam pivot point is located between the sample plane and the rear sample objective. In another example, the beam pivot point is located on the sample plane. For TEM or STEM systems, the beam pivot point can be on the optical axis. The electron beam is tilted within the beam tilt range such that the beam displacement on the sample plane caused by presample lens aberrations is small compared to the beam size. In other words, despite the beam displacement caused by beam tilting, the same selected crystal is still irradiated by the electron beam. The incident angle can range from 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 tilting scheme can be combined with optical aberration correction to minimize beam displacement at the sample and correct for small deviations from the desired tilt increment.

[0053] In one instance, ED patterns can be collected at discrete incident angles. That is, an ED pattern is acquired after tilting the beam by one step angle. In another instance, ED patterns can be collected while the beam is continuously tilted. Therefore, each ED pattern covers a range of incident angles, where the range depends on the beam tilting rate and data acquisition rate for each ED pattern.

[0054] In another instance, step 714 can examine the quality of the acquired diffraction patterns and determine whether to include them in the 3D ED dataset. If the acquired ED patterns are not typical ED patterns for the crystal, method 700 can stop collecting ED patterns at the current position and move to 714.

[0055] At 714, method 700 optionally checks whether ED patterns for all selected crystals in the diffraction detection region have been collected. If the answer is no, method 700 proceeds to 716 to collect the ED pattern for the next selected crystal. If ED patterns for all selected crystals in the current diffraction detection region have been collected, method 700 proceeds to 718 to check if any portion of the ROI has not yet been detected. If any portion of the ROI still has ED patterns to be detected, method 700 moves to 720 to collect ED patterns from another portion of the ROI. For example, if any TEM grid window is left undetected, the next TEM grid window is moved to the diffraction detection region to collect the ED pattern. If all ROIs have been detected, method 700 proceeds to 722. In some instances, if the diffraction detection region is not smaller than the ROI, method 700 may skip steps 714 and 718 and proceed directly from 712 to 722.

[0056] At 722, method 700 checks whether ED pattern acquisition is complete. If ED pattern acquisition is complete, method 700 proceeds to 724. If the answer is no, the ED pattern for the next crystal is acquired. In one instance, the sample can optionally be rotated about a sample rotation axis in the sample plane using a sample holder, and the ED pattern of a selected crystal on the rotating sample is acquired by beam tilting. The sample rotation angle may be greater than the maximum beam tilt angle. For example, the sample rotation angle may be 30–60 degrees. When probing a selected crystal away from the sample holder rotation axis (such as a selected crystal in the TEM grid window), the sample holder can also be adjusted along the optical axis in the z-direction to align the crystal with the sample plane.

[0057] In step 724, ED patterns from all selected crystals are classified, indexed, and categorized based on various parameters (phase, crystal symmetry, pattern SNR, etc.). ED patterns of the same category are merged into a 3D ED dataset, and the molecular 3D structure can be determined using conventional electron crystallography software packages. More importantly, statistical information of the samples can be extracted from multiple 3D ED datasets. Statistical information may include sample purity, sample mixing ratio, polymorphism of a certain crystal, phase distribution, and sample chirality. In some embodiments, step 724 includes centering and distortion correction of the ED patterns due to, for example, beam tilt, before merging the ED patterns.

[0058] This method allows for the efficient and rapid acquisition of ED patterns from a large number of crystals. Multiple 3D ED datasets can be obtained to extract statistically relevant crystallographic information. Compared to using a sample holder with tilted crystals, method 700 does not require setting the crystal at an eccentric height or complex sample tracking. Compared to acquiring an ED pattern for each crystal, using a tilted electron beam allows for the acquisition of ED patterns with precise and fine-angle sampling, probing the 3D cross-section of reciprocal space, thus eliminating the need for prior information on lattice parameters to determine the crystal structure.

[0059] In another embodiment, it can be used Figure 3-5 The bifocal microscope system shown is used to perform method 700. An imaging beam can be used to acquire an image of the sample at the ROI, and a diffraction beam can be used to acquire the ED pattern for each selected crystal. Since switching between sample imaging and ED mode acquisition does not require adjustment of any lenses in the optical column, the frequency of beam position calibration can be reduced.

[0060] Figure 10 Another exemplary method 1000 for acquiring a 3D ED dataset for determining the structure of crystalline samples is shown. Unlike... Figure 7 The method 700 shown, in method 1000, after selecting a crystal based on a sample image, generates a diffraction thermogram by scanning the sample and acquiring an ED pattern at each scanning location. In one example, a TEM system is used with diffraction patterns (such as...) Figure 2 The SA diffraction mode shown is used to scan the sample with an electron beam. In another example, the sample is scanned with the diffraction beam of a bifocal microscope system. At each scanning position, a diffraction fraction representing the quality of the ED pattern is generated. A diffraction thermogram is generated using the diffraction fraction. The crystal can be reselected based on the diffraction thermogram. Furthermore, the coordinates of the selected crystal can be updated based on the diffraction thermogram.

[0061] In 1002 and 1004, similar to Figure 7 In steps 702 and 704, the sample image includes the ROI of the sample acquired by setting the microscope to imaging mode. The sample image can be stitched together from multiple images acquired by translating the sample stage.

[0062] In step 1006, if the diffraction detection area of ​​the microscope is smaller than the region of the area of ​​origin (ROI), a portion of the ROI can be optionally moved into the diffraction detection area. In some instances, after sample movement in step 1006, it can be done as follows: Figure 7 The calibration beam position is shown in step 708.

[0063] At position 1008, a diffraction thermogram is generated. This can be achieved by scanning the diffraction detection region with an electron beam (or the diffraction beam of a bifocal microscope) and / or by moving the sample position within the sample plane using a sample holder. The scan step size can be determined by the beam size on the sample plane. For example, the step size can increase with increasing beam size. The step size can be further determined based on the range of incident angles. At each scan position, the ED pattern is acquired and scored. The diffraction fraction represents the quality of the ED pattern. For example, the diffraction fraction increases with increasing ED pattern intensity. The pixel values ​​of the diffraction thermogram correspond to the diffraction fraction of the ED pattern at the corresponding scan position. Figure 11 An exemplary diffraction thermogram is shown. Pixels with higher intensity (brighter) correspond to higher diffraction fractions.

[0064] In step 1010, the crystal selected in step 1004 is updated based on the thermal map. For example, crystals corresponding to pixels with diffraction fractions below a threshold are removed from a subset of the selected crystals. Furthermore, the beam position can be corrected based on the thermal map. For example, the beam position can be corrected by comparing the coordinates of the selected crystal determined in step 1004 with the thermal map, since the positions of high diffraction fractions in the thermal map are related to the crystal position. Furthermore, the diffraction thermal map can optimize diffraction detection. For example, for large crystals, sub-regions within the crystal with high diffraction fractions can be selected, and multiple ED patterns can be acquired in each sub-region of the crystal.

[0065] In step 1012, the electron beam is guided to one of the selected crystals by beam shifting, and in step 1014, multiple ED patterns of the crystals are acquired by beam tilting. In one example, the coordinates of the selected crystal are determined at step 1004. In another example, the coordinates of the selected crystal are updated coordinates determined at step 1010. In yet another example, the coordinates of the selected crystal are determined based on a diffraction thermogram. The coordinates of the selected crystal can be a location with a high diffraction fraction in the diffraction thermogram, separated from other high diffraction fractions at a distance greater than a threshold distance.

[0066] If the diffraction detection area is smaller than the ROI, then at step 1016, method 1000 checks whether all crystals in the diffraction detection area have been detected. If the answer is no, then at step 1018, the ED pattern for the next selected crystal is acquired. Otherwise, method 1000 moves to step 1020 to check if any portion of the ROI has not yet been detected for ED pattern acquisition. If all ROIs have been detected, method 1000 proceeds to step 1024. If any portion of the ROI still needs to be detected, method 1000 proceeds to step 1022 to acquire the ED pattern from another portion of the ROI.

[0067] In step 1024, method 2000 checks whether ED pattern collection is complete. If the answer is no, then in step 1018, the ED pattern for the next selected crystal is collected. If the answer is yes, then in step 1024, the ED patterns from all selected crystals are merged to form a complete 3D ED dataset, and the crystal structure is determined based on the complete 3D ED dataset.

[0068] In another example, a diffraction thermogram can be generated for the entire ROI, either before or after acquiring a sample image, and crystals can be further selected based on the diffraction thermogram. Furthermore, crystal locations can also be determined based on the diffraction thermogram. In one example, a diffraction thermogram can be acquired by operating the microscope in diffraction mode, such as... Figure 2 The SA diffraction pattern shown.

[0069] In one embodiment, method 1000 can use Figure 3-5 The bifocal microscope system shown is used for this purpose. An imaging beam can be used to acquire images of the sample within the ROI. A diffraction beam can be used to acquire diffraction thermograms and ED patterns for each selected crystal.

[0070] The advantage of acquiring sample images is that it allows for the selection of crystals for ED pattern acquisition and the determination of the selected crystal's position or coordinates. The advantage of guiding the electron beam to each selected crystal for ED pattern acquisition via beam shifting is that beam shifting is faster and introduces less error compared to sample movement. The advantage of tilting the electron beam at each selected crystal location to acquire multiple ED patterns is increased data acquisition speed, which allows for the collection of diffraction tilt series from many crystals (100 to 1000). Therefore, by analyzing the diffraction tilt series, statistically relevant crystallographic information for many crystals can be obtained.

Claims

1. A method for acquiring three-dimensional electron diffraction data, comprising: Acquire sample images; Select multiple crystals from the sample image; Determine the coordinates of multiple selected crystals; An electron beam is directed to each of the plurality of selected crystals and multiple diffraction patterns are acquired from the plurality of selected crystals without rotating or moving the sample, wherein at the location of each selected crystal, the electron beam is adjusted to acquire multiple diffraction patterns of the selected crystal at different incident angles, and the selected crystal is not rotated by the sample holder when the electron beam is directed to the selected crystal; and Crystallographic information is extracted from the plurality of diffraction patterns.

2. The method of claim 1, wherein acquiring multiple diffraction patterns of the selected crystal at different incident angles by tilting the electron beam includes acquiring the multiple diffraction patterns while continuously tilting the electron beam.

3. The method according to claim 1, wherein acquiring multiple diffraction patterns of the selected crystal at different incident angles includes acquiring multiple diffraction patterns of the selected crystal at incident angles relative to the optical axis from -10 degrees to 10 degrees.

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

5. The method according to any one of claims 1 to 4, wherein selecting one or more crystals in the sample image comprises selecting the one or more 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 any one of claims 1 to 4, further comprising calibrating the beam position relative to the sample before directing the electron beam to one of the selected crystals.

7. The method of claim 6, wherein calibrating the beam position comprises acquiring a second sample image having a field of view smaller than that of the sample image, determining a beam displacement by comparing the second sample image with the sample image, and calibrating the beam position based on the beam displacement.

8. The method of claim 1, further comprising: Multiple diffraction patterns are acquired at multiple scanning positions within the field of view of the sample image; Generate a diffraction fraction for each of the plurality of diffraction patterns; A diffraction heatmap is generated based on the diffraction fraction; as well as The coordinates of the selected crystals are further determined based on the diffraction thermogram.

9. The method of claim 8, wherein further determining the coordinates of the plurality of selected crystals based on the diffraction thermogram comprises determining the coordinates of one or more sub-regions of at least one of the plurality of selected crystals based on the diffraction thermogram.

10. The method of claim 1, wherein acquiring multiple diffraction patterns of the selected crystal at different incident angles by tilting the electron beam comprises acquiring the multiple diffraction patterns of the selected crystal by tilting the electron beam in at least one plane orthogonal to the sample plane.

11. The method of claim 1, further comprising: After acquiring the sample image, the sample position is adjusted by translating the sample holder; as well as After adjusting the sample position, the beam position relative to the sample is calibrated, and guiding the electron beam to each selected crystal includes guiding the electron beam based on the coordinates of the selected crystal and the calibrated beam position.

12. A system for acquiring three-dimensional electron diffraction data, comprising: An electron source, which is used to generate an electron beam along the optical axis; A sample holder for holding and adjusting the position of a sample; A deflector for moving the electron beam to illuminate different positions of the sample and tilting the electron beam to adjust the incident angle; a detector for detecting electrons transmitted through the sample; as well as The controller includes a non-transitory memory for storing computer-readable instructions, wherein by executing the computer-readable instructions, the controller is configured to: Acquire sample images of the sample; Select multiple crystals from the sample image; Determine the coordinates of multiple selected crystals; The electron beam is directed to each of the plurality of selected crystals and a plurality of diffraction patterns are acquired from the plurality of selected crystals without rotating or moving the sample, wherein at the position of each selected crystal, the electron beam is adjusted to acquire a plurality of diffraction patterns of the selected crystal at different incident angles, and the selected crystal is not rotated by the sample holder when the electron beam is directed to the selected crystal; Multiple diffraction patterns of the selected crystal are merged into a diffraction dataset; as well as Extract crystallographic information from the diffraction dataset.

13. The system of claim 12, further comprising a second deflector positioned between the sample and the detector for moving and tilting electrons transmitted through the sample back to the optical axis.

14. The system of claim 12, wherein the sample is positioned on a transmission electron microscope (TEM) grid, and the sample holder holds the sample by being attached to the TEM grid.

15. The system of claim 12, further comprising a condenser positioned between the sample and the detector, and wherein the controller is further configured to: Acquire images of the second sample; and The beam position is calibrated by comparing the second sample image with the sample image, and guiding the electron beam into one of the selected crystals includes guiding the electron beam into one of the selected crystals based on the calibrated beam position.

16. The system according to any one of claims 12 to 15, wherein the deflector is a bifocal beamformer that splits the electron beam into a diffraction beam for acquiring the plurality of diffraction patterns and an imaging beam for acquiring an image of the sample, wherein the imaging beam and the diffraction beam have different focal characteristics.

17. The system of claim 16, wherein the bifocal beamformer moves the diffraction beam on the sample plane and tilts the diffraction beam to adjust the incident angle, and wherein guiding the electron beam to one of the selected crystals and acquiring a plurality of diffraction patterns of the selected crystals at different incident angles comprises guiding the diffraction beam to one of the selected crystals within the field of view of the sample image by moving the diffraction beam, and acquiring the plurality of diffraction patterns of the selected crystals by tilting the diffraction beam.

18. The system of claim 16, further comprising a third deflector positioned downstream of the bifocal beamformer, wherein acquiring a plurality of diffraction patterns of the selected crystal at different incident angles comprises acquiring the plurality of diffraction patterns by tilting the diffraction beam using the third deflector.

19. The system of claim 16, further comprising: an accelerator positioned upstream of the dual-focus beamformer; and a multipole element positioned between the dual-focus beamformer and the sample.

20. The system of claim 16, further comprising: an accelerator positioned between the bifocal beamformer and the sample; and a multipole element positioned between the bifocal beamformer and the sample.

21. The system of claim 16, further comprising an aperture for selectively blocking or blanking the diffracted beam or the imaging beam.

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