A ct three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method and system

Abstract

The application relates to the field of electronic information technology and discloses a CT three-dimensional reconstruction coordinate-assisted mass spectrum imaging method and system. The method comprises the following steps: acquiring scanning data of a sample to be measured and reconstructing a three-dimensional digital model; based on the three-dimensional digital model, planning sampling points on the surface of the sample, a laser desorption path and a sample posture angle at each sampling point; controlling the sample to move along the laser desorption path, so that when each sampling point on the sample reaches the focal point of a laser beam of an atmospheric pressure laser desorption / secondary photoionization device, the sample surface at the sampling point is desorbed at the planned sample posture angle, and mass spectrum data is collected through a mass spectrometer; and the collected mass spectrum data is associated with the three-dimensional space coordinates of the corresponding sampling points, and a three-dimensional distribution image of molecules on the surface of the sample to be measured is reconstructed. The application can realize high-spatial-resolution and high-sensitivity mass spectrum imaging on a sample which is too fragile to be sliced and an irregular sample surface.

Description

Technical Field

[0001] This invention relates to the field of electronic information technology, and specifically to a CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method and system. Background Technology

[0002] Mass spectrometry (MSI) is an imaging technique based on desorption-ionization mass spectrometry. It is a powerful method for analyzing and visualizing the spatial distribution of molecular species on the surface of various samples under label-free conditions. MSI can visualize thousands of compounds, such as small metabolites, lipids, peptides, and proteins, in a single imaging experiment. In the past decade or so, MSI technology has developed rapidly and has been widely applied in research in multiple fields, including toxicology, clinical medicine, drug metabolism, and plant metabolism.

[0003] Currently, the most widely used desorption ionization methods are matrix-assisted laser desorption / ionization (MALDI) and electrospray desorption / ionization (DESI), both of which are well-suited for mass spectrometry imaging of biological samples. MALDI-MSI, proposed in 1997, was the earliest mass spectrometry imaging technique, while DESI-MSI, developed in 2006, has different advantages and limitations in terms of desorption ionization apparatus, sample preparation, and imaging performance. MALDI-MSI provides high-throughput, comprehensive, and rapid analysis of composition, relative abundance, and compound distribution in tissues, and can detect peptides and proteins with mass ranges exceeding several thousand Daltons; its best spatial resolution can reach several μm. However, its complex apparatus, cumbersome operation, requirement for vacuum operation, and high cost limit its application. The most important advantages of DESI-MSI technology are its simple structure, minimal sample preparation, and ability to operate under ambient pressure with minimal destructiveness. However, its detection sensitivity is relatively low, making it difficult to detect weakly polar and non-polar compounds, such as high levels of cholesterol in biological tissues. Its spatial resolution is also relatively low, typically around 100–200 μm. Currently, there is a lack of a desorption-ionization mass spectrometry imaging scheme that can simultaneously achieve high spatial resolution and high sensitivity.

[0004] Mass spectrometry imaging data is typically obtained by scanning desorbed compounds on the sample surface pixel by pixel using a microprobe, followed by detection by a mass analyzer. Improving spatial resolution and detection sensitivity is crucial for the further development of mass spectrometry imaging methods. However, increased spatial resolution often leads to decreased detection sensitivity, as higher spatial resolution means a smaller desorption area and fewer desorbed compounds per pixel. The aforementioned ionization methods all have low ionization efficiencies (<1 / 1000). Desorption / secondary ionization is an innovative solution that combines desorption electrospray ionization or laser desorption ionization with secondary ionization techniques (such as electrospray ionization, laser ionization, plasma ionization, or photoionization), offering the unique advantages of two or more ionization methods. Matrix-assisted laser desorption / electrospray ionization (MALDESI), introduced in 2006, was the first desorption / secondary ionization technique to combine laser ablation with electrospray ionization.

[0005] Secondary ionization techniques such as electrospray ionization, laser ionization, plasma ionization, or photoionization can significantly increase the ion signal of desorbed molecules without affecting the primary desorption ionization process. Photoionization is a widely used secondary ionization technique in hybrid ionization methods. As a soft ionization method, photoionization not only produces fewer fragment ions but also has the advantage of non-polarity discrimination against compounds. Vacuum ultraviolet discharge lamps, lasers, or synchrotron radiation accelerators are three common photoionization sources. In 2019, Liu Chengyuan et al. developed a compact secondary photoionization assembly of desorption electrospray ionization / secondary photoionization (DESI / PI) using a vacuum ultraviolet discharge lamp as the ionization source. Compared with DESI, DESI / PI significantly improved the ionization efficiency and signal intensity of nonpolar compounds. Recently, Qi Keke et al. employed a combined back-side laser desorption / ionization (t-AP-LDI / PI) and secondary photoionization method for high-resolution mass spectrometry imaging. This method, named back-side atmospheric pressure laser desorption / ionization (t-AP-LDI / PI), obtained the spatial distribution of various lipids, fatty acids, neurotransmitters, and amino acids in subregions of mouse cerebellar tissue. After careful optimization, the spatial resolution of metabolic mass spectrometry imaging of mouse hippocampal tissue reached as high as 4 μm. Finally, t-AP-LDI / PI mass spectrometry imaging analysis of melanoma tissue sections revealed the metabolic heterogeneity of the melanoma microenvironment and the abnormal proliferation and invasive trends of tumor cells. However, high spatial resolution and high sensitivity mass spectrometry imaging remains impossible for fragile samples that cannot be sectioned; high-resolution mass spectrometry imaging of irregular sample surfaces also remains an unsolved problem.

[0006] In summary, mass spectrometry imaging (MSI) technology can directly acquire spatial distribution information of molecules on the sample surface under label-free conditions. Current mainstream mass spectrometry imaging techniques, such as matrix-assisted laser desorption / ionization (MALDI) and electrospray desorption / ionization (DESI), have significant limitations when dealing with samples with irregular surfaces.

[0007] (1) High requirements for sample preparation. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) technology usually requires analysis of flat, thin-slice samples in a vacuum environment, which is not suitable for thicker, more brittle or unsliceable samples (such as certain biological tissues, rocks, archaeological samples, industrial parts, etc.).

[0008] (2) Spatial resolution and surface adaptability contradiction: Although DESI-MSI technology can be performed under normal pressure, its spatial resolution is low (usually >100μm), and its ionization process is very sensitive to the distance and angle between the nozzle and the sample surface, making it difficult to maintain stable high-resolution imaging on uneven surfaces.

[0009] (3) Lack of multidimensional morphology guidance: Existing technologies lack prior knowledge of the three-dimensional morphology of irregular sample surfaces, which cannot guide the mass spectrometry probe to perform accurate path planning and real-time tracking, resulting in the inability to achieve effective or high-resolution mass spectrometry data acquisition on non-planar samples.

[0010] (4) Although the recently developed back-side atmospheric pressure laser desorption / post-photoionization (t-AP-LDI / PI) technology has achieved high resolution (up to 4μm), its transmission ionization method requires the sample to be a thin slice, and it cannot be applied to irregular surfaces or whole samples.

[0011] Therefore, there is an urgent need in this field for a technical solution that can directly perform high spatial resolution mass spectrometry imaging on irregular surface samples. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention proposes a CT-atmospheric pressure laser desorption / post-photoionization (AP-LDI / PI) mass spectrometry imaging technique assisted by CT three-dimensional reconstruction coordinates. This method uses high-resolution CT scans of samples, obtains the coordinates of the sample surface position after three-dimensional reconstruction, and uses these coordinates to guide the movement of the multi-dimensional moving platform of the laser desorption / post-photoionization / PI mass spectrometry imaging device, thereby achieving high spatial resolution and high sensitivity mass spectrometry imaging of brittle samples that cannot be sliced ​​and irregular sample surfaces.

[0013] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0014] In a first aspect, the present invention provides a CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method, comprising:

[0015] Acquire CT scan data of the sample to be tested and reconstruct a three-dimensional digital model;

[0016] Based on the three-dimensional digital model, the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point are planned.

[0017] The sample is controlled to move along the laser desorption path so that when each sampling point on the sample reaches the laser beam focus of the atmospheric pressure laser desorption / secondary photoionization device, the sample surface at the sampling point is desorbed at the planned sample attitude angle, and mass spectrometry data is collected by a mass spectrometer.

[0018] By correlating the collected mass spectrometry data with the three-dimensional spatial coordinates of the corresponding sampling points, a three-dimensional distribution image of molecules on the surface of the sample to be tested can be reconstructed.

[0019] In one embodiment, the sample orientation angle is such that the incident direction of the laser beam of the atmospheric pressure laser desorption / secondary photoionization device is at a preset optimal angle with the local normal direction of the surface of the sample to be tested at the sampling point.

[0020] In one embodiment, the step of planning the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point based on the three-dimensional digital model specifically includes:

[0021] Based on the user-defined spatial resolution, a set of sampling points is generated on the 3D surface of the region of interest of the 3D digital model.

[0022] The generated set of sampling points is sorted by path, and an approximate solution algorithm for the traveling salesman problem is used to generate the scanning order with the shortest total motion path;

[0023] Calculate the local surface normal vector at each sampling point location;

[0024] Based on the local surface normal vector, the attitude angle of the sample is calculated when each sampling point on the sample reaches the laser beam focus of the atmospheric pressure laser desorption / secondary photoionization device, thereby realizing the planning of the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point.

[0025] In one embodiment, the set of sampling points is generated using a Poisson disk sampling algorithm, such that the sampling points are uniformly distributed on the three-dimensional surface.

[0026] In one embodiment, the method further includes: interpolating and smoothing the planned laser desorption path using a B-spline curve.

[0027] In one embodiment, the sample is driven by a six-axis robot; after the laser desorption path is planned, the mobility accessibility is judged and collision avoidance is detected based on the robot's kinematic model and the three-dimensional digital model of the sample to be tested; if it is determined that the sample is unreachable or there is a risk of collision, the laser desorption path is adjusted or the corresponding sampling point is marked as an invalid point.

[0028] In a second aspect, the present invention provides a mass spectrometry imaging system for implementing the method of any embodiment of the first aspect, comprising:

[0029] The three-dimensional morphology acquisition device acquires a three-dimensional digital model of the sample to be tested through a micro-computed tomography system.

[0030] Mass spectrometry imaging system, including atmospheric pressure laser desorption / secondary photoionization device and mass spectrometer;

[0031] A movable stage is used to support and fix the sample to be tested;

[0032] A six-axis robot used to control a moving platform;

[0033] The control unit is configured to: plan the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point based on the three-dimensional digital model; control the six-axis robot to move the sample to be tested along the planned laser desorption path; synchronously trigger the atmospheric pressure laser desorption / secondary photoionization device to desorb from the sample surface at the sampling point at the planned sample attitude angle; and trigger the mass spectrometer to collect mass spectrometry data and record the pose information to generate a mass spectrometry image.

[0034] In one embodiment, the atmospheric pressure laser desorption / secondary photoionization device includes a laser, a cage structure, a moving stage, and an ion sample introduction and transmission ionization channel; the sample to be tested is fixed on the moving stage, which is controlled by a six-axis robot; the laser beam is emitted from the laser, focused by the cage structure, and then turned by a right-angle optical adjustment frame before being focused from top to bottom onto the sample surface; the ion sample introduction and transmission ionization channel includes a sample introduction tube, a dopant introduction tube, and a transmission ionization tube.

[0035] In one embodiment, one end of the transmission ionization tube is sealed to the glass capillary of the mass spectrometer, and the other end is connected to the vacuum ultraviolet discharge lamp; the sample injection tube and the dopant injection tube are both designed to be located at the end close to the vacuum ultraviolet discharge lamp, respectively on both sides of the transmission ionization tube.

[0036] In one embodiment, the control unit uses a hand-eye calibration algorithm to unify the coordinate system of the three-dimensional shape acquisition device, the coordinate system of the six-axis robot, and the coordinate system of the mass spectrometry imaging system.

[0037] Compared with the prior art, the beneficial technical effects of the present invention are:

[0038] System-level integrated innovation: For the first time, a high-resolution micro-computed tomography (Micro-CT) system is systematically integrated with an ambient pressure laser desorption / secondary photoionization (AP-LDI / PI) mass spectrometry imaging system, forming a complementary joint imaging platform. CT provides the "eyes" to see the surface morphology, while mass spectrometry provides the "nose" to smell the molecular components.

[0039] Three-dimensional guided mass spectrometry (MSI) method: A three-dimensional path planning and real-time laser beam focus tracking desorption method based on prior three-dimensional topographic data is proposed. This is the core of achieving high-resolution MSI on irregular surfaces, ensuring the optimization and stability of detection conditions (such as the relative angle of the laser beam, the consistency of the focus and the sample sampling point position) throughout the entire MSI process, which is impossible to achieve with conventional desorption ionization techniques.

[0040] The combination of atmospheric pressure laser desorption / re-ionization (AP-LDI) and post-photoionization is applied to this scenario: The atmospheric pressure laser desorption / re-ionization (AP-LDI / PI) technology, which has the potential for high-resolution imaging, is successfully applied to the new scenario of irregular surface detection through the three-dimensional navigation system of this invention, giving full play to its comprehensive advantages of atmospheric pressure, high resolution and surface analysis.

[0041] It overcomes the limitations of sample shape: fundamentally solving the problem of high-resolution mass spectrometry imaging for irregular surfaces, monolithic samples, thick samples, or fragile samples. It eliminates the need to prepare samples into thin slices, preserving the original state of the samples to the greatest extent possible. It achieves true three-dimensional surface imaging: extending mass spectrometry imaging from traditional "two-dimensional planar imaging" to "three-dimensional curved surface imaging," accurately reflecting the true distribution of molecules on complex three-dimensional surface structures. Attached Figure Description

[0042] Figure 1 This is a flowchart of the method in an embodiment of the present invention. Detailed Implementation

[0043] A preferred embodiment of the present invention will now be described in detail with reference to the accompanying drawings.

[0044] like Figure 1 As shown, a CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method of the present invention includes the following steps:

[0045] S1: Acquire the scanning data of the sample to be tested and reconstruct a three-dimensional digital model;

[0046] S2, based on a three-dimensional digital model, plans the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point;

[0047] S3, control the sample to move along the laser desorption path, so that when each sampling point on the sample reaches the laser beam focus of the atmospheric pressure laser desorption / secondary photoionization device, the sample surface at the sampling point is desorbed at the planned sample attitude angle, and mass spectrometry data is collected by the mass spectrometer;

[0048] S4 correlates the acquired mass spectrometry data with the three-dimensional spatial coordinates of the corresponding sampling points to reconstruct a three-dimensional distribution image of molecules on the surface of the sample to be tested.

[0049] The system used in this invention mainly includes the following parts:

[0050] Three-dimensional morphology acquisition device: A high-resolution micro-computed tomography (Micro-CT) system is used to scan the irregular sample surface, acquire the three-dimensional morphology data of the sample surface, and reconstruct a high-precision three-dimensional digital model.

[0051] The atmospheric pressure laser desorption / ionization mass spectrometry (AP-LDI / PI-MSI) system is built upon an atmospheric pressure laser desorption / ionization (AP-LDI / PI) device and a high-precision time-of-flight mass spectrometer. The AP-LDI / PI device consists of a laser, a cage structure, a dopant sample introduction system, and an ion introduction and transport ionization channel. The sample is fixed on a moving stage controlled by a six-axis robot. The laser beam is emitted from the laser, focused by the cage structure, and then redirected by a right-angle optical adjustment frame before being focused downwards onto the sample surface. The ion introduction and transport ionization channel consists of a sample introduction tube, a dopant introduction tube, and a transport ionization tube (ionization chamber). One end of the transport ionization tube is sealed to the glass capillary of the mass spectrometer via a graphite ferrule, and the other end is connected to a vacuum ultraviolet discharge lamp via a graphite gasket. Both the sample inlet tube and the dopant inlet tube are designed to be located near the vacuum ultraviolet discharge lamp, forming a 90° angle with the transfer ionization tube, and are situated on opposite sides of the transfer ionization tube. To improve ionization efficiency, a bubble generator introduces the dopant into the ionization chamber. Neutral molecules desorbed by the laser converge with the dopant in the transfer ionization tube, where photons emitted by the vacuum ultraviolet discharge lamp induce an ion-molecule reaction, ionizing the neutral molecules. The mass spectrometer is a sample analysis tool that ionizes substances. Different ions, due to their mass differences, reach the detector in a magnetic field at different times. By measuring the intensity of different ions, the mass spectrum of the sample is obtained.

[0052] A six-axis robot is used to control the movement of a mobile platform.

[0053] Mobile stage: Used to carry and move samples, it can perform translation in the X, Y, and Z directions and rotation in multiple directions under the control of a six-axis robot, achieving three-dimensional motion with micron-level precision.

[0054] Control Unit: This unit is the core of the system and includes a 3D data processing and coordinate system module, a path and attitude planning module, a robot motion control and synchronization module, and a data fusion and imaging module.

[0055] The following describes each module of the control unit.

[0056] The 3D Data Processing and Coordinate System module receives DICOM format tomographic image data from a micro-computed tomography system and generates a 3D digital model (STL format) of the sample surface using a 3D reconstruction algorithm. Subsequently, by unifying the CT coordinate system, the six-axis robot coordinate system, and the mass spectrometry imaging system coordinate system, a precise spatial transformation relationship is established between all devices.

[0057] Path and pose planning module: After the user selects a region of interest (ROI) on the 3D digital model, this module performs the following steps:

[0058] (a) Sampling point generation: On the surface of the 3D digital model of the region of interest, generate a dense set of equally spaced sampling points according to the user-defined spatial resolution (e.g., 50μm). .

[0059] (b) Calculation of local normal vector: for each sampling point Calculate the normal vector of the triangular facet containing that point. This normal vector represents the orientation of the sample surface at that point.

[0060] (c) Calculation of the pose of the moving stage: In this invention, the attitude angle of the sample to be tested is adjusted by the moving stage. To ensure the best detection effect, the pose of the moving stage is calculated. Determined by the following rules:

[0061] Position of the moving platform : Make the laser focus fall exactly on Above. The z-axis coordinate of the moving stage. Depend on The z-axis coordinate plus a preset optimal working distance Decide. It is a constant determined through prior experimental optimization, ensuring the highest ion transport efficiency.

[0062] The attitude of the moving platform : Align the central axis of the moving stage (i.e., the direction of laser emission and ion extraction) with the normal vector calculated in step (b). The laser is either parallel to the sample surface or at a preset optimized angle (when parallel to the sample surface, the preset optimized angle is 0). This ensures that the laser is focused on the sample surface, thereby obtaining the smallest, most regular laser spot and the highest spatial resolution, while ensuring that the ions generated by ionization can be most effectively drawn into the ion inlet. This represents the rotation angle of the moving stage around the x-axis, y-axis, and z-axis at the i-th sampling point. The inlet of the atmospheric pressure laser desorption / secondary photoionization (AP-LDI / PI) device is always kept close to the laser beam focus and the sample sampling point.

[0063] (d) Accessibility and collision detection: After planning the desorption path and sampling points of the laser on the sample surface, the motion trajectory of the six-axis robot is set, and the motion accessibility judgment and collision detection are performed based on the robot kinematic model and the three-dimensional digital model of the sample to be tested. If it is determined that the robot is unreachable or there is a risk of collision, the running path is adjusted or the corresponding sampling point is marked as an invalid point.

[0064] Robot motion control and synchronization module: This module converts the planned pose sequence of the moving stage at each sampling point into motion commands that the six-axis robot can recognize. It controls the robot to move along the planned trajectory and speed, and upon confirming that the robot has reached each sampling point, it sends a hardware trigger signal to the mass spectrometer, commanding it to acquire mass spectrometry data. This position synchronization triggering mechanism ensures that each mass spectrometry data point accurately corresponds to a spatial location on the sample surface.

[0065] Data fusion and imaging module: This module receives and stores the pose of the moving stage in real time. The system uses mass spectrometry data files from the mass spectrometer. After scanning, it extracts the ion intensity at a specific mass-to-charge ratio (m / z) from each mass spectrometry data file and assigns it as a pixel value, along with corresponding spatial coordinates. Finally, using a 3D visualization library (such as VTK), all these 3D pixels are rendered into a 3D distribution image of molecules on the surface of the sample.

[0066] In one embodiment, step S2, which involves planning the sampling points on the sample surface, the laser desorption path, and the sample orientation angle at each sampling point based on a three-dimensional digital model, specifically includes:

[0067] Inputs include: a 3D digital model of the sample (STL file), user-defined spatial resolution, laser operating parameters, and the working distance at which the laser beam is focused on the sample surface. The spatial location of each sampling point on the surface of the sample to be tested.

[0068] Surface discretization: Within the user-selected region of interest, a uniform and random initial set of points is generated on the 3D surface using the Poisson Disk Sampling algorithm. This algorithm avoids the aliasing phenomenon that may occur in regular meshes in certain directions.

[0069] Scanning Path Ordering: The initial point set is unordered. To reduce the robot's idle time and improve scanning efficiency, these points need to be ordered into a continuous and smooth motion path. This invention employs an approximate solution algorithm for the Traveling Salesman Problem (TSP), such as the nearest neighbor algorithm or Christofides' algorithm, to calculate the optimal order for visiting all points with the goal of minimizing the total motion path.

[0070] Pose assignment: For each point on the path, execute steps (b) and (c) in the path and pose planning module to calculate the normal vector, and calculate the position and pose of the moving platform accordingly to form the final six-dimensional pose sequence.

[0071] Trajectory smoothing: Directly sending the six-dimensional pose sequence to the robot may result in unstable motion and jitter. Therefore, it is necessary to interpolate and smooth the path using B-spline curves to generate a continuous and smooth robot end effector trajectory, ensuring the stability and accuracy of the scanning process.

[0072] Output a robot motion trajectory file containing time-pose information, and a scan sequence file for data synchronization.

[0073] On non-planar samples, a fixed-angle moving stage cannot guarantee that the laser beam is perpendicular to and focused on the sample surface at all positions. Tilted incidence leads to ellipticization and increased area of ​​the focal laser spot, severely affecting laser desorption and degrading spatial resolution; changes in the distance between the sample and the inlet cause drastic fluctuations in ion transport efficiency, impacting signal sensitivity and stability. The core processing to solve the problem of "high-resolution acquisition of non-planar samples" requires dynamically maintaining the optimal detection geometry, as follows:

[0074] Vertical incidence control: By calculating the local normal vector at each sampling point and dynamically adjusting the robot's end effector posture accordingly, the laser beam is always focused on the local sample surface. This is crucial for ensuring high spatial resolution.

[0075] Constant working distance control: When planning the laser analysis scanning path, the focus of the laser beam is located on the sample surface at each sampling point, and the two are in the same spatial position. This ensures that the mass spectrometry imaging system inlet is always close to the focus of the laser beam and the sampling point, which is the key to ensuring high and stable detection sensitivity.

[0076] Through the above-mentioned dynamic adjustment of "one position and one posture", the present invention transforms the traditional laser desorption ionization method of moving the sample slice position on a plane into a form of intelligent moving stage based on the CT three-dimensional information of the sample to be tested. This method can intelligently adapt to the AP-LDI / PI MSI of samples with complex curved surfaces, thereby achieving high resolution and sensitivity on non-planar samples that were originally only achievable on flat slices.

[0077] Example:

[0078] The workflow of this invention is as follows:

[0079] Step 1: Place the irregular surface sample to be tested into a micro-computed tomography (Micro-CT) system for scanning to obtain tomographic image data, and generate a high-precision three-dimensional digital model of the sample surface using three-dimensional reconstruction software.

[0080] Step 2: The control unit imports the 3D digital model, and the user sets the region of interest (ROI) for mass spectrometry imaging on the model. A dense, equally spaced array of sampling points is generated on the surface of the ROI, and the motion trajectory required for the moving stage to bring the sample to each point in sequence is calculated to ensure that the laser can be accurately focused on the sample surface at each sampling point.

[0081] Step 3: The sample is transferred to the moving stage.

[0082] Step 4: The system starts up and the moving stage moves according to the preset trajectory, moving the first sampling point of the sample to the laser focus.

[0083] Step 5: The laser emits a pulsed laser to desorb at the point; the resulting gaseous molecules are ionized by a secondary photoionization source; the ions are detected by a mass spectrometer, and the mass spectrum signal at that point is recorded.

[0084] Step 6: The moving stage moves precisely under the control of the six-axis robot to send the next sampling point to the laser focus position, and Step 5 is repeated.

[0085] Step 7: Repeat this process until the entire ROI is scanned. During this process, the moving stage will adjust its height and orientation in real time according to the 3D digital model, so that the laser is focused on the sample surface at the sampling point.

[0086] Step 8: Data Synthesis and Imaging: The control unit maps the spatial coordinates of each sampling point to its corresponding mass spectrometry data. Through software processing, a three-dimensional distribution image of ions with any specific mass-to-charge ratio (m / z) on the irregular surface of the sample can be generated.

[0087] This invention creatively integrates three different technical fields—three-dimensional topography acquisition equipment, a precision motion system with multiple degrees of freedom, and a high-resolution mass spectrometry imaging system—into a unified detection platform. In terms of workflow, this invention first uses a micro-computed tomography (Micro-CT) system to perform non-destructive scanning, acquiring a three-dimensional map of the sample. Then, based on this map, it plans and guides the path and orientation for mass spectrometry detection. This is a new paradigm of guided, intelligent mass spectrometry scanning based on prior three-dimensional topography information. The core motion control of this invention is no longer limited to the XYZ linear movement of a three-axis platform, but extends to precise control of the six-dimensional pose of the moving stage. This allows the moving stage to flexibly adapt to complex surfaces, like a human arm, always maintaining the optimal detection posture. This invention solves detection scenarios that existing mass spectrometry imaging technologies cannot handle, enabling high-resolution, high-sensitivity mass spectrometry imaging of sample surfaces with irregularities and those that are too brittle to slice.

[0088] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0089] It should be understood that although the steps in the flowcharts of the accompanying drawings are shown sequentially as indicated by the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some of the steps in the flowcharts of the accompanying drawings may include multiple steps or stages, which are not necessarily completed at the same time, but may be executed at different times, and the execution order of these steps or stages is not necessarily sequential, but may be performed alternately or in turn with other steps or at least some of the steps or stages of other steps.

[0090] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0091] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0092] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method, characterized in that, include: Acquire CT scan data of the sample to be tested and reconstruct a three-dimensional digital model; Based on the three-dimensional digital model, the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point are planned. The sample is controlled to move along the laser desorption path so that when each sampling point on the sample reaches the laser beam focus of the atmospheric pressure laser desorption / secondary photoionization device, the sample surface at the sampling point is desorbed at the planned sample attitude angle, and mass spectrometry data is collected by a mass spectrometer. By correlating the collected mass spectrometry data with the three-dimensional spatial coordinates of the corresponding sampling points, a three-dimensional distribution image of molecules on the surface of the sample to be tested can be reconstructed.

2. The CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method according to claim 1, characterized in that, The sample orientation angle enables the laser beam incident direction of the atmospheric pressure laser desorption / secondary photoionization device to be at a preset optimal angle with the local normal direction of the sample surface at the sampling point.

3. The CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method according to claim 1, characterized in that, The process of planning sampling points on the sample surface, laser desorption paths, and sample attitude angles at each sampling point based on a three-dimensional digital model specifically includes: Based on the user-defined spatial resolution, a set of sampling points is generated on the 3D surface of the region of interest of the 3D digital model. The generated set of sampling points is sorted by path, and an approximate solution algorithm for the traveling salesman problem is used to generate the scanning order with the shortest total motion path; Calculate the local surface normal vector at each sampling point location; Based on the local surface normal vector, the attitude angle of the sample is calculated when each sampling point on the sample reaches the laser beam focus of the atmospheric pressure laser desorption / secondary photoionization device, thereby realizing the planning of the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point.

4. The CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method according to claim 3, characterized in that, The set of sampling points is generated using the Poisson disk sampling algorithm, so that the sampling points are uniformly distributed on the three-dimensional curved surface.

5. The CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method according to claim 3, characterized in that, Also includes: The planned laser desorption path is smoothed by interpolation using B-spline curves.

6. The CT three-dimensional reconstruction coordinate-assisted mass spectrometry imaging method according to claim 3, characterized in that, The sample is driven by a six-axis robot; after the laser desorption path is planned, the mobility accessibility and collision avoidance detection are performed based on the robot's kinematic model and the three-dimensional digital model of the sample to be tested; if it is determined that it is unreachable or there is a risk of collision, the laser desorption path is adjusted or the corresponding sampling point is marked as an invalid point.

7. A system for implementing the method according to any one of claims 1 to 6, characterized in that, include: The three-dimensional morphology acquisition device acquires a three-dimensional digital model of the sample to be tested through a micro-computed tomography system. Mass spectrometry imaging system, including atmospheric pressure laser desorption / secondary photoionization device and mass spectrometer; A movable stage is used to support and fix the sample to be tested; A six-axis robot used to control a moving platform; The control unit is configured to: plan the sampling points on the sample surface, the laser desorption path, and the sample attitude angle at each sampling point based on the three-dimensional digital model; control the six-axis robot to move the sample to be tested along the planned laser desorption path; synchronously trigger the atmospheric pressure laser desorption / secondary photoionization device to desorb from the sample surface at the sampling point at the planned sample attitude angle; and trigger the mass spectrometer to collect mass spectrometry data and record the pose information to generate a mass spectrometry image.

8. The system according to claim 7, characterized in that, The atmospheric pressure laser desorption / secondary photoionization device includes a laser, a cage structure, and an ion sample introduction and transmission ionization channel; the sample to be tested is fixed on a moving stage, which is controlled by a six-axis robot; the laser beam is emitted from the laser, focused by the cage structure, and then turned by a right-angle optical adjustment frame before being focused from top to bottom onto the sample surface; the ion sample introduction and transmission ionization channel includes a sample introduction tube, a dopant introduction tube, and a transmission ionization tube.

9. The system according to claim 8, characterized in that, One end of the transmission ionization tube is sealed to the glass capillary of the mass spectrometer, and the other end is connected to the vacuum ultraviolet discharge lamp; the sample injection tube and the dopant injection tube are both designed to be located at the end close to the vacuum ultraviolet discharge lamp, respectively on both sides of the transmission ionization tube.

10. The system according to claim 7, characterized in that, The control unit uses a hand-eye calibration algorithm to unify the coordinate system of the three-dimensional shape acquisition device, the coordinate system of the six-axis robot, and the coordinate system of the mass spectrometry imaging system.

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