Method and system for determining the absolute structure of a crystal

Abstract

The crystal structure is determined based on one or more second electron diffraction patterns acquired at selected zone axes where the strength of the dynamic effect is strong, and the zone axes are selected by ranking accessible zone axes determined from the plurality of first electron diffraction patterns.

Description

[Technical Field]

[0001] This specification relates generally 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 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; ranking the plurality of zone axes based on a strength of a dynamic effect in each zone axis among the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the ranking; 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.

[0003] In another embodiment, a charged particle microscope system includes 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 directing the electron beam toward the sample, a detector for detecting electron diffraction patterns, and a controller, wherein the controller includes a processor and a non-transitory memory for storing computer-readable instructions, and 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 crystal of the plurality of crystals; align an electron optical axis of the electron beam with at least one of selected zone axes, wherein the selected zone axis is determined based on a ranking of a plurality of zone axes of the at least one crystal, the plurality of zone axes being identified by indexing the first EDP, and the plurality of zone axes being ranked based on the strength of dynamic effects in each of the plurality of zone axes; and acquire one or more second EDPs using the aligned electron beam to determine an absolute structure of the crystal.

[0004] It should be understood that the foregoing summary is provided to introduce a selection of concepts in a simplified form 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 defined uniquely by the claims that follow the detailed description. Moreover, the claimed subject matter is not limited to implementations that solve any disadvantages discussed above or in any part of this disclosure. [Brief explanation of the drawings]

[0005] [Figure 1] 1 shows a transmission electron microscopy (TEM) system operating in a first mode. [Figure 2] 2 shows the TEM system of FIG. 1 operating in another mode. [Figure 3] This is a method for determining the absolute structure of a crystal. [Figure 4A] 1 is a simulated electron diffraction pattern (EDP) of the enantiomers in the first zone axis. [Figure 4B] 1 is a simulated electron diffraction pattern (EDP) of the enantiomers in the first zone axis. [Figure 5A] Simulated EDP of the enantiomers in the second zone axis. [Figure 5B] Simulated EDP of the enantiomers in the second zone axis. [Figure 6A] 1 is a graph showing the intensity of the different reflections of the enantiomers versus the crystal thickness. [Figure 6B] 1 is a graph showing the intensity of the different reflections of the enantiomers versus the crystal thickness.

[0006] Like reference numerals refer to corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION OF THE INVENTION

[0007] Crystallographic information of a crystal can be obtained based on electron diffraction patterns (EDPs) obtained with a charged particle microscope. In particular, a structural model can be obtained by analyzing the EDPs. The structural model can be further refined based on kinematic methods to obtain a more accurate crystal structure. For example, the structural model can be optimized by least-squares refinement of calculated crystal structure factors against the observed diffraction data.

[0008] One of the main limitations of the above-mentioned kinetic methods for structural mode refinement is their inability to determine the absolute configuration of atomic positions in a crystal. Absolute configuration includes chirality, piezoelectricity, and polarity. For example, in chiral molecules, i.e., molecules without a center of symmetry, molecular crystals may have the same chemical composition and similar structural features but two different absolute configurations, left-handed or right-handed (i.e., enantiomers). Without considering dynamic diffraction effects, kinetic refinement has a 50% ambiguity regarding the absolute configuration of a crystal, and therefore, chiral identification is impossible. Furthermore, current methods for determining the absolute configuration of a crystal require large amounts of diffraction data, which are difficult to obtain from dose-sensitive samples.

[0009] The following description relates to systems and methods for determining the absolute structure of a crystal using electron diffraction. Absolute structure includes the structure of the crystal as well as the absolute configuration of the crystal, which includes the non-centrosymmetric properties of the crystal, such as chirality. While the description focuses on chirality, the same methods and systems can be extended to determine other non-centrosymmetric properties, such as piezoelectricity and polarity.

[0010] The method includes obtaining 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; ranking the plurality of zone axes based on a strength of a dynamic effect in each zone axis among the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the ranking; aligning an electron optical axis of an electron beam with the selected at least one zone axis; obtaining 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.

[0011] In this way, high-quality EDPs with the strongest dynamic effects can be obtained at low total electron doses. This process requires a large amount of diffraction data, which is important for determining absolute structure, because the quality of the EDP can rapidly deteriorate over time as the electron dose increases. By using the methods disclosed herein, the absolute structure of a crystal can be determined reliably and quickly with less diffraction data than conventional methods.

[0012] In one example, acquiring a plurality of first EDPs from at least one crystal includes directing an electron beam at each of the plurality of crystals and acquiring a plurality of EDPs from each of the plurality of crystals at different angles of incidence. The acquisition of the first EDPs may 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 may be directed to each of the plurality of crystals based on an electron microscope image of the sample area including the plurality of crystals collected prior to acquiring the first EDPs.

[0013] In another example, all of the multiple first EDPs can be obtained from a single crystal, which can be selected from an electron microscope image acquired prior to acquisition of the first EDPs.

[0014] Multiple first EDPs can be obtained based on the method disclosed in U.S. Patent Application No. 17 / 217,103 by Buijsse et al., filed March 30, 2021, which is incorporated herein by reference in its entirety for all purposes. Multiple first EDPs for a particular crystal can be obtained by adjusting the direction of the electron beam so that each first EDP is obtained at a different angle of incidence. The number of first EDPs obtained for each crystal of the multiple 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 kinetic model of the crystal structure based on the first EDPs. Therefore, simulated EDPs can be generated via dynamic simulation based on a structural model solved from the first EDPs.

[0015] In one example, the electron beam for obtaining 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 for obtaining the first EDP is a focused beam. The first EDP can be a convergent beam electron diffraction (CBED) pattern. For electron-sensitive samples, SAED and PED patterns may be preferred over CBED patterns.

[0016] At least one crystal for acquiring 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 (i.e., the ice thickness) close to the crystal. The ice thickness can be estimated based on the image contrast in the TEM image. A crystal located in a sample region having an ice thickness below a threshold can be selected for acquiring the first EDP. In another example, the crystal can additionally or alternatively be 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 from a zero-loss peak extracted from the EELS spectrum in a specific energy range.

[0017] The multiple first EDPs can be manually or automatically indexed to identify multiple zone axes 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 of the crystal information, including the crystal orientation, unit cell parameters, and the experimental geometry, i.e., the beam direction, the stage (or sample holder) rotation axis, and the detector position and orientation. Based on the index output, one or more zone axis orientations of the crystal that are accessible by the imaging system can be identified. A particular zone axis of the crystal is accessible by the imaging system when the electron optical axis of the electron beam can be aligned with the zone axis of the crystal under the current system configuration using beam direction adjustment and / or sample stage movement (translation and rotation).

[0018] In one example, a structural model of a crystal can be solved based on the first EDP. The structural model of a crystal represents the complete / whole 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 following crystal parameters: crystal type, crystal shape, unit cell dimensions, and atomic positions. If sufficient data is collected from the first EDP, a structural model and absolute configuration can be obtained from the first EDP. However, for most samples, due to limitations in the maximum electron dose, it is difficult to obtain high-quality diffraction data sufficient to determine the absolute configuration. In other words, the crystal may be destroyed before an EDP exhibiting strong dynamic effects can be obtained. Therefore, in this specification, the structural model solved from the first EDP is refined using a second EDP to obtain the absolute configuration of the crystal, and the second EDP is obtained at or near a zone axis exhibiting strong dynamic effects. In one example, the absolute configuration can be determined using a 1-second EDP.

[0019] In another example, the structural model may be obtained from an external source.

[0020] The strength of the dynamic effect at each of the identified zone axes can be determined through dynamic simulation based on crystal information. The crystal information can include one or more of the crystal's chemical composition, crystal symmetry, and electron-sample interaction dynamics. The crystal information can be obtained from prior sample knowledge and / or by analyzing the first EDP. For example, the strength of the dynamic effect at each of the identified zone axes can be determined based on a structural model solved from the first EDP. The strength of the dynamic effect at a particular zone axis can be defined by the asymmetry of the amplitude of the Bijvoet pair. In one example, simulated EDPs at each of the multiple zone axes can be generated through simulation. The strength of the dynamic effect at a zone axis can be determined based on the asymmetry of the amplitude of the Bijvoet pair in the simulated EDP. The simulated EDP can include a subset of all reflections observed at a particular zone axis. For example, the simulated EDP can include only Bijvoet pairs with asymmetric amplitudes (i.e., asymmetric Bijvoet pairs). Therefore, the strength of the dynamic effect can be determined based on simulated asymmetric Biojvoet pairs.

[0021] As shown in Figures 6A and 6B, the strength of the dynamic effect is affected by the crystal thickness. The strength of the dynamic effect in each of the multiple zone axes can be further determined based on the crystal thickness estimated during indexing the first EDP. For example, the simulation can utilize the estimated crystal thickness to determine the strength of the dynamic effect.

[0022] The identified zone axes may be ranked based on the strength of the dynamic effect calculated from the simulated EDP. One or more higher-ranked zone axes may be selected to obtain a second EDP. In one example, the process of ranking the zone axes may generate a list of ranked zone axes with corresponding beam and stage manipulations to align the beam with the axis.

[0023] The identified zone axes can be further ranked based on their accessibility by the electron beam. Zone axes that can reliably align the electron optical axis of the electron beam can be ranked higher. For example, a first zone axis of one crystal that is accessible only by beam tilt and deflection is ranked higher than a second zone axis of the same or a different crystal that is accessible through both beam tilt and deflection and sample stage movement.

[0024] In some cases, crystal imperfections make complete dynamic modeling of the intensity too difficult. For this reason, certain strong zone axes are typically removed from the experimental data before processing. In these cases, some of the ranked accessible zone axes can be adjusted to remove them from the ranking. As a result, in one example, the second EDP is not acquired at these zone axes. In another example, the positions of these zone axes to be aligned are adjusted, for example, within the list of ranked zone axes, so that the second EDP is acquired at a non-zero angle (e.g., an angle greater than a threshold angle) from the zone axis.

[0025] Aligning the electron optical axis of the electron beam with the selected zone axis includes adjusting at least the direction of the electron beam so 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 may include adjusting the electron beam and / or sample position according to output from indexing the first EDP. Alignment of the electron beam's electron optical axis with the selected zone axis may be derived and / or confirmed based on further acquired EDPs.

[0026] The electron beam for acquiring the second EDP may be parallel, quasi-parallel, or focused. The second EDP may be one of an SAED pattern, a PED pattern, and a CBED pattern. For electron-sensitive samples, SAED and PED patterns may be preferred over CBED patterns. On the other hand, CBED patterns are more sensitive to non-centrosymmetric structures and are therefore preferred when possible. For each selected zone axis, an SAED or PED pattern may be acquired by tilting the electron beam relative to the selected zone axis.

[0027] The structural model solved from the first EDP can be dynamically refined based on the second EDP to obtain an absolute structure. Dynamic refinement takes into account the dynamic effects of the electron beam interacting with the crystal lattice. For example, the structural model can be dynamically refined by comparing the structural model, which takes into account dynamic 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 the combined data set. For example, in each iteration, the structural model is refined using one of the EDPs. Refinement can be completed in response to an R-factor (a measure of agreement between the crystallographic model and experimental diffraction data) being greater than a threshold R-factor value.

[0028] In one example, the structural model can be refined iteratively. For example, in each iteration, the structural model is refined using one of the first or second EDPs. In some examples, the crystal thickness can be an output of the refinement process. The crystal thickness can be estimated during each iteration and used to refine the structural model in the next iteration.

[0029] In one example, the second EDPs may be acquired at a higher beam energy compared to the first EDPs. In another example, if the first EDPs and the second EDPs are the same type of electron diffraction pattern, e.g., SAED patterns or PED patterns, the number of second EDPs may be greater than the number of first EDPs.

[0030] In this way, the absolute structure of a crystal, including its absolute configuration, can be reliably determined. By dynamically refining the structural model using a second EDP acquired at a specific orientation (e.g., along or near a selected zone axis), the structural model can rapidly converge to reveal the absolute structure of the crystal. Throughout the data collection process, the total electron dose to the sample can be kept to a minimum by collecting only the diffraction data critical to elucidating the absolute structure.

[0031] The first and second EDPs may be acquired in a charged particle microscope (CPM) in either transmission or reflection mode. The CPM may be any one of a TEM system, a SEM system, or a STEM (scanning electron microscope) system. The first and second EDPs may be processed by a controller including at least a processor. The controller may be a controller located locally within the CPM. Alternatively, the controller may include one or more processors remotely connected to the CPM via a network. The acquired EDPs may be sent to a remotely located processor for processing. Computationally intensive portions of the method, such as indexing the EDPs, solving the structural model, ranking the zone axes, and refining the structural model, may be performed by a remotely located processor, such as in a cloud server. In one example, the first EDP may be sent to a remotely located processor for indexing and zone axis ranking. The ranked zone axes may be sent back to the CPM to acquire the second EDP.

[0032] In some examples, after acquiring the first and second EDPs, the electron diffraction data may be stored in non-transitory memory and post-processed by a processor to determine the absolute configuration of the crystal.

[0033] 1-2, a transmission electron microscope (TEM) system 100, an example of a CPM, is shown in different operating modes. The TEM system may be a cryogenic electron microscope system for imaging a cryogenically cooled sample. The TEM system 100 includes an electron source 10 that emits an electron beam 11 along an optical axis 110. The electron beam is directed along the electron optical axis via an electron optical column 113 to a sample 14 held by a sample holder 13. The electron optical axis may be aligned with the optical axis 110. The electron optical column may include one or more of focusing optics, a deflector, and an objective lens. In some embodiments, the condenser optics 12 may include one or more condenser lenses and one or more apertures. A deflector 19 positioned downstream of the condenser optics 12 shifts and / or tilts the electron beam relative to the optical axis 110. A pre-specimen objective lens 16 positioned downstream of the deflector 19 collimates the electron beam and directs it onto the sample 14. The sample 14 can be held in the sample plane 111 by a sample holder 13. 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 sample plane. Scattered electrons transmitted through the sample 14 pass sequentially through a post-sample objective lens 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 to amplify the signal before sending it 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.

[0034] FIG. 1 shows a TEM system 100 operating in low magnification (LM) imaging mode. Dashed line 22 indicates the beam path of scattered electrons from a point on the sample to detector 25 in LM imaging mode, with post-sample objective lens 123 either off or operated at a low excitation voltage to acquire sample images with a large FOV and low resolution. A beam stopper 17 can be used to block the intense unscattered beam. Projection system 21 is operated differently in imaging mode (e.g., LM imaging mode or SA imaging mode) and diffraction mode (e.g., SA diffraction mode). LM imaging mode can be used to inspect large sample areas and locate sample regions for collecting EDPs.

[0035] FIG. 2 shows the TEM system 100 operating in SA imaging mode and SA diffraction mode. Dashed line 41 indicates 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 post-specimen objective lens 123 onto the detector 25. A beam stopper 17 is inserted into the electron-optics system 110 to block the unscattered beam. Dashed line 42 indicates 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 stopper 17 is retracted from the electron-optics system 110. Sample images acquired in SA imaging mode may have a smaller FOV and higher magnification compared to sample images acquired in LM imaging mode. In one example, an SA aperture may be inserted into the beam path. The SA aperture may be positioned within the SA plane 44. Alternatively, an aperture in the condenser optics 12 can function as the beam-limiting aperture. In another example, an image deflector 45 can be positioned between the sample and the detector to shift and tilt the electrons transmitted through the sample back to the electron optics, so that the ED pattern remains centered on the detector during beam tilt and the image remains centered on the detector during beam shift. An image deflector 45 can be positioned between the back focal plane 43 and the SA plane 44.

[0036] The controller 30 may control the operation of the TEM system 100 manually in response to operator commands or automatically in accordance with computer-readable instructions stored in a non-transitory memory (or computer-readable medium) 32. The controller 30 may include a processor and be configured to execute computer-readable instructions to control various components of the TEM system 100 to implement any of the methods described herein. For example, the controller may adjust the TEM system to operate in any one of an LM imaging mode, an SA imaging mode, and an SA diffraction mode by adjusting one or more of the SA aperture 18, the excitation of the objective lens 123, the beam stopper 17, and the projector system 21. The controller 30 may adjust the beam position and / or the beam incidence angle relative to the sample by adjusting the deflector 19. The controller 30 may further be coupled to a display 31 to display notifications and / or signals detected by the detector 25. The controller 30 may control the TEM system to acquire any of an SAED pattern, a PED pattern, and a CBED pattern. The controller 30 may 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 the crystal based on the acquired dataset.

[0037] Although a TEM system is described as an example, it should be understood that the sample image and diffraction pattern may be acquired with other CPMs. For example, the CPM may be a scanning transmission electron microscopy (STEM) system. In that case, the sample image may be created in a scanning STEM mode, and the diffraction image may be obtained with a (quasi-)parallel beam. In another example, the CPM is an SEM. This discussion of a TEM system is provided merely as an example of one suitable imaging modality.

[0038] FIG. 3 illustrates a method 300 for determining the absolute structure of a crystal using electron diffraction data. Multiple first EDPs are acquired for indexing. Through the indexing process, accessible zone axes are identified. The identified zone axes are then ranked to determine the sequence in which second EDPs are collected. Second EDPs are acquired with higher-ranked zone axes (i.e., zone axes with stronger dynamic effects) and collected first to ensure successful structural model refinement to obtain the absolute structure of the crystal.

[0039] In 302, after loading the sample into the microscope, one or more electron microscope images of the sample are first acquired. The sample may be a crystal held by a cryogenically frozen TEM grid. For example, TEM images of the sample may be acquired in either or both of the LM imaging mode and SA imaging mode shown in FIGS. 1-2. The electron microscope images can be used to select and locate crystals for acquiring a first EDP. Crystals in the electron microscope images can be selected based on the crystal thickness estimated in the electron microscope images.

[0040] In 304, the crystal thickness is optionally estimated. In one example, the crystal thickness can be estimated based on the sample image acquired in 302. The estimated crystal thickness can be used to determine the strength of the dynamic effect in a particular zone axis. In one example, the ice thickness is estimated based on image contrast in different sample regions. Crystals located in sample regions with a preferred ice thickness can be selected to collect a first EDP. Sample regions with a preferred ice thickness can have an imaging contrast lower than a threshold contrast, indicating a thin ice thickness. In another example, the crystal thickness can be estimated based on the contrast of the crystals in the sample image. Low contrast of crystals in a TEM image can indicate thinner crystals. In yet another example, crystals can be selected based on their size and shape estimated in an electron microscope image. Crystals with a size smaller than a threshold size can be selected to acquire a second EDP. In some examples, the crystal thickness can be estimated based on other signals, such as an EELS signal.

[0041] In 306, multiple first EDPs of one or more crystals are acquired. In 302, crystals for collecting the first EDPs can be selected and positioned within the electron microscope image. In one embodiment, the first EDPs are three-dimensional ED data collected using a parallel or quasi-parallel electron beam. For example, the electron beam is directed at each of the selected crystals. For a particular crystal, the electron beam is tilted relative to the crystal to acquire SAED patterns at different angles of incidence. For example, as described in U.S. Patent Application No. 17 / 217,103 by Buijsse et al., filed March 30, 2021, multiple EDP patterns are acquired by tilting the electron beam over a small angle range (e.g., from -10 to 10 degrees). In another example, a PED pattern is acquired as the first EDP.

[0042] In 308, the first EDP is indexed and an accessible zone axis is identified. In one example, each of the first EDPs can be individually indexed. The EDP indexing process can output one or more of the crystal information, including the crystal orientation, unit cell parameters, beam direction, stage (or sample holder) rotation axis, and detector position and orientation. Based on the index output, the zone axis of the current crystal arrangement can be calculated. Based on the microscope configuration, the accessible zone axis of the crystal that can be aligned with the electron beam is determined. The microscope configuration may include one or more of the beam tilt range, beam deflection range, and stage movement (translation and rotation) range. The first EDP can be indexed using DIALS software. The accessible zone axis may be the zone axis of a different crystal.

[0043] 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 the crystal type, crystal shape, and kinematic structure. The structural model can also be solved using DIALS software. In another example, the structural model can be obtained from an external source, such as a database or from previous experiments.

[0044] At 310, the zone axes identified at 306 are ranked based on the strength of the dynamic effects observable at the zone axes. The output of the ranking may be a ranked list of accessible zone axes, with zone axes corresponding to stronger dynamic effects being ranked higher and selected first for acquiring the second EDP. The list may include the coordinates of the crystal corresponding to each zone axis and the orientation of the zone axis relative to the reference frame.

[0045] The dynamic effect may be chirality-dependent, causing different intensities of Bijvoet pairs in EDPs acquired at some zone axes. For example, Figures 4A and 4B show simulated CBED patterns at the

[0110] zone axis for D-GLA and L-GLA, respectively. Figures 5A and 5B show simulated CBED patterns at the

[0012] zone axis for D-GLA and L-GLA, respectively. The arrows in the figures point to one of the Bijvoet pairs, indicating the asymmetry in intensity at each CBED.

[0046] The strength of the dynamic effect can be measured by the asymmetry or difference in the intensity of the Bijvoet pairs in the simulated EDPs along the zonal axis. In one example, the asymmetry can be calculated as a normalized contrast based on the intensity of the Bijvoet pairs.

number

[0047] In one example, the identified zone axes may be further ranked based on the estimated crystal thickness. For example, simulations can take the estimated crystal thickness into account to determine the intensity of the Bijvoet pair. Figures 6A and 6B show that the intensity of Bijvoet pairs in crystals with different chiralities is affected differently by crystal thickness. Figures 6A and 6B show the simulated intensities of the (020) and (0-20) reflections of glutamic acid (GLA) in the two enantiomers D(-) and L(+): D-GLA and L-GLA, respectively, in the

[0110] orientation. The difference in the contrast between the reflection intensities of the two enantiomers varies with increasing crystal thickness. Therefore, the estimated crystal thickness can be used to more accurately determine the intensity of dynamic effects in a particular zone axis of the crystal.

[0048] In another example, the identified zone axes may be further ranked based on the accessibility of the zone axis, e.g., a zone axis that can be reliably accessed (aligned) through a combination of stage and beam movement is ranked higher.

[0049] At 312, one of the zone axes is selected based on the ranking at 310. Selecting a zone axis includes selecting a corresponding crystal having the selected zone axis.

[0050] In 316, the electron beam is aligned with the selected zone axis of the selected crystal. The electron beam may be automatically aligned with the selected zone axis by actuating one or more of the deflectors and the sample holder based on the orientation matrix generated in 306. 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 either beam direction adjustment or combined beam and sample holder adjustment. Beam direction adjustment includes tilting the electron beam by operating one or more deflectors. Beam direction adjustment may also include shifting the beam in the XY 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 that holds the sample.

[0051] At 318, one or more of the second EDPs may be acquired. The second EDPs may be one or more CBED patterns acquired by tilting the beam about a selected zone axis, or multiple EDPs (SAED or PED patterns). If the second EDPs are acquired using a parallel or quasi-parallel beam, the dose of each second EDP may be higher than the dose of each first EDP. Because CBED patterns are more sensitive to dynamic effects, they may be preferred over SAED or PED patterns for the second EDPs, if the system configuration allows.

[0052] In 320, method 300 determines whether another zone axis needs to be selected. If second EDPs have been acquired for all accessible zone axes, another zone axis is not selected. If the absolute configuration of the crystal cannot be determined with certainty, another zone axis can be selected. For example, if the R-factor is higher than a threshold R-factor, another zone axis can be selected. In some examples, if the quality of the second EDP is low, method 300 can determine to select another zone axis in 322. 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 asymmetry of the intensity of the Bijvoet pair. In one example, if the second EDP shows high radiation damage (e.g., disappearing reflections), a zone axis of another crystal can be selected. In another example, if the intensity of the dynamic effect is not strong in the second EDP, a zone axis of the same or a different crystal can be selected. If another zone axis needs to be selected, method 300 moves to 312 to select another zone axis. If a zone axis does not need to be selected, method 300 proceeds to 322.

[0053] 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 on a second EDP or a combined data set from both the first and second EDPs. The structural model can be adjusted so that an R-factor calculated from the observed and calculated structure factors is minimized. The refinement can take into account both kinetic and dynamic effects of the electron-sample interaction. If the second EDP is an SAED pattern or a PED pattern, the structural model can be refined on the combined second EDP. If the second EDP is a CBED pattern, the absolute configuration can be determined using a single second EDP. In another example, the structural model can be solved and refined using only the second EDP. In this example, the first EDP is used only to determine the accessible zone axes and not to solve the structural model.

[0054] In some embodiments, the refinement may be performed simultaneously while acquiring the second EDPs. For example, as soon as one or more of the second EDPs are acquired, they may be sent to a processor for refinement. Based on the results of the refinement, it may be determined whether more second EDPs need to be acquired.

[0055] In this way, the absolute structure of a crystal, including its non-centrosymmetric structure, can be determined efficiently and automatically. The technical effect of acquiring the first EDP is to determine the accessible zone axes. The technical effect of ranking multiple accessible zone axes based on the strength of the dynamic effect in each zone axis is to minimize the number of second EDPs required to refine the structural model. Therefore, the absolute structure of a radiation-sensitive crystal can be rapidly determined using high-quality diffraction data acquired at a relatively low total electron dose.

[0056] Example 1 is a method for crystallography, comprising: 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; ranking the plurality of zone axes based on a strength of a dynamic effect in each zone axis among the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the ranking; 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.

[0057] Example 2 includes any of the subject matter of Example 1, further including determining through simulation a strength of the dynamic effect in each zone axis of the plurality of zone axes, wherein a simulated EDP is generated in each zone axis based on a structural model of the crystal.

[0058] Example 3 includes any subject matter of example 2, further including determining a strength of the dynamic effect in each zone axis of the plurality of zone axes based on the estimated crystal thickness.

[0059] Example 4 includes any subject matter of example 2, further specifying that the structural model is determined based on the first EDP.

[0060] Example 5 is the method of claim 4, further specifying that the first EDP has an integrity less than a threshold integrity.

[0061] Example 6 includes any subject matter of example 2, further specifying that the strength of the dynamic effect in each zone axis is determined based on the asymmetry of the amplitudes in the Bijvoet pairs in the simulated EDPs.

[0062] Example 7 includes any subject matter of Example 2, further specifying 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 a structural model based on the one or more second EDPs.

[0063] Example 8 includes the subject matter of any of Examples 1-7, further specifying that aligning an electronic optical axis of the electron beam with the selected zone axis includes adjusting at least a direction of the electron beam.

[0064] Example 9 includes the subject matter of any of Examples 1-8, further specifying that indexing the first EDP includes determining one or more of a crystal orientation, a unit cell parameter, a beam direction, a stage rotation axis, a detector position, and a detector orientation.

[0065] Example 10 includes the subject matter of any of Examples 1-9, further specifying that the first EDP is obtained using a quasi-parallel electron beam.

[0066] Example 11 includes the subject matter of any of Examples 1-10, further specifying that the one or more second EDPs are one or more CBEDs obtained using a focused electron beam.

[0067] Example 12 includes the subject matter of any of Examples 1-11, further including acquiring a sample image including a plurality of crystals, and selecting at least one crystal from the plurality of crystals in the sample image for acquiring an EDP.

[0068] Example 13 includes any subject matter of example 12, further including estimating a crystal thickness from the sample image; and selecting the at least one crystal based on the estimated crystal thickness.

[0069] Example 14 is a 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 directing the electron beam toward the sample; a detector for detecting electron diffraction patterns; and a controller, wherein the controller includes a processor and a non-transitory memory for storing computer-readable instructions, and 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 crystal of the plurality of crystals; align an electron optical axis of the electron beam with at least one of selected zone axes, wherein the selected zone axis is determined based on a ranking of a plurality of zone axes of the at least one crystal, the plurality of zone axes being identified by indexing the first EDP, and the plurality of zone axes being ranked based on the strength of dynamic effects in each of the plurality of zone axes; and acquire one or more second EDPs using the aligned electron beam to determine the absolute structure of the crystal.

[0070] Example 15 includes any of the subject matter of Example 14, further specifying that determining the absolute structure of the crystal includes determining the non-centrosymmetry of the crystal.

[0071] Example 16 includes any of the subject matter of example 14, further including adjusting the electron optical column to direct a quasi-parallel electron beam at the sample to obtain the first EDP, and adjusting the electron optical column to direct a focused electron beam at the sample to obtain the second EDP.

[0072] Example 17 includes the subject matter of any of Examples 14-16, further specifying that an electron optical axis of the electron beam is aligned with at least one of the selected zone axes by adjusting one or more components in the electron optical column.

[0073] Example 18 includes any of the subject matter of example 17, further specifying that the sample holder is further adjusted to align an electron optical axis of the electron beam with at least one of the selected zone axes.

[0074] Example 19 includes the subject matter of any of Examples 14-18, further specifying that obtaining a plurality of first EDPs from at least one crystal of the plurality of crystals includes directing an electron beam at each of the plurality of crystals and obtaining a plurality of EDPs at different angles of incidence from each of the plurality of crystals.

[0075] Example 20 includes any of the subject matter of example 19, further including adjusting the angle of incidence by adjusting the direction of the electron beam.

Claims

1. 1. A method for crystallography comprising: obtaining a plurality of first electron diffraction patterns (EDPs) from at least one crystal; indexing the first EDP to identify multiple zone axes of the at least one crystal; ranking the plurality of zone axes based on a strength of dynamic effects in each of the plurality of zone axes; selecting at least one zone axis from the plurality of zone axes based on the ranking; 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 the absolute structure of the crystal based on the one or more second EDPs.

2. 2. The method of claim 1, further comprising determining the intensity of the dynamic effect at each of the plurality of zone axes through simulation, wherein a simulated EDP is generated at each zone axis based on a structural model of the crystal.

3. The method of claim 2 , further comprising determining a strength of the dynamic effect in each of the plurality of zone axes based on an estimated crystal thickness.

4. The method of claim 2 , wherein the structural model is determined based on the first EDP.

5. The method of claim 4 , wherein the first EDP has an integrity less than a threshold integrity.

6. The method of claim 2 , wherein the strength of the dynamic effect in each zonal axis is determined based on amplitude asymmetry in Bijvoet pairs in the simulated EDP.

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

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

9. 9. The method of claim 1, wherein indexing the first EDP comprises determining one or more of a crystal orientation, a unit cell parameter, a beam direction, a stage rotation axis, a detector position, and a detector orientation.

10. The method according to any one of claims 1 to 9, wherein the first EDP is obtained using a quasi-parallel electron beam.

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

12. 12. The method of claim 1, further comprising: acquiring a sample image including a plurality of crystals; and selecting the at least one crystal from the plurality of crystals in the sample image for acquiring the EDP.

13. 13. The method of claim 12, 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. 1. A 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 directing the electron beam toward the sample; a detector for detecting the electron diffraction pattern; a controller, the controller including a processor and a non-transitory memory for storing computer readable instructions, the execution of the instructions in the processor causing the charged particle microscope system to: obtaining a plurality of first electron diffraction patterns (EDPs) from at least one crystal of the plurality of crystals; aligning an electron optical axis of the electron beam with at least one selected zone axis, the selected zone axis being determined based on a ranking of a plurality of zone axes of the at least one crystal, the plurality of zone axes being identified by indexing the first EDP, and the plurality of zone axes being ranked based on a strength of a dynamic effect in each of the plurality of zone axes; and and acquiring one or more second EDPs using the aligned electron beam to determine the absolute structure of the crystal.

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

16. 16. The charged particle microscope system of claim 14 or 15, further comprising: adjusting the electron optical column to direct a quasi-parallel electron beam at the sample to obtain the first EDP; and adjusting the electron optical column to direct a focused electron beam at the sample to obtain the second EDP.

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

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

19. 19. The charged particle microscope system of claim 14, wherein acquiring a plurality of first EDPs from at least one of the plurality of crystals comprises directing 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 angles of incidence.

20. 20. The charged particle microscope system of claim 19, further comprising adjusting the angle of incidence by adjusting the direction of the electron beam.

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