Method and system for determining absolute structure of crystal

Abstract

The crystal structure is determined based on one or more second electron diffraction patterns acquired at a selected ribbon axis with very strong kinetic effect intensity. The zone axis is selected by sorting accessible zone axes determined from the plurality of first electron diffraction patterns.

Description

Technical Field

[0001] This specification generally relates to methods and systems for crystallography, and more particularly, to determining the absolute structure of a crystal using electron diffraction. Summary of the Invention

[0002] In one embodiment, a method for crystallography includes: collecting a plurality of first electron diffraction patterns (EDPs) from at least one crystal; indexing the first EDPs to identify a plurality of zone axes of the at least one crystal; sorting the plurality of zone axes based on the intensity of the dynamical effect at each of the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the sorting; aligning an electron optical axis of an electron beam with the selected at least one zone axis; collecting one or more second EDPs using the aligned electron beam; and determining the absolute structure of the crystal based on the one or more second EDPs.

[0003] In another embodiment, a charged particle microscope system includes: a sample holder configured to hold a sample including a plurality of crystals; an electron source configured to generate an electron beam; an electron optical column configured to direct the electron beam towards the sample; a detector configured to detect an electron diffraction pattern; and a controller including a processor and a non-transitory memory configured to store computer-readable instructions, wherein by executing the instructions in the processor, the charged particle microscope system is configured to: collect a plurality of first electron diffraction patterns (EDPs) from at least one of the plurality of crystals; align the electron optical axis of the electron beam with at least one of the selected zone axes, wherein the selected zone axes are determined based on a sorting of a plurality of zone axes of the at least one crystal, the plurality of zone axes are identified by indexing the first EDPs, and the plurality of zone axes are sorted based on the intensity of the dynamical effect at each of the plurality of zone axes; and collect one or more second EDPs using the aligned electron beam to determine the absolute structure of the crystal.

[0004] It should be understood that the above summary is provided to introduce in a simplified form some concepts that are further described in the detailed description. It is not intended to identify key or essential features of the claimed subject matter, the scope of which is uniquely defined by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to implementations that solve any disadvantages noted above or in any part of this disclosure. Brief Description of the Drawings

[0005] Figure 1 Illustrates a transmission electron microscope (TEM) system operating in a first mode.

[0006] Figure 2 illustrates a TEM system operating in other modes Figure 1 .

[0007] Figure 3 is a method for determining the absolute structure of a crystal.

[0008] Figure 4A and Figure 4B are simulated electron diffraction patterns (EDPs) of enantiomers at a first zone axis.

[0009] Figure 5A and Figure 5B are simulated EDPs of enantiomers at a second zone axis.

[0010] Figure 6A and Figure 6B is a graph showing the relationship between the different reflection intensities of enantiomers and the crystal thickness.

[0011] In several views of the drawings, like reference numerals refer to corresponding parts. DETAILED DESCRIPTION

[0012] Crystallographic information of a crystal can be obtained based on an electron diffraction pattern (EDP) acquired in a charged particle microscope. In particular, a structural model can be obtained by analyzing the EDP. The structural model can be further refined based on a kinematic method to obtain a more accurate crystal structure. For example, the structural model can be optimized by performing least-squares refinement of the calculated crystal structure factors against the observed diffraction data.

[0013] One of the main limitations of the above kinematic method for structural model refinement is the inability to determine the absolute configuration of atomic positions in a crystal. The absolute configuration includes chirality, piezoelectricity, and polarity. For example, in chiral molecules (i.e., molecules without a center of symmetry), molecular crystals can have the same chemical composition and similar structural features, but have two different absolute configurations, left-handed or right-handed (i.e., enantiomers). Without considering dynamical diffraction effects, kinematic refinement has a 50% ambiguity with respect to the absolute configuration of a crystal, and thus chiral recognition cannot be performed. In addition, current methods for determining the absolute configuration of a crystal require a large amount of diffraction data, which are difficult to obtain from dose-sensitive samples.

[0014] The following description relates to systems and methods for using electron diffraction to determine the absolute structure of a crystal. The absolute structure includes the structure of the crystal and the absolute configuration of the crystal, where the absolute configuration includes non-centrosymmetric properties of the crystal, such as chirality. Although the description focuses on chirality, the same methods and systems can also be extended to determine other non-centrosymmetric properties, such as piezoelectricity and polarity, etc.

[0015] The method includes: collecting a plurality of first EDPs from at least one crystal; indexing the first EDPs to identify a plurality of zone axes of the at least one crystal; sorting the plurality of zone axes based on the intensity of the dynamical effect at each of the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the sorting; aligning the electron optical axis of an electron beam with the selected at least one zone axis; collecting one or more second EDPs using the aligned electron beam; and determining the absolute structure of the crystal based on the one or more second EDPs.

[0016] In this way, high-quality EDPs with the strongest dynamical effects can be collected with a lower total electron dose. This is crucial for determining the absolute structure because the process requires a large amount of diffraction data, but as the electron dose increases, the quality of the EDPs may rapidly degrade over time. By using the method disclosed herein, the absolute structure of a crystal can be reliably and rapidly determined with less diffraction data compared to conventional methods.

[0017] In one example, collecting a plurality of first EDPs from at least one crystal includes: directing an electron beam to each of a plurality of crystals, and collecting a plurality of EDPs from each of the plurality of crystals at different incident angles. The collection of the first EDPs can be guided by an electron microscope image of the sample, such as a transmission electron microscope (TEM) image or a scanning electron microscope (SEM) image. The electron beam can be directed to each of the plurality of crystals based on the electron microscope image of the sample area, which includes the plurality of crystals collected before collecting the first EDPs.

[0018] In another example, all of the first EDPs among the plurality of first EDPs can be collected from a single crystal. The single crystal can be selected from the electron microscope image collected before collecting the first EDPs.

[0019] The plurality of first EDPs can be collected based on the method disclosed in U.S. Patent Application 17 / 217,103 filed on March 30, 2021 by Buijsse et al., the entire content of which is incorporated herein by reference and used for all purposes. The plurality of first EDPs at a specific crystal can be collected by adjusting the direction of the electron beam such that each first EDP is collected at a different incident angle. The number of first EDPs collected at each of the plurality of crystals can be small to avoid radiation damage to the sample. The number of first EDPs can be determined by the maximum electron dose allowed for the sample. In some examples, the first EDPs provide sufficient completeness to solve a complete kinematic model of the crystal structure based on the first EDPs. In this way, simulated EDPs can be generated via dynamical simulation based on the structural model solved from the first EDPs.

[0020] In one example, the electron beam used to collect the first EDP is parallel or quasi-parallel. The first EDP can be a selected area electron diffraction (SAED) pattern or a precession electron diffraction (PED) pattern. In another example, the electron beam used to collect the first EDP is a focused beam. The first EDP can be a convergent beam electron diffraction (CBED) pattern. For electron-sensitive samples, SAED patterns and PED patterns may be superior to CBED patterns.

[0021] At least one crystal for collecting the first EDP can be selected based on an electron microscope image. The crystal can be selected based on an estimated crystal thickness. In one example, the crystal thickness can be estimated based on the thickness of the vitreous ice adjacent to the crystal (i.e., the ice thickness). The ice thickness can be estimated based on the image contrast in the TEM image. Crystals located in a sample region where the ice thickness is below a threshold can be selected to collect the first EDP. In another example, the crystal can be additionally or alternatively selected based on the size and / or shape of the crystal in the electron microscope image. In yet another example, the crystal thickness can be estimated based on an electron energy loss spectroscopy (EELS) signal. For example, the crystal thickness can be estimated according to the zero-loss peak extracted from the EELS spectrum in a specific energy range.

[0022] Multiple first EDPs can be indexed manually or automatically to identify the zone axes of multiple crystals of at least one crystal. The indexing process can be performed using, for example, DIALS software. The EDP indexing process can output one or more types of crystal information in the crystal information, which includes crystal orientation, unit cell parameters, and the EDP indexing process outputs the experimental geometry, i.e., the beam direction, the rotation axis of the stage (or sample holder), the detector position and orientation. Based on the indexing output, the zone axis orientation of one or more crystals in the crystal that can be accessed by the imaging system can be identified. In the current system configuration, when the electron optical axis of the electron beam can be aligned with the zone axis of the crystal, the imaging system can access a specific zone axis of the crystal through beam direction adjustment and / or sample stage movement (translation and rotation).

[0023] In one example, the structural model of a crystal can be solved based on a first EDP. The structural model of the crystal represents the complete / all structure of the molecule. That is, each molecule can be uniquely defined by its structural model. The structural model can include one or more of the crystal parameters, including crystal type, crystal geometry, unit cell size, and atomic positions. If sufficient data is collected from the first EDP, the structural model and the absolute configuration can be obtained from the first EDP. However, for most samples, due to the limitation of the maximum electron dose, it is difficult to collect sufficient high-quality diffraction data for determining the absolute configuration. In other words, the crystal may be damaged before an EDP showing strong dynamical effects can be obtained. Therefore, in this article, a second EDP is used to refine the structural model solved from the first EDP to obtain the absolute configuration of the crystal, where the second EDP is collected at or near the zone axis showing strong dynamical effects. In one example, one second EDP can be utilized to determine the absolute configuration.

[0024] In another example, the structural model can be obtained from an external source.

[0025] The intensity of the dynamical effect at each of the identified zone axes can be determined by kinetic simulations based on crystal information. The crystal information can include one or more of the chemical composition of the crystal, crystal symmetry, and the kinetics of the electron-sample interaction. The crystal information can be obtained from prior sample knowledge and / or by analyzing the first EDP. For example, based on the structural model solved from the first EDP, the intensity of the dynamical effect at each of the identified zone axes is determined. The intensity of the dynamical effect at a specific zone axis can be defined by the amplitude asymmetry of the Bijvoet pairs. In one example, simulated EDPs for each of the zone axes can be generated by simulation. The intensity of the dynamical effect at the zone axis can be determined based on the amplitude asymmetry of the Bijvoet pairs in the simulated EDP. The simulated EDP can include a subset of all the reflections observed at a specific zone axis. For example, the simulated EDP can include only the Bijvoet pairs with asymmetric amplitudes (i.e., asymmetric Biojvoet pairs). Therefore, the intensity of the dynamical effect can be determined based on the simulated asymmetric Biojvoet pairs.

[0026] As Figure 6A and Figure 6B shown, the intensity of the dynamical effect is affected by the crystal thickness. The intensity of the dynamical effect at each of the zone axes can be further determined based on the crystal thickness estimated during the indexing of the first EDP. For example, the simulation can utilize the estimated crystal thickness to determine the intensity of the dynamical effect.

[0027] The identified zone axes can be sorted based on the intensity of the dynamical effects calculated from the simulated EDP. One or more of the higher sorted zone axes can be selected to acquire a second EDP. In one example, the process of sorting the zone axes can generate a list of sorted zone axes that includes the corresponding beam and stage operations for aligning the beam with the axis.

[0028] The identified zone axes can also be sorted based on the accessibility of the electron beam. Zone axes with which the electron optical axis of the electron beam can be reliably aligned can have a higher sort. For example, a first zone axis of a crystal that can be accessed only by beam tilting and deflection has a higher sort than a second zone axis of the same or a different crystal that can be accessed by both beam tilting and deflection and sample stage movement.

[0029] In some examples, due to defects in the crystal, it becomes very difficult to perform a full dynamical modeling of the intensity. For this reason, certain strong zone axes are typically removed from the experimental data before processing. In these cases, some of the sorted accessible zone axes can be adjusted to be removed from the sort. Thus, in one example, the second EDP will not be acquired at these zone axes. In another example, for instance, in the list of sorted zone axes, the positions of these zone axes to be aligned are adjusted such that the second EDP is acquired at a non-zero angle (e.g., an angle greater than a threshold angle) with respect to the zone axis.

[0030] Aligning the electron optical axis of the electron beam with the selected zone axis includes at least adjusting the direction of the electron beam such that the electron optical axis of the electron beam is substantially aligned with the selected zone axis. In some examples, mechanical movement of the sample stage (or sample holder) may also be required to align the electron optical axis of the electron beam. Aligning the electron beam with the selected zone axis can include adjusting the electron beam and / or sample position according to the output indexing the first EDP. The alignment of the electron optical axis of the electron beam with the selected zone axis can be guided and / or confirmed based on further acquired EDPs.

[0031] The electron beam used to acquire the second EDP can be parallel, quasi-parallel, or focused. The second EDP can be one of an SAED pattern, a PED pattern, and a CBED pattern. For electron-sensitive samples, the SAED pattern and the PED pattern may be preferred over the CBED pattern. On the other hand, the CBED pattern is more sensitive to non-centrosymmetric properties and is thus more preferred when possible. At each selected zone axis, an SAED pattern or a PED pattern can be acquired by tilting the electron beam with respect to the selected zone axis.

[0032] The structural model solved from the first EDP can be dynamically refined based on the second EDP to obtain the absolute structure. The dynamic refinement takes into account the dynamical effects of the interaction between the electron beam and the lattice. For example, the structural model can be dynamically refined by comparing the structural model that takes into account the dynamical diffraction effects with the observed second EDP. In some examples, the second EDP and the first EDP can be combined into a combined data set, and the structural model can be dynamically refined based on this combined data set. For example, in each iteration, one of the EDPs in the EDP is used to refine the structural model. The refinement can be completed in response to the R factor (a measure of the consistency between the crystallographic model and the experimental diffraction data) being greater than a threshold R factor value.

[0033] In one example, the structural model can be refined iteratively. For example, in each iteration, one of the first or second EDPs is used to refine the structural model. In some examples, the crystal thickness can be the output of the refinement process. The crystal thickness can be estimated during each iteration and used in the next iteration to refine the structural model.

[0034] In one example, the second EDP can be collected at a higher beam energy compared to the first EDP. In another example, if the first EDP and the second EDP are of the same type of electron diffraction pattern, such as SAED pattern or PED pattern, the number of the second EDPs may be greater than that of the first EDP.

[0035] In this way, the absolute structure of the crystal, including the absolute configuration of the crystal, can be reliably determined. By dynamically refining the structural model using the second EDP collected at a specific orientation (e.g., along or near the selected zone axis), the structural model can converge quickly, thus revealing the absolute structure of the crystal. Throughout the data collection process, the total electron dose used for the sample can be minimized by collecting only the diffraction data that is crucial for solving the absolute structure.

[0036] The first EDP and the second EDP can be acquired in a transmission mode or a reflection mode in a charged particle microscope (CPM). The CPM can be any one of a TEM system, a SEM system, or a STEM (scanning electron microscope) system. The first EDP and the second EDP can be processed by a controller that includes at least a processor. The controller can be a local controller located within the CPM. Alternatively, the controller can include one or more processors remotely connected to the CPM via a network. The acquired EDP can be sent to a remotely located processor for processing. For example, computationally intensive portions of the method, such as indexing the EDP, solving the structural model, sorting the zone axes, and refining the structural model, can be performed by a remotely located processor, such as a processor in a cloud server. In one example, the first EDP can be sent to a remotely located processor for indexing and zone axis sorting. The sorted zone axes can be sent back to the CPM to acquire the second EDP.

[0037] In some examples, after the first EDP and the second EDP are acquired, the electron diffraction data can be stored in non-transitory memory and post-processed by a processor to determine the absolute configuration of the crystal.

[0038] Go to Figures 1 to 2, an example of a CPM, transmission electron microscope (TEM) system 100 is shown in different operating modes. The TEM system can be a cryogenic electron microscope system for imaging a sample cooled to cryogenic temperatures. The TEM system 100 includes an electron source 10 that emits an electron beam 11 along an optical axis 110. The electron beam is guided along an electron optical axis through an electron column 113 toward a sample 14 that is held by a sample holder 13. The electron optical axis can be aligned with the optical axis 110. The electron column can include one or more of a condenser system, a deflector, and an objective lens. In some embodiments, the condenser system 12 can include one or more condenser lenses and one or more apertures. A deflector 19 positioned downstream of the condenser system 12 moves and / or tilts the electron beam relative to the optical axis 110. A pre-sample objective 16 positioned downstream of the deflector 19 collimates the electron beam and directs the electron beam onto the sample 14. The sample 14 can be held by the sample holder 13 in a specimen plane 111. In some examples, 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 electron optical axis and / or translating the sample within the specimen plane. Scattered electrons transmitted through the sample 14 sequentially pass through a post-sample objective 123 and a 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 a signal to an image processor 24 to form an image. The detector 25 can include an amplifier for amplifying the signal before sending the signal to the image processor 24. In one example, the detector 25 can be a CCD camera or a CMOS camera. In some embodiments, different detectors can be used for diffraction pattern acquisition and sample image acquisition.

[0039] Figure 1 The TEM system 100 operating in a low magnification (LM) imaging mode is shown. A dashed line 22 illustrates the beam path of scattered electrons from a point on the sample to the detector 25 in the LM imaging mode, where the post-sample objective 123 is turned off or operated at a low excitation voltage to acquire a sample image with a large FOV and low resolution. A beam blocker 17 can be used to intercept the strong unscattered beam. The projection system 21 operates differently in imaging modes (such as the LM imaging mode or the SA imaging mode) and diffraction modes (such as the SA diffraction mode). The LM imaging mode can be used to examine large sample areas and to locate sample regions for collecting EDPs.

[0040] Figure 2Shows a TEM system 100 operating in SA imaging mode and SA diffraction mode. The dashed line 41 illustrates the beam path of scattered electrons from the sample 14 to the detector 25 in SA diffraction mode. In SA diffraction mode, the projector system 21 images the back focal plane 43 of the sample post-objective lens 123 onto the detector 25. The beam blocker 17 is inserted into the electron optical axis 110 to block the unscattered beam. The dashed line 42 illustrates the beam path of scattered electrons from the sample 14 to the detector 25 in SA imaging mode. In SA imaging mode, the specimen 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 blocker 17 is retracted from the electron optical axis 110. Compared with the sample image acquired in LM imaging mode, the sample image acquired in SA imaging mode may have a smaller FOV and a higher magnification. In one example, an SA aperture may be inserted into the beam path. The SA aperture may be positioned in the SA plane 44. Alternatively, the aperture in the condenser system 12 may be used as a beam limiting aperture. In another example, an image deflector may be positioned between the sample and the detector for moving and tilting the electrons transmitted through the sample back onto the electron optical axis so that the ED pattern remains centered on the detector during beam tilting and the image remains centered on the detector during beam movement. The image deflector 45 may be positioned between the back focal plane 43 and the SA plane 44.

[0041] The controller 30 can control the operation of the TEM system 100 manually in response to operator instructions or automatically according to computer-readable instructions stored in a non-transitory memory (or computer-readable medium) 32. The controller 30 may include a processor and is configured to execute the 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 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 to operate in any of the LM imaging mode, SA imaging mode, and SA diffraction mode. The controller 30 can adjust the beam position and / or the beam incident angle on the sample by adjusting the deflector 19. The controller 30 may also be coupled to a display 31 to display notifications and / or signals detected by the detector 25. The controller 30 can control the TEM system to acquire any of the SAED pattern, PED pattern, and CBED pattern. 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.

[0042] 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 CPMs. For example, the CPM is a scanning transmission electron microscope (STEM) system. In this case, a sample image can be made in the scanning STEM mode, and a diffraction image can be obtained with a (quasi)-parallel beam. In another example, the CPM is a SEM. The present discussion of the TEM system is provided only as an example of a suitable imaging form.

[0043] Figure 3 Method 300 for determining the absolute structure of a crystal using electron diffraction data is shown. A plurality of first EDPs are acquired for indexing. Accessible zone axes are identified through an indexing process. Then the identified zone axes are sorted to determine the order in which the second EDPs are collected. The second EDPs collected at zone axes with higher sorting (i.e., zone axes with stronger dynamical effects) are collected first to ensure successful refinement of the structural model, thereby obtaining the absolute structure of the crystal.

[0044] At 302, after loading the sample into the microscope, one or more electron microscope images of the sample are acquired first. The sample can be a crystal held by a cryo-cooled TEM grid. For example, the TEM image of the sample can be acquired in either or both of the LM imaging mode and the SA imaging mode as shown. The electron microscope images can be used to select and position the crystal for acquiring the first EDP. The crystal in the electron microscope image can be selected based on the estimated crystal thickness in the electron microscope image. Figures 1 to 2 The electron microscope images can be used to select and position the crystal for acquiring the first EDP. The crystal in the electron microscope image can be selected based on the estimated crystal thickness in the electron microscope image.

[0045] At 304, the crystal thickness is optionally estimated. In one example, the crystal thickness can be estimated based on the sample image acquired at 302. The estimated crystal thickness can be used to determine the strength of the dynamical effect at a particular zone axis. In one example, the ice thickness is estimated based on the image contrast in different sample regions. Crystals located in sample regions with a preferred ice thickness are selected for collecting the first EDP. The sample regions with a preferred ice thickness may have an imaging contrast below a threshold contrast, indicating a thinner ice thickness. In another example, the crystal thickness can be estimated based on the contrast of the crystal in the sample image. A low contrast of the crystal in the TEM image may indicate a thinner crystal. In yet another example, the crystal can be selected based on the estimated size and shape of the crystal in the electron microscope image. Crystals with a size below a threshold size can be selected for acquiring the second EDP. In some examples, the crystal thickness can be estimated based on other signals such as EELS signals.

[0046] At 306, multiple first EDPs of one or more crystals are collected. At 302, a crystal for collecting the first EDP can be selected and positioned in an electron microscope image. In one embodiment, the first EDP is three-dimensional ED data collected using a parallel or quasi-parallel electron beam. For example, the electron beam is directed to each of the selected crystals. At a particular crystal, the electron beam is tilted relative to the crystal to collect SAED patterns with different incident angles. For example, as described in U.S. Patent Application 17 / 217,103 filed on March 30, 2021 by Buijsse et al., multiple EDP patterns are collected by tilting the electron beam within a small angle range (e.g., -10 degrees to 10 degrees). In another example, a PED pattern is collected as the first EDP.

[0047] At 308, the first EDPs are indexed and accessible zone axes are identified. In one example, each of the first EDPs can be indexed individually. The EDP indexing process can output one or more types of crystal information in the crystal information, which includes crystal orientation, unit cell parameters, beam direction, rotation axis of the stage (or sample holder), detector position and orientation. Based on the index output, the zone axis of the current crystal configuration can be calculated. Based on the configuration of the microscope, the accessible zone axes of the crystals that can be aligned with the electron beam are determined. The configuration of the microscope can include one or more of a beam tilt range, a beam deflection range, and a stage movement (translation and rotation) range. The DIALS software can be used to index the first EDPs. The accessible zone axes can be the zone axes of different crystals.

[0048] At 309, a structural model of the crystal is obtained. In one example, the structural model can be obtained by solving the structural model using the first EDP. The first EDPs from different crystals can be combined to determine one or more of crystal type, crystal geometry, and kinematic structure. The DIALS software can also be used to solve the structural model. In another example, the structural model can be obtained from an external source, such as from a database or a previous experiment.

[0049] At 310, the zone axes identified at 306 are ranked based on the intensity of the dynamical effects observable at the zone axes. The output of the ranking can be a list of ranked accessible zone axes, where the zone axes corresponding to stronger dynamical effects have higher rankings and will be selected first for collecting the second EDP. The list can include the coordinates of the crystals corresponding to each zone axis and the orientation of the zone axis relative to the reference frame.

[0050] The dynamical effect can be a chirality-dependent dynamical effect, which results in different intensities of the Bijvoet pairs in the EDPs collected at some zone axes. For example, Figure 4A andFigure 4B Simulated CBED patterns of D-GLA and L-GLA at the

[110] zone axis are shown respectively. Figure 5A and Figure 5B Simulated CBED patterns of D-GLA and L-GLA at the

[012] zone axis are shown respectively. The arrows in the figures point to one of the Bijvoet pairs, and these Bijvoet pairs show intensity asymmetry in each CBED.

[0051] The intensity of the kinetic effect can be measured by the asymmetry or difference in the intensity of the Bijvoet pairs in the simulated EDP at the zone axis. In one example, the asymmetry can be calculated as the normalized contrast based on the intensity of the Bijvoet pairs:

[0052]

[0053] where D and L are the intensities of the Bijvoet pairs in the simulated EDP. The intensities of the Bijvoet pairs in the simulated EDP at the zone axis can be generated by simulation based on crystal information. The crystal information can include one or more of the chemical composition of the crystal, crystal symmetry, and the kinetics of electron-sample interaction. At 302 and 308, some known information can be obtained from the indexing results and the solved structural model. For example, crystal symmetry information can be obtained from the indexing results. In one example, through this simulation, the kinetic interaction between the electron beam and the crystal structure described by the structural model obtained at 309 is simulated. The simulated EDP including the intensities of the Bijvoet pairs is generated.

[0054] In one example, the identified zone axes can also be sorted based on the estimated crystal thickness. For example, the simulation can consider the estimated crystal thickness to determine the intensity of the Bijvoet pairs. Figure 6A and Figure 6B show that the intensities of the Bijvoet pairs in crystals with different chiralities are affected differently by the crystal thickness. Figure 6A and Figure 6B are the simulated intensities of the (020) reflection and (0-20) reflection in two enantiomers D(-) and L(+) of glutamic acid (GLA) in the

[110] orientation: D-GLA and L-GLA. The contrast difference in the reflection intensities of the two enantiomers oscillates as the crystal thickness increases. Therefore, using the estimated crystal thickness, the intensity of the kinetic effect at a specific zone axis of the crystal can be determined more accurately.

[0055] In another example, the identified zone axes can also be sorted based on the accessibility of the zone axis. For example, a zone axis that can be reliably accessed (aligned) through a combination of stage and beam movement has a higher ranking.

[0056] At 312, one zone axis among the zone axes is selected based on the sorting at 310. Selecting a zone axis includes selecting a corresponding crystal that has the selected zone axis.

[0057] At 316, the electron beam is aligned with the selected zone axis of the selected crystal. Based on the orientation matrix generated at 306, the electron beam can be automatically aligned with the selected zone axis by actuating one or more of the deflector and the sample holder. Aligning the electron beam with the selected zone axis includes aligning the electron optical axis of the electron beam with the selected zone axis by beam direction adjustment or by a combination of beam adjustment and sample holder adjustment. Beam direction adjustment includes tilting the electron beam by operating one or more deflectors. Beam direction adjustment may also include moving the beam in the X - Y plane without changing the angle between the electron optical axis and the sample plane. Sample holder adjustment may include translating and rotating the sample holder for holding the sample.

[0058] At 318, one or more second EDPs in the second EDP can be acquired. The second EDP can be one or more CBED patterns, or a plurality of EDPs (SAED patterns or PED patterns) acquired by tilting the beam around the selected zone axis. If a parallel or quasi - parallel beam is used to acquire the second EDP, the dose of each second EDP may be higher than the dose of each first EDP. Since CBED patterns are more sensitive to dynamical effects, if the system configuration allows, CBED patterns may be preferred over SAED patterns or PED patterns for the second EDP.

[0059] At 320, method 300 determines whether another zone axis needs to be selected. If second EDPs have been acquired for all accessible zone axes, then there is no need to select another zone axis. If the absolute configuration of the crystal cannot be determined confidently, then another zone axis can be selected. For example, if the R - factor is higher than a threshold R - factor, then another zone axis can be selected. In some examples, if the quality of the second EDP is low, then method 300 can determine at 322 to select another zone axis. The quality of the EDP can be evaluated based on one or more of the number of reflections in the EDP, the intensity of the reflections, and the intensity asymmetry of the Bijvoet pairs. In one example, if the radiation damage shown in the second EDP is high (e.g., reflections disappear), then the zone axis of another crystal can be selected. In another example, if the intensity of the dynamical effect in the second EDP is not strong, then another zone axis of the same or a different crystal can be selected. If another zone axis needs to be selected, then method 300 proceeds to 312 to select another zone axis. If no zone axis needs to be selected, then method 300 proceeds to 322.

[0060] At 322, the absolute structure of the crystal is determined. Determining the absolute structure includes determining the absolute configuration of the crystal. In one example, the absolute structure of the crystal can be determined by refining a structural model according to a second EDP or a combined data set from both the first EDP and the second EDP. The structural model can be adjusted such that the R factor calculated based on the observed structure factors and the calculated structure factors is minimized. This refinement can take into account both the kinematic and dynamic effects of the electron-sample interaction. If the second EDP is a SAED pattern or a PED pattern, the structural model can be refined according to the integrated second EDP. If the second EDP is a CBED pattern, a single second EDP can be used to determine the absolute configuration. In another example, only the second EDP can be used to solve and refine the structural model. In this example, the first EDP is only used to determine the accessible zone axes and not for solving the structural model.

[0061] In some embodiments, the refinement can be performed while the second EDP is being acquired. For example, once one or more second EDPs in the second EDP are acquired, they can be sent to a processor for refinement. Based on the refinement results, it can be determined whether more second EDPs need to be acquired.

[0062] In this way, the absolute structure of the crystal, including non-centrosymmetric properties, can be determined effectively and automatically. The technical effect of acquiring the first EDP is to determine the accessible zone axes. The technical effect of sorting the multiple accessible zone axes based on the intensity of the dynamic effect at each zone axis is to minimize the number of second EDPs required to refine the structural model. Thus, high-quality diffraction data can be acquired at a relatively low total electron dose, enabling the rapid determination of the absolute structure of radiation-sensitive crystals.

[0063] Example 1 is a method for crystallography that includes: acquiring a plurality of first electron diffraction patterns (EDPs) from at least one crystal; indexing the first EDPs to identify a plurality of zone axes of the at least one crystal; sorting the plurality of zone axes based on the intensity of the dynamic effect at each zone axis of the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the sorting; aligning the electron optical axis of an electron beam with the selected at least one zone axis; acquiring one or more second EDPs using the aligned electron beam; and determining the absolute structure of the crystal based on the one or more second EDPs.

[0064] Example 2 includes the subject matter of any of Example 1 and further includes determining the intensity of the dynamic effect at each zone axis of the plurality of zone axes by simulation, wherein the simulated EDPs are generated at each zone axis based on a structural model of the crystal.

[0065] Example 3 includes the subject matter according to any one of Example 2, and further includes determining the intensity of the dynamical effect at each of the plurality of zone axes based on an estimated crystal thickness.

[0066] Example 4 includes the subject matter according to any one of Example 2, and further specifies that the structural model is determined based on the first EDP.

[0067] Example 5. The method according to claim 4, and further specifies that the integrity of the first EDP is lower than a threshold integrity.

[0068] Example 6 includes the subject matter according to any one of Example 2, and further specifies that the intensity of the dynamical effect at each zone axis is determined based on the amplitude asymmetry of the Bijvoet pairs in the simulated EDP.

[0069] Example 7 includes the subject matter according to any one of Example 2, and further specifies that determining the absolute structure of the crystal based on the one or more second EDPs includes determining the absolute configuration of the crystal by refining the structural model based on the one or more second EDPs.

[0070] Example 8 includes the subject matter according to any one of Examples 1-7, and further specifies that aligning the electron optical axis of the electron beam with a selected zone axis includes at least adjusting the direction of the electron beam.

[0071] Example 9 includes the subject matter according to any one of Examples 1-8, and further specifies that indexing the first EDP includes determining one or more of crystal orientation, unit cell parameters, beam direction, rotation axis of the stage, detector position, and detector orientation.

[0072] Example 10 includes the subject matter according to any one of Examples 1 to 9, and further specifies that the first EDP is acquired using a quasi-parallel electron beam.

[0073] Example 11 includes the subject matter according to any one of Examples 1-10, and further specifies that the one or more second EDPs are one or more CBEDs acquired using a focused electron beam.

[0074] Example 12 includes the subject matter according to any one of Examples 1-11, and further includes: acquiring an image of a sample including a plurality of crystals; and selecting at least one crystal from the plurality of crystals in the sample image for acquiring the EDP.

[0075] Example 13 includes the subject matter according to any one of Example 12, and further includes estimating the crystal thickness from the sample image, and wherein the at least one crystal is selected based on the estimated crystal thickness.

[0076] Example 14 is a charged particle microscope system, the charged particle microscope system comprising: a sample holder for holding a sample including a plurality of crystals; an electron source for generating an electron beam; an electron optical column for guiding the electron beam towards the sample; a detector for detecting an electron diffraction pattern; and a controller including a processor and a non-transitory memory for storing computer-readable instructions, wherein by executing the instructions in the processor, the charged particle microscope system is configured to: acquire a plurality of first electron diffraction patterns (EDPs) from at least one of the plurality of crystals; align an electron optical axis of the electron beam with at least one of the selected zone axes, wherein the selected zone axes are determined based on a ranking of a plurality of zone axes of the at least one crystal, the plurality of zone axes are identified by indexing the first EDPs, and the plurality of zone axes are ranked based on an intensity of a dynamical effect at each of the plurality of zone axes; and acquire one or more second EDPs with the aligned electron beam to determine an absolute structure of the crystal.

[0077] Example 15 includes the subject matter according to any one of Examples 14, and further specifies that determining the absolute structure of the crystal includes determining non-centrosymmetric characteristics of the crystal.

[0078] Example 16 includes the subject matter according to any one of Examples 14, and further includes: adjusting the electron optical column to guide a quasi-parallel electron beam to the sample to acquire the first EDP; and adjusting the electron optical column to guide a focused electron beam to the sample to acquire the second EDP.

[0079] Example 17 includes the subject matter according to any one of Examples 14 - 16, and further specifies that by adjusting one or more components in the electron optical column, the electron optical axis of the electron beam is aligned with at least one of the selected zone axes.

[0080] Example 18 includes the subject matter according to any one of Examples 17, and further specifies that by further adjusting the sample holder, the electron optical axis of the electron beam is aligned with at least one of the selected zone axes.

[0081] Example 19 includes the subject matter according to any one of Examples 14 - 18, and further specifies that acquiring a plurality of first EDPs from at least one of the plurality of crystals includes: guiding the electron beam to each of the plurality of crystals; and acquiring a plurality of EDPs from each of the plurality of crystals at different incident angles.

[0082] Example 20 includes the subject matter according to any one of Examples 19, and further includes adjusting the incident angle by adjusting the direction of the electron beam.

Claims

1. A method for crystallography, the method comprises: acquiring a plurality of first electron diffraction patterns (EDPs) from at least one crystal; indexing the first EDPs to identify a plurality of zone axes of the at least one crystal; sorting the plurality of zone axes based on the intensity of the dynamical effect at each of the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the sorting; aligning an electron optical axis of an electron beam with the selected at least one zone axis; acquiring one or more second EDPs using the aligned electron beam; and determining an absolute structure of the crystal based on the one or more second EDPs.

2. The method according to claim 1, the method further comprising determining the intensity of the dynamical effect at each of the plurality of zone axes by simulation, wherein simulated EDPs are generated at each zone axis based on a structural model of the crystal.

3. The method according to claim 2, the method further comprising determining the intensity of the dynamical effect at each of the plurality of zone axes based on an estimated crystal thickness.

4. The method according to claim 2, wherein the structural model is determined based on the first EDPs.

5. The method according to claim 4, wherein the first EDPs have an integrity below a threshold integrity.

6. The method according to claim 2, wherein the intensity of the dynamical effect at each zone axis is determined based on the amplitude asymmetry in the Bijvoet pairs in the simulated EDPs.

7. The method according to claim 2, wherein determining the absolute structure of the crystal based on the one or more second EDPs comprises determining an absolute configuration of the crystal by refining the structural model based on the one or more second EDPs.

8. The method according to any one of claims 1-7, wherein aligning the electron optical axis of the electron beam with the selected zone axis comprises at least adjusting a direction of the electron beam.

9. The method according to any one of claims 1-8, wherein indexing the first EDPs comprises determining one or more of crystal orientation, unit cell parameters, beam direction, rotation axis of a stage, detector position, and detector orientation.

10. The method according to any one of claims 1-9, wherein the first EDPs are acquired using a quasi-parallel electron beam.

11. The method according to any one of claims 1-10, wherein the one or more second EDPs are one or more CBEDs acquired using a focused electron beam.

12. The method according to any one of claims 1-11, the method further comprising acquiring a sample image comprising a plurality of crystals, and selecting at least one crystal from the plurality of crystals in the sample image for acquiring the EDPs.

13. The method according to claim 12, the method further comprising estimating a crystal thickness from the sample image, and wherein the at least one crystal is selected based on the estimated crystal thickness.

14. A charged particle microscope system, the charged particle microscope system Comprising: A sample holder for holding a sample including a plurality of crystals; An electron source for generating an electron beam; An electron optical column for guiding the electron beam towards the sample; A detector for detecting an electron diffraction pattern; And A controller including a processor and a non-transitory memory for storing computer-readable instructions, wherein by executing the instructions in the processor, the charged particle microscope system is configured to: Acquire a plurality of first electron diffraction patterns (EDPs) from at least one of the plurality of crystals; Align the electron optical axis of the electron beam with at least one of the selected zone axes, wherein the selected zone axis is determined based on the ranking of a plurality of zone axes of the at least one crystal, the plurality of zone axes are identified by indexing the first EDPs, and the plurality of zone axes are ranked based on the intensity of the dynamical effect at each of the plurality of zone axes; And Acquire one or more second EDPs with the aligned electron beam to determine the absolute structure of the crystal.

15. The charged particle microscope system according to claim 14, wherein determining the absolute structure of the crystal includes determining the non-centrosymmetric property of the crystal.

16. The charged particle microscope system according to any one of claims 14 - 15, the charged particle microscope system further includes adjusting the electron optical column to guide a quasi-parallel electron beam to the sample to acquire the first EDP, and adjusting the electron optical column to guide a focused electron beam to the sample to acquire the second EDP.

17. The charged particle microscope system according to any one of claims 14 - 16, wherein by adjusting one or more components in the electron optical column, the electron optical axis of the electron beam is aligned with at least one of the selected zone axes.

18. The charged particle microscope system according to claim 17, wherein by further adjusting the sample holder, the electron optical axis of the electron beam is aligned with at least one of the selected zone axes.

19. The charged particle microscope system according to any one of claims 14 - 18, wherein acquiring a plurality of first EDPs from at least one of the plurality of crystals Comprises: Guiding the electron beam to each of the plurality of crystals; And acquiring a plurality of EDPs from each of the plurality of crystals at different incident angles.

20. The charged particle microscope system according to claim 19, the charged particle microscope system further includes adjusting the incident angle by adjusting the direction of the electron beam.

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