A double diffractometer system for single crystal diffraction and in-situ crystal plate of a synchrotron radiation device and a single crystal diffraction detection method thereof

Abstract

The present application relates to a kind of for single crystal diffraction and in-situ crystal plate double diffractometer system of synchrotron radiation device and its single crystal diffraction detection method, system includes vertical diffractometer module, horizontal crystal plate diffractometer module, coaxial observation collimating module and bearing base, two kinds of diffraction module are installed in bearing base and is equipped with independent translation bearing respectively, can be moved to incident X ray optical path center alternately, coaxial observation collimating module is arranged on the incident light path upstream of two sets of module and is shared by two, incident X ray is irradiated to single crystal sample in vertical diffractometer module or horizontal crystal plate diffractometer module after passing through coaxial observation collimating module.The present application adopts double diffraction module integrated layout and shared optical assembly, solves the defects that traditional equipment cannot be compatible with two kinds of single crystal detection, needs to disassemble and reassemble when experiment is changed, optical path is recalibrated, hardware cost is high, and debugging is time-consuming, two kinds of diffraction experiments can be switched once optical path is calibrated, reduce optical hardware investment, shorten beamline debugging working hours.

Description

Technical Field

[0001] This invention belongs to the technical field of synchrotron radiation devices and experimental equipment for biological macromolecular crystallography, and more specifically relates to a dual diffractometer system for single-crystal diffraction and in-situ crystallization plates in synchrotron radiation devices, and a method for detecting single-crystal diffraction. Background Technology

[0002] Synchrotron radiation sources, with their superior beam characteristics of high brightness, high collimation, and broad energy spectrum, are the core experimental platform for analyzing the three-dimensional structures of biological macromolecules using crystallography. X-ray single-crystal diffraction experiments based on synchrotron radiation beamlines have become the mainstream technique for resolving the atomic-level structures of biological macromolecules such as proteins and nucleic acids. Currently, there are two non-substitutable single-crystal diffraction experimental modes in the industry, each corresponding to two completely different diffractometers. The applicable scenarios, equipment structures, sample placement methods, and experimental requirements of these two types of experiments differ significantly.

[0003] The first type is low-temperature single-crystal diffraction experiments, which is the most common data acquisition scheme for the analysis of biomolecular structures and is suitable for most common protein single-crystal samples. This experiment uses a high-precision vertical air-floating turntable diffractometer with the main axis vertically arranged. The single-crystal sample is pre-adsorbed onto a micron-sized magnetic sample carrier made of nylon and placed in a liquid nitrogen cryogenic gas flow environment to suppress crystal radiation damage caused by X-ray irradiation. The sample is irradiated with horizontally incident X-rays, and the vertical air-floating turntable drives the crystal to rotate 360° with high precision. With the help of a two-dimensional surface detector at the back end, a complete diffraction dataset is collected, which can achieve high-resolution three-dimensional structure analysis.

[0004] The second type is room-temperature in-situ crystallization plate diffraction experiments, developed for special, high-protection-level samples, and possessing irreplaceable application value. For microcrystals, radiation-sensitive crystals, and biological samples requiring maintenance of physiological room temperature and a pristine liquid-phase environment, cryogenic freezing would destroy the original crystal lattice structure. For pathogenic microorganisms and infectious protein samples, BSL-2 or higher biosafety protection requirements must be met. The crystals must be completely sealed within the crystallization plate droplets and cannot be removed by opening the lid; in-situ diffraction testing can only be performed directly on the entire crystallization plate. This experiment requires the crystallization plate to be placed vertically to keep the internal crystallization droplets stable and prevent leakage. X-rays must penetrate the crystallization plate film and the internal crystals horizontally; therefore, a horizontally arranged air-floating rotary stage diffractometer with a horizontally positioned main axis is necessary to complete room-temperature diffraction data acquisition.

[0005] Existing synchrotron radiation beamline stations for biomacromolecule crystallography have inherent limitations in equipment configuration, preventing the simultaneous online use of two types of diffractometers. The mainstream implementation methods in the industry fall into two categories, both of which have significant drawbacks:

[0006] The first implementation method involves equipping a single beamline with only a single type of diffractometer, enabling only the corresponding single experimental mode. If researchers need to switch experimental types, the entire existing diffractometer setup must be completely disassembled and moved before installing a new diffractometer. After reassembly, the entire optical path must be recalibrated. This entire disassembly, assembly, and calibration process can take several hours to several days. Synchrotron radiation facility time resources are scarce, and repeated disassembly and assembly would result in a significant waste of valuable experimental time. Furthermore, frequent disassembly, assembly, and relocation would alter the reference positions of the air-bearing turntable and optical components, reducing the long-term operational stability of the system and hindering the implementation of automated high-throughput data acquisition processes.

[0007] The second compromise solution involves some beamlines maintaining the equipment and directly tilting and fixing the crystallization plate onto a vertical air-floating turntable diffractometer to conduct in-situ crystallization plate experiments. However, this temporary modification has several fatal flaws: tilting the crystallization plate causes internal crystal droplets to flow and the crystals to shift, resulting in sample position deviation; the incident and scattered X-ray paths are distorted, the experimental background scattering increases significantly, the signal-to-noise ratio of the diffraction data decreases, and the quality of the final acquired data cannot meet the requirements for high-precision structural analysis, leading to a low experimental success rate. Summary of the Invention

[0008] To address the shortcomings of existing synchrotron radiation beamline diffractometer configurations, which cannot simultaneously support the rapid and efficient execution of both low-temperature single-crystal diffraction and room-temperature in-situ crystallization plate diffraction experiments, resulting in long experiment switching cycles, poor equipment stability, difficulty in ensuring data quality, high hardware investment costs, and inability to adapt to the detection of high-biosafety-level sealed samples, this invention aims to provide a dual diffractometer system for single-crystal diffraction and in-situ crystallization plate diffraction in synchrotron radiation facilities, along with a single-crystal diffraction detection method.

[0009] The dual diffractometer system for single-crystal diffraction and in-situ crystallization plate diffraction in a synchrotron radiation facility according to the present invention includes a vertical diffractometer module, a horizontal crystallization plate diffractometer module, a coaxial observation collimation module, and a support base. Both the vertical diffractometer module and the horizontal crystallization plate diffractometer module are mounted on the support base, each equipped with an independent translational support stage capable of alternating movement to the center of the incident X-ray path. The coaxial observation collimation module is located upstream of the incident light path of both diffractometer modules and is shared by both. The incident X-rays pass through the coaxial observation collimation module and then irradiate the single-crystal sample position of either the vertical diffractometer module or the horizontal crystallization plate diffractometer module. This invention overcomes the industry limitations of existing single diffraction instruments, which cannot simultaneously detect two types of single crystals and require disassembly and reassembly of equipment and repeated optical path calibration when changing experiments, by integrating two types of diffraction modules on the same support base and providing an independent translation stage, and setting up a set of shared front coaxial observation collimation modules. It can switch between two diffraction experiments with only a single optical path calibration, greatly reducing the debugging time of synchrotron radiation beamlines, and at the same time reducing the procurement cost of a set of independent optical hardware.

[0010] In a preferred embodiment, the vertical diffractometer module includes a three-axis translation stage, a vertical air-bearing turntable, a two-dimensional fine-tuning platform, and a magnetic sample carrier. The three-axis translation stage has three degrees of freedom of movement in the X, Y, and Z directions, driving the entire vertical diffractometer module into and out of the optical path. The rotation axis of the vertical air-bearing turntable is arranged along the Z direction, and the rotation axis is perpendicular to the incident X-ray. The two-dimensional fine-tuning platform is located above the vertical air-bearing turntable and has X and Y fine-tuning degrees of freedom. The magnetic sample carrier uses magnetic force to adsorb the single crystal sample. This invention achieves overall optical path switching of the entire vertical diffractometer module through the three-axis translation stage. Combined with the vertically arranged air-bearing turntable, two-stage X / Y fine-tuning structure, and magnetic sample carrier, it can achieve high-precision three-dimensional alignment of low-temperature single crystal samples, ensuring that the sample remains stably within the X-ray spot throughout the 360° rotation process. The magnetic structure is compatible with automated robotic arms for sample loading and unloading, improving the data resolution and automation level of low-temperature diffraction experiments.

[0011] In a preferred embodiment, the horizontal crystallizer module includes a dual-axis translation stage, a horizontal air-bearing turntable, a hollow lightweight adapter plate, a two-dimensional scanning stage, and a crystallizer clamping mechanism. The dual-axis translation stage has X and Z-axis degrees of freedom, driving the entire horizontal crystallizer module into and out of the optical path. The rotation axis of the horizontal air-bearing turntable is arranged along the X-axis. The hollow lightweight adapter plate is assembled between the horizontal air-bearing turntable and the two-dimensional scanning stage, and the crystallizer clamping mechanism is installed on the two-dimensional scanning stage and maintains the vertical orientation of the crystallizer. This invention achieves lateral optical path switching of the crystallizer module through the X / Z dual-axis translation stage, weight reduction design of the hollow lightweight adapter plate, and the clamping mechanism maintaining the vertical orientation of the crystallizer, thereby reducing background scattering noise in diffraction experiments. The lightweight adapter plate also reduces the interference of load on the rotational accuracy of the horizontal air-bearing turntable.

[0012] In a preferred embodiment, the coaxial observation collimation module includes a coaxial microscopic imaging component and a pneumatic backlight avoidance component. The optical path of the coaxial microscopic imaging component is coaxial with the incident X-ray and has a through-hole to allow the incident X-ray to pass through unobstructed. The pneumatic backlight avoidance component has a rotating structure to rotate the backlight component out of the incident optical path, preventing the backlight component from blocking the crystal plate and interfering with the diffraction signal acquisition. This invention, by combining a coaxial microscopic imaging component with a through-hole with a rotatable pneumatic backlight avoidance structure, enables the sharing of the microscopic observation optical path and the X-ray incident optical path, eliminating the need for two independent observation devices. Under crystal plate detection conditions, the backlight automatically rotates out to avoid blocking the sample and generating stray diffraction signals, adapting to fully automatic switching operation of two types of diffraction experiments.

[0013] In a preferred embodiment, the coaxial observation collimation module is configured with a beam blocker with a concave light-facing end face and a beam-limiting collimator with a long, straight through-hole. The beam blocker is equipped with a multi-dimensional adjustment stage, which has X, Y, and Z-axis translational and roll-rotational degrees of freedom, used to move the beam blocker entirely into and out of the incident light path. The beam-limiting collimator is equipped with a dual-axis adjustment stage, which has X and Z-axis translational degrees of freedom, used to adjust the coaxial alignment of the beam-limiting collimator with the incident X-ray. This invention, by using a concave end face beam blocker with a four-dimensional adjustment stage and a long, straight through-hole beam-limiting collimator with an X / Z dual-axis adjustment stage, can completely absorb the direct main X-ray to protect the back-end detector, suppress stray light generated by the beam-limiting structure, and simultaneously achieve rapid and accurate coaxial calibration of the beam blocker and beam-limiting components, improving the diffraction signal-to-noise ratio.

[0014] In a preferred embodiment, the support base is equipped with a two-stage linked pneumatic avoidance cooling assembly. This assembly includes a short-stroke pneumatic sliding stage and a long-stroke pneumatic sliding stage. The short-stroke stage carries a cooling airflow output component, while the long-stroke stage moves the entire cooling assembly away from the optical path. This invention, through the avoidance cooling assembly composed of short-stroke and long-stroke two-stage linked pneumatic sliding stages, can output cooling airflow at close range to suppress sample radiation damage during low-temperature single-crystal experiments. During room-temperature experiments with the crystallization plate, the entire cooling assembly can be moved out of the optical path without manual disassembly and assembly of the cooling equipment. It is compatible with dual-mode automatic switching conditions and is a dedicated structure for dual-diffraction integrated systems.

[0015] In a preferred embodiment, the supporting base is an integrated marble vibration-damping base that simultaneously supports the vertical diffractometer module, the horizontal crystallizer module, and the coaxial observation collimation module while isolating them from ground vibration. This invention employs an integrated marble vibration-damping base that simultaneously supports all optical and rotational modules, effectively reducing the interference of ground vibration on the coaxial rotation accuracy of the two air-bearing turntables, preventing vibration from causing single-crystal samples to deviate from the X-ray focus, and maintaining stable, high-precision system operation over the long term.

[0016] The single-crystal diffraction detection method of the above-mentioned dual diffractometer system according to the present invention includes the following steps: S1, removing the beam blocker and the beam collimator, completing the beam coaxial calibration by relying on the coaxial microscopic imaging component, and then moving in the beam collimator for secondary calibration, respectively calibrating the rotation centers of the two sets of air-bearing turntables to coincide with the beam center; S2, the three-axis translational support stage sends the vertical diffractometer module into the optical path, and the two-stage linkage pneumatic avoidance cooling component approaches the sample to complete data acquisition; S3, the long-stroke pneumatic sliding stage removes the cooling component and the pneumatic avoidance backlight component to rotate out of the optical path, and the dual-axis translational support stage sends the horizontal crystal plate diffractometer module into the optical path to collect single-crystal diffraction data in the crystal plate. This invention overcomes the shortcomings of existing equipment that requires separate independent calibration of the optical path for two types of experiments by unifying the optical path calibration once and switching the acquisition of two types of diffraction modes step by step. After the entire calibration process is completed, low-temperature single crystal and crystal plate single crystal detection can be carried out alternately without secondary calibration, which greatly improves the efficiency of beamline equipment. This collaborative operation process cannot be simply derived from the conventional operation of existing single diffraction equipment.

[0017] In a preferred embodiment, in step S1, the magnetic sample carrier is loaded with a calibration tip, and the vertical air-bearing turntable is rotated twice to acquire image coordinates. The average value of the coordinates is then used to lock the reference focal length of the coaxial microscopic imaging component. This invention, through the focal length calibration process of acquiring coordinates by rotating the turntable twice and averaging the values, can eliminate depth-of-field deviation in microscopic imaging, ensuring that the position of the single-crystal sample within the observation field completely corresponds to the actual X-ray irradiation position, avoiding alignment errors caused by parallax, and improving sample positioning accuracy.

[0018] In a preferred embodiment, in step S3, the entire area of ​​the crystallization plate is traversed to identify single-crystal apertures. The target single crystal is aligned with the incident beam, and a horizontal air-bearing turntable is driven to collect multiple sets of diffraction data and stitch together a complete dataset. This invention, through a grid-based full-domain scanning process to identify single crystals and a multi-angle data stitching acquisition process, can automatically screen single-crystal locations within the crystallization plate that possess effective diffraction signals. After acquiring data from multiple angles, a complete three-dimensional diffraction pattern is obtained by stitching together the data, improving the efficiency and data integrity of batch sample testing of the crystallization plate.

[0019] This invention integrates two types of diffraction modules that can be independently translated and switched onto the same support base and is equipped with a shared front coaxial observation collimation optical path. Relying on the integrated layout, the independent translation and switching structure of the modules, and the synergistic cooperation of the shared optical components, it solves the technical shortcomings of existing diffraction equipment: it cannot simultaneously perform two types of single-crystal detection experiments on the same beamline; it requires disassembling and reassembling the equipment and repeatedly performing optical path calibration when changing experimental types; it incurs high costs for repeated configuration of optical hardware; and it is time-consuming for equipment switching and debugging. Only one unified optical path calibration is required to alternately perform two types of single-crystal diffraction acquisition, reducing the hardware investment cost of optical components and significantly shortening the time required for equipment switching and debugging. The optical path debugging process of the synchrotron radiation beamline is equipped with automatic avoidance optics and cooling structures adapted to two types of experimental conditions, realizing fully automatic switching between two detection modes without manual intervention. It takes into account the different usage requirements of high-precision rotational acquisition of low-temperature single crystals and batch sample detection of crystal plates in vertical orientation. It effectively reduces experimental background scattering noise, improves sample alignment accuracy and diffraction data resolution, and simultaneously suppresses the interference of ground vibration on the coaxial accuracy of the rotating components. It ensures that the entire system maintains stable and high-precision operation in the long term. The dedicated collaborative calibration and step-by-step acquisition and detection process further improves the efficiency of beamline equipment and the integrity of single crystal diffraction data. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall structure of the dual diffractometer system according to the present invention;

[0021] Figure 2 for Figure 1 A schematic diagram of the vertical diffractometer module;

[0022] Figure 3 for Figure 1 A schematic diagram of the structure of a horizontal crystallizer module;

[0023] Figure 4 for Figure 1 A schematic diagram of the coaxial observation collimation module;

[0024] Figure 5 for Figure 4 A schematic diagram of the structure of a coaxial microscopic imaging component;

[0025] Figure 6 for Figure 4 A schematic diagram of the collimation subsystem;

[0026] Figure 7 for Figure 1 A structural diagram of the auxiliary equipment;

[0027] Figure 8 for Figure 7 A schematic diagram of the structure of a two-stage linkage aerodynamic avoidance refrigeration component. Detailed Implementation

[0028] The preferred embodiments of the present invention are given below with reference to the accompanying drawings and described in detail.

[0029] like Figure 1 As shown, the dual diffractometer system according to the present invention includes a vertical diffractometer module 1, a horizontal crystallizer module 2, a coaxial observation collimation module 3, and auxiliary equipment 4.

[0030] Auxiliary equipment 4 provides simultaneous shock absorption support, a low-temperature sample environment, and a supporting structure for fluorescence signal detection, serving as the foundation for the entire system. The vertical diffractometer module 1, the horizontal crystallizer module 2, and the coaxial observation and collimation module 3 are all supported and fixed on auxiliary equipment 4. Vertical diffractometer module 1 is used for low-temperature single-crystal diffraction experiments on single-crystal samples of biological macromolecules (e.g., protein crystals), while horizontal crystallizer module 2 is used for room-temperature in-situ diffraction experiments on crystallizer modules of single-crystal samples. Both are mounted on the support base 41 (e.g., a marble stage) of auxiliary equipment 4 and can independently move over a large range using their respective support platforms, alternately entering the center of the synchrotron radiation incident X-ray beam path without manual disassembly. The coaxial observation and collimation module 3 is used to achieve micron-level (1μm precision) visualization of single-crystal samples and precise alignment of the incident X-ray beam with the single-crystal sample. It is positioned in front of the vertical diffractometer module 1 and the horizontal crystallizer module 2 at the front end of the incident X-ray beam path. Figure 1 In this configuration, the incident X-ray beam travels horizontally from left to right along the positive Y-axis. The beam first passes through the coaxial observation collimation module 3, and then illuminates the sample position of either the vertical diffractometer module 1 or the horizontal crystal plate diffractometer module 2. The coaxial observation collimation module 3 is shared by both the vertical diffractometer module 1 and the horizontal crystal plate diffractometer module 2, eliminating the need for separate optical observation and collimation components for each diffractometer.

[0031] Compared to existing technologies where each beamline station can only be equipped with a single diffractometer and an independent optical system, this invention integrates two completely different diffractometers (i.e., vertical diffractometer module 1 and horizontal crystal plate diffractometer module 2) into the same device (dual diffractometer system), sharing a common coaxial observation collimation module 3. This eliminates the cost of purchasing a complete set of optical hardware. At the same time, relying on the respective carrier platforms of the two diffractometers, the optical path can be automatically switched in seconds to minutes without recalibrating the X-ray optical path, significantly saving scarce synchrotron radiation experimental time.

[0032] like Figure 2As shown, the vertical diffractometer module 1 includes a three-axis translation stage 11, a vertical air-bearing turntable 12, a two-dimensional fine-tuning platform 13, and a magnetic sample carrier 14. The magnetic sample carrier 14 is detachably mounted above the two-dimensional fine-tuning platform 13. The two-dimensional fine-tuning platform 13 is mounted above the vertical air-bearing turntable 12 via a connecting structure. The vertical air-bearing turntable 12 is mounted above the three-axis translation stage 11. The three-axis translation stage 11 is entirely placed on the support base 41 of the auxiliary equipment 4, see [reference]. Figure 1 It can achieve a wide range of X, Y, and Z axis translation, and can move the entire vertical diffractometer module 1 into and out of the X-ray optical path. The support base 41 can reduce the impact of ground vibration on the vertical air-bearing turntable 12 and improve the axial eccentricity of the air-bearing turntable.

[0033] The magnetic sample carrier 14 relies on magnetic adsorption of the single crystal sample holder, which is suitable for conventional single crystal sample loading operations in a low-temperature liquid nitrogen environment, and can be used with a robotic arm to achieve automated batch sample loading.

[0034] The vertical air-bearing turntable 12 has its rotation axis vertically arranged along the Z-axis, perpendicular to the direction of the horizontally incident X-rays. It is a gravity-type ultra-high-precision industrial air-bearing turntable with an axial eccentricity (SOC) better than ±0.3 μm, capable of rotating single-crystal samples 360° at high speed. Compared to existing horizontally arranged air-bearing turntables, the vertically arranged turntable has a smaller rotational eccentricity error, allowing for continuous and stable operation for months without secondary calibration. This ensures that the tiny micron-sized crystal remains within the X-ray focus during rotation, improving diffraction data resolution.

[0035] The two-dimensional fine-tuning platform 13 is a high-precision two-dimensional motion platform with an accuracy better than 0.02μm, providing fine-tuning in the X and Y planes. The three-axis translational bearing stage 11 has a large load-bearing capacity and provides three-dimensional precision translational adjustment functions in X, Y, and Z directions. Its Z-axis adjustment is used for precise vertical centering of single-crystal samples, while its X and Y-axis adjustments can achieve sub-micron level precise centering of single-crystal samples relative to the X-ray focus. Compared to existing technologies that require manual axis-by-axis calibration after disassembly and assembly, this invention relies on the large stroke motion of the three-axis translational bearing stage 11 to achieve rapid entry / exit of the vertical air-floating turntable. After switching modes, the sample positioning repeatability is better than 0.1μm.

[0036] like Figure 3 As shown, the horizontal crystallizer module 2 includes a biaxial translational support stage 21, a horizontal air-float turntable 22, a hollow lightweight adapter plate 23, a two-dimensional scanning stage 24, and a crystallizer clamping mechanism 25. The crystallizer clamping mechanism 25 is detachably mounted on the two-dimensional scanning stage 24. The two-dimensional scanning stage 24 is mounted on the horizontal air-float turntable 22 via the hollow lightweight adapter plate 23. The horizontal air-float turntable 22 is mounted on the biaxial translational support stage 21. The biaxial translational support stage 21 is entirely placed on the support base 41 of the auxiliary equipment 4, see [reference]. Figure 1It can achieve large-scale translation in the X and Z directions, and the entire horizontal crystallizer module 2 can be automatically translated to the center of the X-ray beam path. The support base 41 can reduce the impact of ground vibration on the horizontal air-bearing turntable 22 and improve the axial eccentricity of the air-bearing turntable.

[0037] The crystallization plate clamping mechanism 25 includes a clamping mechanism and a crystallization plate. The clamping mechanism is used to support and fix the crystallization plate, enabling rapid replacement of the crystallization plate. The crystallization plate is sealed with a low-scattering thin film, maintaining a sealed state throughout the process. It is suitable for detecting infectious, radiation-sensitive, and physiological single-crystal samples with biosafety protection levels of BSL-2 and above, overcoming the limitation of existing low-temperature diffractometers that cannot directly detect sealed crystallization plate samples.

[0038] The 2D scanning stage 24 is a high-speed, high-precision 2D motion stage with a maximum motion speed of no less than 20 mm / s. It can quickly traverse all the holes in the crystal plate, automatically scan and find the holes with crystals, improve the utilization efficiency of X-ray beams, and is suitable for high-throughput batch testing of crystal plates.

[0039] The hollow lightweight adapter plate 23 has a hollow lightweight structure and connects the horizontal air-bearing turntable 22 and the two-dimensional scanning stage 24. By reducing weight, it reduces the interference of the load on the axial accuracy of the horizontal air-bearing turntable.

[0040] The horizontal air-bearing turntable 22 has a horizontal (X-axis) rotation axis, providing a large radial load capacity to support the entire assembly of the crystallization plate and scanning stage. Its rotation angle range is ±45°, meeting the in-situ diffraction data acquisition angle requirements of the crystallization plate. Compared to the compromise solution of tilting and fixing the crystallization plate to a vertical turntable in existing technologies, this invention uses a dedicated horizontal rotation axis to keep the crystallization plate vertical, avoiding droplet flow and crystal displacement within the plate, reducing background scattering, and significantly improving the signal-to-noise ratio of the diffraction data.

[0041] The dual-axis translational support stage 21 is a high-load motion platform that provides a wide range of translation in the horizontal X-axis and vertical Z-axis. It is used for the overall relocation of the horizontal crystal plate diffractometer module 2 and realizes automatic switching of the optical path with the vertical diffractometer module 1.

[0042] like Figure 4 As shown, the coaxial observation collimation module 3 includes a beam blocking subsystem (beam blocker support stage 31, four-dimensional motion stage 32, and beam blocker 33), a sample observation subsystem (pneumatic backlight assembly 34, sample observation system support stage 35, five-dimensional motion stage 36, coaxial microscope clamping mechanism 37, and coaxial microscopic imaging assembly 38), and a collimation subsystem 39. The beam blocker 33, pneumatic backlight assembly 34, and coaxial microscopic imaging assembly 38 are collinear and coaxial with the incident X-rays.

[0043] The beam blocker support platform 31 is fixed to the bearing base 41 of the auxiliary equipment 4, the four-dimensional motion stage 32 is installed on the support platform 31, and the beam blocker 33 is fixed to the end of the four-dimensional motion stage 32.

[0044] The beam blocker 33 is made of tungsten or tantalum heavy metal and is cylindrical with a concave surface facing the light. This concave structure completely absorbs the direct X-ray main beam, protecting the downstream two-dimensional surface detector from damage caused by strong light. This concave structure also reduces X-ray scattering and lowers experimental background noise. Compared to ordinary planar beam blockers, the concave structure has a larger beam absorption area, significantly improving the signal-to-noise ratio in the small-angle diffraction region.

[0045] The four-dimensional motion stage 32 provides four-dimensional adjustment of X, Y, and Z-axis translation and roll rotation. The X, Y, and Z three-dimensional motion adjustment can precisely align the beam blocker 33 with the X-ray main beam, while the roll adjustment is used to move the beam blocker 33 into and out of the X-ray optical path.

[0046] The sample observation system support platform 35 is fixed to the bearing base 41 of the auxiliary equipment 4. The five-dimensional motion stage 36 is mounted on the support platform 35. The coaxial microscope clamping mechanism 37 is mounted on the five-dimensional motion stage 36. The coaxial microscopic imaging component 38 is clamped and fixed to the clamping mechanism 37. The pneumatic backlight assembly 34 passes through the backlight adapter 42 of the auxiliary equipment 4 (see...). Figure 7 It is fixedly arranged behind the coaxial microscopic imaging component 38.

[0047] The five-dimensional motion stage 36 provides three-axis translation (X, Y, Z), two-dimensional rotation (tilt and yaw), and five-dimensional adjustment, which is used to precisely adjust the optical axis of the coaxial microscopic imaging component 38 to be completely aligned with the incident X-ray, eliminating observation parallax.

[0048] The pneumatic obstacle avoidance backlight assembly 34 includes a backlight component and a pneumatic turntable. The backlight component is a surface light source with adjustable brightness, and its light emission center coincides with the center of the coaxial microscopic imaging assembly 38, providing illumination for sample observation and improving the observation accuracy of single-crystal samples. The pneumatic turntable rotates at an angle greater than 90°, allowing for rapid movement of the backlight component into the optical path for sample observation, or automatic rotation out of the optical path to avoid obstructing the crystal plate and interfering with diffraction signals. Existing single-set diffractometer backlights are mostly fixedly installed, requiring manual disassembly when switching crystal plate experiments. This invention employs a pneumatic automatic obstacle avoidance structure, enabling seamless switching between two experimental modes without manual intervention.

[0049] like Figure 5As shown, the coaxial microscopy imaging assembly 38 includes an objective lens 381, a 45° deflector 382, ​​a zoom lens 383, an eyepiece 384, and an electronic camera 385. The objective lens 381 is used to observe single-crystal samples, with a working distance of 35 mm and a 2.0 mm through-hole in its center to ensure complete X-ray passage. The 45° deflector 382 also has a 2.0 mm through-hole in its center, allowing horizontally incident X-rays to pass through completely to the single-crystal sample. The 45° deflector 382 vertically deflects the horizontal observation light from the objective lens 381 into the zoom lens 383. The zoom lens 383 is used to adjust the microscope's magnification and field of view, employing an electronic zoom structure for faster and more stable operation. The eyepiece 384 is connected to the electronic camera 385 at its rear end for real-time acquisition and display of single-crystal sample images, enabling remote image acquisition and display.

[0050] This invention employs a coaxial optical path design, ensuring that the microscope's observation optical axis and the X-ray incident optical axis are completely aligned, achieving "what you see is what you are photographing" and completely eliminating parallax alignment errors. Existing equipment often uses side-view microscopes, resulting in a misalignment between the observation optical path and the X-ray path, leading to poor sample alignment accuracy. This coaxial structure can improve sample alignment accuracy to the sub-micron level.

[0051] like Figure 6 As shown, the collimation subsystem 39 includes a beam-limiting collimator 391 (e.g., a nine-hole collimator) and a dual-axis adjustment stage 392. The dual-axis adjustment stage 392 is fixed to the support base 41 of the auxiliary equipment 4, and the beam-limiting collimator 391 is mounted on the dual-axis adjustment stage 392.

[0052] The dual-axis adjustment stage 392 provides X and Z-axis translation, and precisely adjusts the collimator 391 to be coaxial with the X-ray beam.

[0053] The 391 beam collimator is a matrix-type multi-specification beam-limiting aperture. The entrance end is made of tungsten metal, which is resistant to high temperature and radiation. The rear end is a 20mm thick through-hole to reduce stray scattering generated by the beam-limiting aperture. Different aperture spot sizes can be switched according to the crystal size to adapt to various single crystal samples from micrometer to hundreds of micrometer scale.

[0054] like Figure 7 As shown, the auxiliary device 4 includes a support base 41, a backlight adapter 42, a two-stage linkage pneumatic avoidance cooling assembly 43, an adjustable fluorescence detector 44, and a support frame 45. The adjustable fluorescence detector 44 is used to collect the fluorescence absorption signal of the crystal to assist in positioning the single-crystal sample; it is fixedly mounted on the support base 41. The support frame 45 is also fixedly mounted on the support base 41. The backlight adapter 42, used to mount the pneumatic avoidance backlight assembly 34, is fixedly mounted on the support frame 45. The two-stage linkage pneumatic avoidance cooling assembly 43 is also fixedly mounted on the support frame 45.

[0055] like Figure 8As shown, the two-stage linkage pneumatic obstacle avoidance cooling assembly 43 includes a cooling airflow output component 431, a clamping mechanism 432, a short-stroke pneumatic sliding stage 433, and a long-stroke pneumatic sliding stage 434. The cooling airflow output component 431 is fixed to the short-stroke pneumatic sliding stage 433 by the clamping mechanism 432. The short-stroke pneumatic sliding stage 433 is integrally mounted on the long-stroke pneumatic sliding stage 434, and the long-stroke pneumatic sliding stage 434 is fixed to the support frame 45. (See attached image) Figure 7 .

[0056] The cooling airflow output component 431 can output a minimum low-temperature airflow of 80K, maintaining a low-temperature environment of 100K for samples in routine experiments, suppressing radiation damage to single-crystal samples by X-rays, and is only used in low-temperature single-crystal experiments with a vertical diffractometer.

[0057] The short-stroke pneumatic sliding stage 433 has a stroke of 4mm and can quickly extend and retract. When the robotic arm automatically loads samples, the nozzle can quickly retract to avoid the robotic arm gripper mechanism. When collecting data, it can quickly extend to align with the single crystal sample, thereby quickly moving the cooling airflow output component 431 closer to or away from the sample position.

[0058] The long-stroke pneumatic sliding stage 434 has a stroke of about 20cm. When conducting room temperature experiments on crystallization plates, the entire set of cooling airflow output components 431 is moved out of the optical path to avoid the low-temperature nozzles blocking the crystallization plate and interfering with diffraction acquisition.

[0059] Compared to existing equipment with fixed cryogenic nozzles, this invention uses a two-stage pneumatic obstacle avoidance platform with varying lengths. When switching between the two diffractometers, there is no need to manually disassemble the cryogenic device, achieving automated mode switching. It is a dedicated matching structure adapted to the dual diffractometer integration scheme.

[0060] The working process of the dual diffractometer system of the present invention is briefly described below.

[0061] First, the optical path unification and collimation steps in the early stage of the experiment.

[0062] 1) Basic optical path calibration: Control the four-dimensional motion stage 32 to completely remove the beam blocker 33 from the optical path, and control the dual-axis adjustment stage 392 to move the beam collimator 391 out of the optical path; place an ionization chamber at the end of the beam to monitor the X-ray intensity in real time, and open the X-ray shutter. Adjust the five-dimensional motion stage 36 to scan the X, Z, pitch, and yaw dimensions, and use the maximum light intensity of the ionization chamber as a reference to make the optical axis of the coaxial microscopic imaging component 38 completely coincide with the incident X-ray; then move the beam collimator 391 into the optical path, adjust the dual-axis adjustment stage 392, and perform X and Z direction scans respectively. Similarly, based on the maximum light intensity signal measured in real time by the ionization chamber at the end, adjust the beam collimator 391 to be coaxial with the incident X-ray beam to complete the coaxial calibration of the collimator. At this time, the incident X-ray accurately passes through the coaxial microscopic imaging component 38 and the beam collimator 391, and the main X-ray beam is consistent with the field center of the coaxial microscopic imaging component 38. Once the entire optical path is calibrated once, switching between two diffractometers does not require secondary calibration. Compared to the existing solution where the optical path must be recalibrated when replacing a diffractometer, this significantly saves debugging time.

[0063] 2) Coaxial Microscope Focal Length Calibration: Activate the coaxial microscopic imaging assembly 38 and remotely display the microscope field of view image in real time. Install a magnetic tip with a diameter of approximately 0.5 μm and a length of approximately 18 mm on the magnetic sample carrier 14. Adjust the Y-axis of the five-dimensional motion stage 36 until the image is clear and record the coordinate Y1. Rotate the vertical air-bearing stage 12 180°, readjust the five-dimensional motion stage 36 to focus and record Y1′. Adjust the Y-axis of the five-dimensional motion stage to the average of the two values ​​(Y1 + Y1′) / 2, and use this as the focal length of the coaxial microscopic imaging assembly 38. To improve accuracy, the above steps need to be repeated multiple times. While keeping the Y-direction constant, determining the focal length only needs to be performed once. This completes the focusing setting of the coaxial microscopic imaging assembly 38. The sample or tip image observed through the microscope thereafter will be the true morphology and position.

[0064] 3) Coaxial calibration of the air-bearing turntable rotation center: First, mark the center of the field of view of the coaxial microscopic imaging component 38 as a crosshair. After beam collimation, this mark coincides with the incident X-ray axis, thus it can replace the X-ray axis position. With the vertical air-bearing turntable 12 at any starting angle, by adjusting the X and Y directions of the two-dimensional fine-tuning platform 13 and the Z direction of the three-axis translational support stage 11, move the magnetic tip to the crosshair mark at the center of the field of view of the coaxial microscopic imaging component 38. Rotate the vertical air-bearing turntable 12 180°. At this time, the tip will deviate from the crosshair mark at the center of the field of view. Record the tip's X-direction offset X′. By adjusting the X direction of the three-axis translational support stage 11, move the magnetic tip in the opposite direction by a distance of X′ / 2. Adjust the Y and Z directions of the two-dimensional fine-tuning platform 13 again to move the tip back to the crosshair mark. Repeat the operation until the tip of the vertical air-bearing turntable 12 always remains at the crosshair mark at any rotation angle. After this operation is completed, the rotation center of the vertical air-bearing turntable 12 will precisely coincide with the field of view center (i.e., the incident X-ray optical axis) of the coaxial microscopic imaging component 38. The same procedure is used to complete the coaxial calibration of the horizontal air-bearing turntable 22, ensuring that the rotation centers of both diffractometers coincide with the X-ray focal point.

[0065] Second, low-temperature single-crystal diffraction experiment

[0066] 1) Sample loading: After the single crystal sample is scooped onto the nylon ring, it is adsorbed onto the magnetic sample carrier 14. The magnetic sample carrier 14 is then installed onto the two-dimensional fine-tuning platform 13 by manual or robotic arm. Using data collection software, the three-axis translational carrier stage 11, the vertical air-bearing turntable 12, and the two-dimensional fine-tuning platform 13 can be remotely observed and operated in real time via the electronic camera 385. Initially, when the single crystal sample is selected, the software automatically adjusts the single crystal sample to the crosshair mark at the center of the field of view of the coaxial microscopic imaging component 38. Then, the vertical air-bearing turntable 12 is rotated 90°, and the position of the single crystal sample changes. When the single crystal sample is selected again at this angle, the software automatically adjusts the single crystal sample to the crosshair mark at the center of the field of view of the coaxial microscopic imaging component 38. The above 90° rotation and single crystal sample selection operations are repeated multiple times to eliminate the depth-of-field error of the coaxial microscopic imaging component 38 and complete the precise alignment of the single crystal sample.

[0067] 2) Low-temperature environment setup: The long-stroke pneumatic sliding stage 434 and the short-stroke pneumatic sliding stage 433 are extended, and the nozzle of the cooling gas output component 431 is brought close to the crystal to maintain a low-temperature environment of approximately 100K for the sample, suppressing radiation damage to the crystal. Typically, the cooling gas tip of the cooling gas output component 431 is approximately 10mm from the single-crystal sample, with a center temperature of approximately 100K, which meets the experimental requirements for single-crystal samples.

[0068] 3) Data acquisition: Set the X-ray energy to 12658 eV, exposure to 0.1 s, swing angle to 1°, and sample detector distance to 200 mm. The vertical air-floating turntable 12 drives the single crystal sample to rotate 360° continuously to acquire the complete diffraction pattern and complete the reconstruction of the three-dimensional structure of the biomolecule.

[0069] Third, room temperature in-situ crystallization plate diffraction experiment

[0070] 1) Sample clamping: The sealed crystallization plate with single crystal droplets is fixed by the crystallization plate clamping mechanism 25 to keep the crystallization plate in a vertical position; the long-stroke pneumatic sliding stage 434 is controlled to move the cooling airflow output component away from the optical path as a whole, and the pneumatic avoidance backlight assembly 34 is controlled to turn out of the optical path to avoid blocking the crystallization plate.

[0071] 2) Automatic Scanning and Positioning: Synchrotron X-rays are used to rapidly scan each preset grid point of the crystal plate, mapping the diffraction intensity (number of diffraction points) to a two-dimensional diffraction pattern, visually presenting the diffraction capability and mass distribution of each region of the crystal plate. The software automatically identifies the location of the strongest diffraction signal, determines the precise coordinates of the single-crystal sample, and the control system moves the crystal plate to the corresponding position via the two-dimensional scanning stage 24, placing the crystal in the X-ray path. The two-dimensional scanning stage 24 then rapidly traverses all the pores of the crystal plate, and the software automatically identifies the coordinates of the crystal with the strongest diffraction signal, moving the target crystal to the center of the X-ray path. Alternatively, the crystal target can be manually selected in the microscope image, i.e., manually selected using the electronic camera 385.

[0072] 3) Data acquisition: Due to the geometric limitations of the crystallization plate, the horizontal air-bearing turntable 22 rotates slightly within a range of ±30° to acquire diffraction data; multiple crystals in the same crystallization plate acquire small-angle datasets separately, which are then merged and spliced ​​to obtain complete diffraction data. The crystallization plate is kept sealed throughout the process, which can meet the requirements of BSL-2 biosafety experiments.

[0073] The technical advantages of the dual diffractometer system according to the present invention are briefly described below.

[0074] 1) Integrated dual diffractometer and independent large-stroke translation platform structure: For the first time, the vertical low-temperature diffractometer and the horizontal crystallization plate diffractometer are integrated into the same line station equipment. Automatic optical path switching is achieved by relying on two independent load-bearing platforms. No disassembly or re-adjustment of the optical path is required. The switching time is reduced from several hours in the existing technology to several minutes. The positioning repeatability is ≤0.1μm, which solves the industry pain point of a lot of waste in synchrotron radiation machines.

[0075] 2) A coaxial optical system can adapt to two sample postures in both directions: it shares a coaxial microscope, collimator and beam blocker, and a single optical structure can adapt to two sample forms: low temperature single crystal and vertical sealed crystal plate. This saves the cost of purchasing a complete set of optical equipment and produces a low-cost and high-efficiency effect that cannot be achieved by two existing independent sets of equipment.

[0076] 3) Equipped with a dedicated pneumatic automatic obstacle avoidance mechanism: The backlit pneumatic rotary table and the dual-stage pneumatic cryogenic moving platform are designed specifically for the dual diffractometer switching scenario. A single diffractometer device does not require this structure, which automatically avoids spatial interference between the two sets of equipment, achieving full automation without manual intervention.

[0077] 4) Simultaneously covers the experimental needs of all types of biological crystals: It retains the high-precision low-temperature diffraction capability of the vertical air-floating turntable, and can also carry out room temperature in-situ detection of sealed crystal plates. It is compatible with a variety of samples, including ordinary room temperature protein crystals, radiation-sensitive crystals, and BSL-2 level infectious pathogen crystals, achieving the goal that existing single diffractometer equipment cannot simultaneously support all experimental scenarios.

[0078] 5) Improved mechanical structure precision and long-term stability: The vertical air-bearing turntable has an SOC of ≤ ±0.3μm. The entire set of equipment does not require frequent disassembly and assembly, and there is no optical path deviation during long-term operation. It supports fully automatic high-throughput sample loading and acquisition by robotic arms, which greatly improves the experimental throughput of synchrotron radiation beamlines.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. That is, all simple and equivalent changes and modifications made based on the claims and description of this invention fall within the protection scope of the claims. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A dual diffractometer system for single-crystal diffraction and in-situ crystallization plates in a synchrotron radiation facility, characterized in that, It includes a vertical diffractometer module (1), a horizontal crystal plate diffractometer module (2), a coaxial observation collimation module (3), and a support base (41). The vertical diffractometer module (1) and the horizontal crystal plate diffractometer module (2) are both mounted on the support base (41). Each of them is equipped with an independent translation support stage and can be moved alternately to the center of the incident X-ray beam path. The coaxial observation collimation module (3) is located upstream of the incident light path of the two diffractometer modules and is shared by both. After the incident X-ray passes through the coaxial observation collimation module (3), it irradiates the single crystal sample position of the vertical diffractometer module (1) or the horizontal crystal plate diffractometer module (2).

2. The dual diffractometer system according to claim 1, characterized in that, The vertical diffractometer module (1) includes a three-axis translational support stage (11), a vertical air-bearing turntable (12), a two-dimensional fine-tuning platform (13), and a magnetic sample carrier (14). The three-axis translational support stage (11) has three-axis movement freedom in the X, Y, and Z directions, driving the entire vertical diffractometer module (1) to enter and exit the optical path. The rotation axis of the vertical air-bearing turntable (12) is arranged along the Z direction, and the rotation axis is perpendicular to the incident X-ray. The two-dimensional fine-tuning platform (13) is located above the vertical air-bearing turntable (12) and has X and Y-axis fine-tuning freedom. The magnetic sample carrier (14) relies on magnetic force to adsorb single crystal samples.

3. The dual diffractometer system according to claim 1, characterized in that, The horizontal crystal plate diffractometer module (2) includes a dual-axis translational support stage (21), a horizontal air-bearing turntable (22), a hollow lightweight adapter plate (23), a two-dimensional scanning stage (24), and a crystal plate clamping mechanism (25). The dual-axis translational support stage (21) has X-axis and Z-axis movement degrees of freedom, driving the entire horizontal crystal plate diffractometer module (2) to enter and exit the optical path. The rotation axis of the horizontal air-bearing turntable (22) is arranged along the X-axis. The hollow lightweight adapter plate (23) is assembled between the horizontal air-bearing turntable (22) and the two-dimensional scanning stage (24). The crystal plate clamping mechanism (25) is installed on the two-dimensional scanning stage (24) and keeps the crystal plate in a vertical position.

4. The dual diffractometer system according to claim 1, characterized in that, The coaxial observation collimation module (3) includes a coaxial microscopic imaging component (38) and a pneumatic backlight component (34). The optical path of the coaxial microscopic imaging component (38) is coaxial with the incident X-ray and has a through hole to allow the incident X-ray to pass through without obstruction. The pneumatic backlight component (34) is provided with a rotating structure to drive the backlight component to rotate out of the incident optical path to prevent the backlight component from blocking the crystal plate and interfering with the diffraction signal acquisition.

5. The dual diffractometer system according to claim 4, characterized in that, The coaxial observation collimation module (3) is equipped with a beam blocker (33) with a concave light-facing end face and a beam-limiting collimator (391) with a long straight through-hole. The beam blocker (33) is equipped with a multi-dimensional adjustment stage (32), which has X-axis, Y-axis, and Z-axis translation and roll angle adjustment degrees of freedom, used to move the beam blocker (33) into and out of the incident light path as a whole. The beam-limiting collimator (391) is equipped with a dual-axis adjustment stage (392), which has X-axis and Z-axis translation degrees of freedom, used to adjust the beam-limiting collimator (391) to be coaxially aligned with the incident X-ray.

6. The dual diffractometer system according to claim 1, characterized in that, The support base (41) is provided with a two-stage linkage pneumatic avoidance cooling assembly (43); the two-stage linkage pneumatic avoidance cooling assembly (43) includes a short-stroke pneumatic sliding stage (433) and a long-stroke pneumatic sliding stage (434). The short-stroke pneumatic sliding stage (433) is equipped with a cooling airflow output component (431), and the long-stroke pneumatic sliding stage (434) drives the entire cooling assembly away from the optical path.

7. The dual diffractometer system according to claim 1, characterized in that, The bearing base (41) is an integrated marble shock-absorbing base that simultaneously supports the vertical diffractometer module (1), the horizontal crystallization plate diffractometer module (2), and the coaxial observation collimation module (3) while isolating ground vibration.

8. A single-crystal diffraction detection method based on the dual diffractometer system according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1, remove the beam blocker (33) and the beam collimator (391), complete the beam coaxial calibration by relying on the coaxial microscopic imaging component (38), and then move in the beam collimator (391) for secondary calibration, respectively calibrating the rotation center of the two sets of air-bearing turntables to coincide with the beam center; S2, the three-axis translational support stage (11) sends the vertical diffractometer module (1) into the optical path, and the two-stage linkage pneumatic avoidance cooling component (43) approaches the sample to complete data acquisition; S3, the long-stroke pneumatic sliding stage (434) moves away the cooling component and pneumatically avoids the backlight component (34) to turn out of the optical path, and the dual-axis translational carrier stage (21) sends the horizontal crystal plate diffractometer module (2) into the optical path to collect single crystal diffraction data in the crystal plate.

9. The single-crystal diffraction detection method according to claim 8, characterized in that, In step S1, the magnetic sample carrier (14) is loaded with the calibration needle tip, the vertical air-bearing turntable (12) is rotated twice to collect image coordinates, and the average value of the coordinates is used to lock the reference focal length of the coaxial microscopic imaging component (38).

10. The single-crystal diffraction detection method according to claim 8, characterized in that, In step S3, the single crystal aperture is identified by traversing the entire area of ​​the crystal plate, the target single crystal is aligned with the incident beam, and the horizontal air-bearing turntable (22) is driven to collect multiple sets of diffraction data and splice the complete dataset.

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