Vacuum in-situ laser desorption post-ionization mass spectrometry and fluorescence dual-mode imaging device and method

Abstract

This invention discloses a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device and method. The device includes: a vacuum chamber; an electrode assembly consisting of 6-12 electrodes with central through holes, providing a gradient decreasing electric field through resistive voltage division; an ion grid; deflection electrode plates; an MCP ion detector; a fluorescence imaging module connected via a second window optical path; a desorption laser module introduced through a first window; an ionization laser module introduced through a magnesium fluoride lens; a three-dimensional sample displacement stage; and a timing controller for controlling timing synchronization. The device of this invention can fully utilize the advantages of fluorescence imaging and mass spectrometry imaging, providing precise fluorescence localization while achieving high-throughput molecular visualization analysis. It is mainly used in the field of mass spectrometry imaging technology.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry imaging technology, specifically to a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device and method. Background Technology

[0002] Fluorescence imaging provides high spatial resolution morphological localization information of biological tissues, with resolution reaching the sub-micron level, but it relies on fluorescent labeling and cannot directly detect unknown molecules. Mass spectrometry imaging requires no labeling and can simultaneously detect lipids, metabolites, peptides, drugs, and their metabolites, obtaining molecular weight information and spatial distribution, but its spatial resolution is lower and it lacks tissue morphology information. The combined use of fluorescence imaging and mass spectrometry imaging can simultaneously obtain morphological localization and molecular distribution information, making it an important direction for research on drug metabolism and metabolomics in biological tissues. Secondary ion mass spectrometry (SIMS) uses a high-energy ion beam to bombard the sample, offering high spatial resolution, but it involves hard ionization, causing severe fragmentation of organic molecules, making it difficult to obtain intact molecular ions. It is generally only suitable for small molecules below 1000 Da and is not suitable for drug metabolism tracking and metabolite visualization; it also requires a 10... -7 Ultra-high vacuum below Pa involves complex equipment, high cost, and high maintenance costs. Laser desorption-ionization after desorption (LDPI) separates the desorption and ionization processes. Neutral molecules are first generated by gentle laser desorption, followed by post-ionization. This is a soft ionization method that preserves molecular integrity and is suitable for matrix-free mass spectrometry imaging of biological tissues, but it lacks tissue morphology information.

[0003] Therefore, how to design and construct devices to achieve in-situ synchronization of fluorescence localization and mass spectrometry detection, make up for the shortcomings of single technologies, and broaden the detection range of multiple components in biological tissues is a technical issue that urgently needs to be studied in the industry. Summary of the Invention

[0004] This invention provides a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device and method to solve one or more technical problems existing in the prior art, and to at least provide a beneficial option or create conditions.

[0005] This invention provides a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device, comprising: a vacuum cavity, an electrode assembly, an ion grid, a deflection electrode plate, an MCP ion detector, a fluorescence imaging module, a desorption laser module, an ionization laser module, a sample displacement stage, and a timing controller; The vacuum chamber is provided with: a first window, a second window, a mechanical pump interface, a vacuum pump interface, a sample injection interface, and an ionization light introduction interface; The electrode assembly consists of 6 to 12 electrodes arranged sequentially and spaced apart in the horizontal direction, with the centers of each electrode coaxial; the first electrode is a closed electrode, and each of the remaining electrodes has a through hole in its center; equal resistors are connected in series between each electrode and between the final electrode and the ion grid to form a resistive voltage divider, so as to provide a gradient-decreasing accelerating electric field. The electrode assembly provides an accelerating electric field. The ion grid is located at the rear end of the ion emission direction of the electrode assembly and is used to filter neutral particles and particulate impurities. The deflection electrode is located at the rear end of the ion grid and is used to adjust the ion flight direction. The MCP ion detector is located at the rear end of the deflection electrode and is connected to an external data acquisition card to convert the ion signal into a voltage signal. The fluorescence imaging module includes: an excitation light source, a CMOS imaging unit, an excitation filter, an emission filter, a reflector, a dichroic mirror, and an imaging lens. The fluorescence imaging module is connected to the vacuum cavity optical path via the second window and is used for fluorescence excitation and imaging of the sample. The desorption laser module includes: a high-power pulsed laser source for generating desorption laser. The desorption laser passes through the first window and a focusing lens in sequence and is focused on the sample surface. The ionization laser module includes an ionization laser source and a four-wave mixer. The four-wave mixer is connected to the vacuum cavity via the ionization light inlet interface. A magnesium fluoride lens is sealed at the ionization light inlet interface. The ionization laser source generates a 355nm pulsed laser. After passing through the four-wave mixer, the pulsed laser generates 118nm vacuum ultraviolet ionization light. The vacuum ultraviolet ionization light is focused above the sample via the ionization light inlet interface. The sample displacement stage includes a sample plate, a three-dimensional displacement stage, and a sample feed rod. The sample plate is fixed to the sample feed rod, and the sample feed rod is mounted on the three-dimensional displacement stage to support the sample and achieve three-dimensional spatial positioning. The timing controller is electrically connected to the desorption laser module, the ionization laser module, and the data acquisition card, respectively, and is used to control the timing synchronization of desorption light emission, ionization light emission, and mass spectrometry data acquisition.

[0006] Furthermore, in the electrode assembly, the distance between the first electrode and the second electrode is 15 mm, and the distance between the remaining adjacent electrode plates is 8 mm; the diameter of the through hole in the second electrode is 2 mm, and the diameter of the through hole in each of the remaining electrode plates is 5 mm.

[0007] Furthermore, the deflection electrode plate consists of two horizontally symmetrically arranged electrode plates; the deflection electrode plate is used to reduce the velocity of ions in the vertical direction by applying an adjustable DC voltage from 0V to 500V; an electric field-free flight zone is provided between the deflection electrode plate and the MCP ion detector, and the length of the electric field-free flight zone is 0.8m to 1.5m.

[0008] Furthermore, both the first window and the second window are sealed with ultraviolet-fused planar quartz glass.

[0009] Furthermore, both the mechanical pump interface and the vacuum pump interface are equipped with gate valves.

[0010] Furthermore, the magnesium fluoride lens is used to transmit vacuum ultraviolet ionization light and maintain a vacuum seal.

[0011] Furthermore, the sample-carrying surface of the sample plate is horizontally set, and the distance between the sample-carrying surface and the axis of the electrode assembly is adjustable within the range of 0mm to 5mm by the three-dimensional displacement stage.

[0012] Furthermore, the sample plate is a transparent insulating material component with a thickness of 3 mm and a light transmittance greater than 95%.

[0013] On the other hand, a method for vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging is provided. This method is applied to the vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device described in any of the above technical solutions. The method includes: Step 1, fixing a fluorescently labeled biological tissue sample onto a sample plate, inserting it into a vacuum chamber, and sequentially evacuating the sample to a working pressure of 5 × 10⁻⁶ using a mechanical pump and a molecular pump. -5 Below Pa; Step 2: Use a fluorescence imaging module to excite and acquire fluorescence images of biological tissue samples to obtain high-resolution fluorescence images with a resolution ≤1 μm; define the region of interest on the fluorescence image to obtain the target fluorescence image, and generate a two-dimensional mass spectrometry scanning coordinate array based on the target fluorescence image; Step 3: Based on the two-dimensional scanning coordinate matrix of the mass spectrometer, control the three-dimensional displacement stage (31) to move the sample to the positions of each point in the preset scanning matrix. The step size of the scanning matrix is ​​5μm to 10μm. The following operations are performed sequentially: the desorption laser module emits a desorption pulse laser with a working wavelength of 1064nm or 532nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. The laser is incident through the first window and focused on the sample surface with a focused spot diameter of 5μm to 10μm, causing the sample to desorb and form neutral molecular clusters. After a delay of 2μs to 20μs, the ionization laser source generates a pulse laser with a working wavelength of 355nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. The pulsed laser, after being mixed with four waves, generates 118nm vacuum ultraviolet ionizing light, which is then incident on the sample through the ionization light introduction interface and focused above the sample, ionizing neutral molecules into ions. The timing controller, after controlling the desorption laser module to output the desorption pulsed laser, synchronously triggers the ionization laser source and data acquisition card via a delay, achieving time synchronization of desorption, ionization, and data acquisition. Ions are accelerated by the electrode assembly, filtered by the ion grid, and oriented by the deflection electrode plates before entering the field-free flight region. They are detected by the MCP ion detector and converted into voltage signals, which are then recorded by the data acquisition card to obtain mass spectrometry data for each scanning point. Step 4: Based on the mass spectrometry data of each scanning point, stitch the data according to the spatial position of the scanning point array to generate a two-dimensional mass spectrometry image corresponding to each mass-charge ratio. Step 5: Map the two-dimensional mass spectrometry image onto the target fluorescence image according to coordinates to achieve pixel-level registration error, and simultaneously obtain the morphological information of the sample, the spatial distribution information of fluorescently labeled molecules, and the spatial distribution information of each mass spectrometry-detected molecule; wherein, the target fluorescence image contains the biological tissue morphological information and fluorescently labeled molecule distribution information of the sample, and the two-dimensional mass spectrometry image contains the distribution information of mass spectrometry-detected molecules.

[0014] Furthermore, the thickness of the biological tissue sample slices is 5 μm to 10 μm.

[0015] This invention offers at least the following advantages: The device of this invention fully leverages the advantages of fluorescence imaging and mass spectrometry imaging, providing precise fluorescence localization while achieving high-throughput molecular visualization analysis. This technical solution can simultaneously acquire morphological images and multi-component distribution information of the sample on a single imaging platform, overcoming the limitations of limited throughput in fluorescence imaging and the inability to obtain morphological information in mass spectrometry imaging; it also overcomes the localization inaccuracies and information variation defects caused by traditional secondary imaging. Furthermore, this invention also provides a corresponding method, the benefits of which are similar to those of the method itself, and will not be repeated here. This invention is primarily applicable to the field of mass spectrometry imaging technology. Attached Figure Description

[0016] The accompanying drawings are provided to further understand the technical solutions of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the technical solutions of the present invention, and do not constitute a limitation on the technical solutions of the present invention.

[0017] Figure 1 This is a frontal schematic diagram (black and white) of a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device. Figure 2 This is a top-view schematic diagram (black and white) of a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device. Figure 3 This is a frontal schematic diagram (in color) of a dual-mode imaging device for ionization mass spectrometry and fluorescence after vacuum in-situ laser desorption / ionization mass spectrometry. Figure 4 This is a top-view schematic diagram (in color) of a dual-mode imaging device for vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence imaging. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] It should be noted that although functional modules are divided in the system diagram and the logical order is shown in the flowchart, in some cases, the steps shown or described may be performed in a different order than the module division in the system or the order in the flowchart. The terms "first," "second," etc., in the specification, claims, and the aforementioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0020] The following is an explanation of some terms: LDPI: Laser Desorption Post-Ionization; MCP: Microchannel Plate Ion Detector. CMOS: Complementary Metal-Oxide-Semiconductor Imaging Unit; SIMS: Secondary Ion Mass Spectrometry; TOF-MS: Time-of-flight mass spectrometry; LDPI (Laser Desorption-Ionization) is a two-step ionization mass spectrometry method. Its basic principle is to first irradiate the sample surface with a pulsed laser, causing the molecules in the sample to desorb and volatilize. These neutral molecules then enter the post-ionization region, where they are further ionized into ions through electron bombardment, chemical ionization, or photoionization, and finally enter the mass analyzer for detection. This design of separating the desorption and ionization processes gives LDPI good ionization controllability, reducing molecular debris generated by direct laser ionization and thus better preserving the integrity of the analyte. It is particularly suitable for spatial distribution imaging of relatively large molecules such as drugs and their metabolites. Furthermore, LDPI technology has low matrix dependence; some variants can even directly analyze biological tissue sections without a matrix. The laser beam can be focused to the micrometer level to achieve high spatial resolution mass spectrometry imaging, making it valuable for the visualization of lipids, drugs, and metabolites in tissue sections.

[0021] SIMS (Secondary Ion Mass Spectrometry) is a surface analysis mass spectrometry method based on ion beam bombardment. Its working principle involves bombarding the sample surface with a high-energy primary ion beam (such as gallium ions, cesium ions, bismuth cluster ions, or argon ions). The impact energy of the primary ions causes atoms or molecules on the sample surface to sputter, with a very small fraction emitted as charged ions (i.e., secondary ions). These secondary ions are collected and separated and detected by a mass analyzer according to their mass-to-charge ratio, thus obtaining information about the elemental or molecular composition of the sample surface. SIMS technology can be divided into two modes based on the primary ion dose: static SIMS and dynamic SIMS. The former uses a low-dose ion beam to analyze only a single surface layer, suitable for imaging organic molecules; the latter uses a high-dose ion beam to continuously sputter the sample, enabling layer-by-layer profiling and three-dimensional imaging in the depth direction. Modern time-of-flight secondary ion mass spectrometry (ToF-SIMS) uses a pulsed primary ion beam, combining high quality resolution with a wide mass detection range. The most prominent advantage of SIMS technology lies in its extremely high spatial resolution, reaching sub-micron or even nanometer levels with an information depth of only about 1 to 2 nanometers. It is a true surface-sensitive analysis technology, capable of simultaneously providing elemental and molecular fragmentation information and possessing excellent three-dimensional imaging capabilities. However, because primary ion bombardment is a hard ionization process, it leads to severe molecular fragmentation, making the analysis of intact macromolecules difficult, and the mass detection range is typically limited to below 1000 Daltons. In the field of drug metabolism tracking in biological tissues, SIMS technology is particularly suitable for drug distribution studies requiring single-cell or sub-cell level spatial resolution, as well as applications requiring simultaneous acquisition of elemental distribution information and three-dimensional depth analysis.

[0022] Mass spectrometry imaging (MSI) is an imaging method based on mass spectrometry. This method directly scans biological samples using mass spectrometry, allowing simultaneous analysis of the spatial distribution characteristics of hundreds of molecules on the same tissue slice or tissue chip. The working principle of MSI technology is as follows: First, the sample to be tested is acquired and prepared in an appropriate manner. The mass spectrometer scans the sample according to a pre-set acquisition program using lasers or high-energy ion beams, desorbing and ionizing molecules or ions on its surface. Then, a mass analyzer obtains the mass-to-charge ratio and ion intensity of ions at each pixel on the sample surface. Using mass spectrometry imaging software, the mass spectrum peaks of ions with any specified mass-to-charge ratio are searched in the mass spectrometry data of each pixel. Combining the signal intensity of the corresponding ion with its position on the sample surface, a two-dimensional distribution map of the corresponding molecule or ion on the sample surface is drawn. Subsequently, the same software is used to further process the two-dimensional distribution map of the continuous sample slices to obtain the three-dimensional spatial distribution of the analyte in the sample.

[0023] Research has revealed that drug metabolism tracking and metabolite visualization require the detection of metabolites, lipids, peptides, and proteins in biological tissues. Current technologies utilize secondary ion mass spectrometry (SIMS) for mass spectrometry imaging. SIMS imaging primarily uses a beam of primary ions (such as Ga+, Bi+, Bi3+, etc.) to spray onto the surface of the sample, charging the surface atoms, which are then analyzed and imaged using magnetic and electric fields. However, because the primary ion beam typically has high energy, it easily breaks covalent bonds, often resulting in a large number of fragment ions during testing. SIMS is generally suitable for elemental analysis, inorganic analysis, and the detection of small molecules (less than 1000 Da). When SIMS technology is applied to organic samples, especially biomolecules, the high-energy ion bombardment often severely damages the molecular structure of drugs / metabolites, generating a large number of small fragment ions, while producing almost no large molecular fragments that provide chemical structural information. Therefore, it is impossible to obtain complete molecular ion peaks, accurately track drug metabolism, accurately characterize metabolites, or identify unknown metabolites. Furthermore, this technology can only achieve visualization analysis of single molecules, making it difficult to meet the requirements of simultaneous visualization analysis of multiple molecules in drug metabolism tracking and metabolite visualization applications. SIMS technology requires maintaining an ultra-high vacuum environment, with a typical operating pressure of 1×10⁻⁶. -7 Below Pa, the equipment is expensive, complex to maintain, and has poor stability.

[0024] Therefore, the inventive concept of this application is to separate the desorption and ionization processes by employing laser desorption combined with post-ionization technology. The laser desorption process can more gently vaporize intact analyte molecules from biological tissues without ionizing or excessively fragmenting them. Subsequently, post-ionization is performed using 118 nm vacuum ultraviolet (VUV) light. VUV photon energy is sufficiently high (10.5 eV) to cause single-photon ionization of many organic molecules, but its energy is relatively mild, unlikely to cause deep molecular fragmentation, thus generating richer molecular ion signals. This "soft ionization" characteristic is crucial for the integrity analysis of complex organic molecules such as drugs and their metabolites. It can provide clearer and more complete mass spectra for various biomolecules, thereby enabling more accurate molecular identification and spatial distribution imaging. The vacuum level required for laser desorption / ionization technology generally only needs to be maintained at 10... -4 Pa to 10 -7 Between Pa, specifically, the entire device only needs to be maintained at 5 × 10 Pa. -5 Normal signal acquisition can be achieved in a vacuum environment of Pa. This reduces the vacuum requirement by two orders of magnitude compared to secondary ion mass spectrometry. This invention fully leverages the advantages of fluorescence imaging and mass spectrometry imaging, providing precise fluorescence localization while enabling high-throughput molecular visualization analysis. It can well meet the needs of simultaneous visualization analysis of multiple components in drug metabolism tracking and metabolite visualization studies.

[0025] The specific implementation methods of this technical solution are described in detail below.

[0026] refer to Figure 1 , Figure 2 , Figure 3 and Figure 4 In order to solve the problems existing in the prior art, this application discloses a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device and method.

[0027] Among them, a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device is provided, including: a vacuum cavity 19, an electrode assembly, an ion grid 16, a deflection electrode plate 17, an MCP ion detector 18, a fluorescence imaging module, a desorption laser module, an ionization laser module, a sample displacement stage, and a timing controller.

[0028] The vacuum chamber 19 is provided with: a first window 20, a second window 21, a mechanical pump interface 22, a vacuum pump interface 23, a sample injection interface 24, and an ionization light introduction interface 25.

[0029] The electrode assembly is used to provide an accelerating electric field. Specifically, the electrode assembly consists of 6 to 12 electrodes arranged at intervals along the horizontal direction, with the centers of each electrode coaxial; the first electrode 10 is a closed electrode, and each of the remaining electrodes has a through hole in its center; equal-value resistors are connected in series between each electrode and between the final electrode and the ion grid 16 to form a resistive voltage divider, thereby providing an accelerating electric field with a decreasing gradient.

[0030] The ion grid 16 is disposed at the rear end of the ion emission direction of the electrode assembly and is used to filter neutral particles and particulate impurities; the deflection electrode plate 17 is disposed at the rear end of the ion grid 16 and is used to adjust the ion flight direction.

[0031] The MCP ion detector 18 is located at the rear end of the deflection electrode plate 17 and is connected to an external data acquisition card to convert ion signals into voltage signals.

[0032] The fluorescence imaging module includes: an excitation light source 1, a CMOS imaging unit 2, an excitation filter 3, an emission filter 4, a reflector 5, a dichroic mirror 6, and an imaging lens 7. The fluorescence imaging module is optically connected to the vacuum cavity 19 via the second window 21 and is used for fluorescence excitation and imaging of the sample.

[0033] The desorption laser module includes a high-power pulsed laser source 8 and a focusing lens 9. The high-power pulsed laser source 8 is used to generate desorption laser, which passes through the first window 20 and the focusing lens 9 in sequence and is focused on the sample surface.

[0034] The ionization laser module includes an ionization laser source 29 and a four-wave mixer 26. The four-wave mixer 26 is connected to the vacuum cavity 19 via the ionization light inlet interface 25. A magnesium fluoride lens 27 is sealed at the ionization light inlet interface 25. The ionization laser source 29 generates a 355nm pulsed laser. After passing through the four-wave mixer 26, the pulsed laser generates 118nm vacuum ultraviolet ionized light. The vacuum ultraviolet ionized light is focused above the sample through the ionization light inlet interface 25. An ultraviolet fused silica lens 28 is provided at the light inlet of the four-wave mixer 26.

[0035] The sample displacement stage includes a sample plate 30, a three-dimensional displacement stage 31, and a sample feed rod 32. The sample plate 30 is fixed on the sample feed rod 32, and the sample feed rod 32 is installed on the three-dimensional displacement stage 31 to carry the sample and achieve three-dimensional spatial positioning.

[0036] The timing controller is electrically connected to the desorption laser module, the ionization laser module, and the data acquisition card, respectively, and is used to control the timing synchronization of desorption light emission, ionization light emission, and mass spectrometry data acquisition.

[0037] In the electrode assembly, the distance between the first electrode and the second electrode is 15 mm, and the distance between the remaining adjacent electrode plates is 8 mm; the diameter of the through hole in the second electrode is 2 mm, and the diameter of the through hole in each of the remaining electrode plates is 5 mm.

[0038] The deflection electrode plate 17 consists of two horizontally symmetrically arranged electrode plates. The deflection electrode plate 17 is used to reduce the velocity of ions in the vertical direction by applying an adjustable DC voltage from 0V to 500V. A field-free flight region is provided between the deflection electrode plate 17 and the MCP ion detector 18, and the length of the field-free flight region is 0.8m to 1.5m.

[0039] Both the first window 20 and the second window 21 are sealed with ultraviolet-fused planar quartz glass. Both the mechanical pump interface 22 and the vacuum pump interface 23 are equipped with gate valves. The magnesium fluoride lens 27 is used to transmit vacuum ultraviolet ionization light and maintain the vacuum seal.

[0040] The sample-carrying surface of the sample plate 30 is horizontally set, and the distance between the sample-carrying surface and the axis of the electrode group is adjustable within the range of 0 mm to 5 mm by the three-dimensional displacement stage 31.

[0041] The sample plate 30 is a transparent insulating material component with a thickness of 3 mm and a light transmittance of more than 95%.

[0042] The mass spectrometer ion source and analyzer consist of an electrode assembly, an ion grid 16, a deflection electrode plate 17, an MCP ion detector 18, and a vacuum chamber 19. All components are sequentially arranged inside the vacuum chamber 19, with their centers coaxial. The electrode assembly contains 6 to 12 electrodes; in this embodiment, 6 electrodes are used. The six electrodes are designated as the first electrode 10, the second electrode 11, the third electrode 12, the fourth electrode 13, the fifth electrode 14, and the sixth electrode 15.

[0043] The distance between the first and second electrode plates is 15 mm, and the distance between subsequent electrode plates is 8 mm. Except for the first electrode plate 10, each of the other electrode plates has a through hole in its center. The diameter of the through hole in the second electrode plate 12 is 2 mm, and the diameter of the through hole in the other electrode plates is 5 mm. All electrode plates are coaxial.

[0044] The voltage applied to the first electrode is 6000V. The ion grid 16 is grounded. Equivalent resistors are connected in series between the electrodes of the electrode group and between the ion grid 16, forming a resistive voltage divider. The voltage gradient applied to each electrode of the electrode group and the ion grid 16 decreases progressively, providing an accelerating electric field for the ions. The ion source can operate in positive ion mode or negative ion mode: in positive ion mode, a positive high voltage is applied to the first electrode; in negative ion mode, a negative high voltage is applied to the first electrode. The ion grid 16 is used to filter neutral particles and particulate impurities. The deflection electrode 17 consists of two horizontally symmetrically arranged electrode plates, with an adjustable DC voltage of 0-500V applied to reduce the vertical velocity of the ions and improve the mass spectrometry signal intensity. A field-free flight region without electric field is set between the deflection electrode 17 and the MCP ion detector 18. Preferably, the length of the field-free flight region is 0.8-1.5m. The MCP ion detector is connected to a data acquisition card, which can convert the ion signal into a voltage signal for the data acquisition card to collect. The vacuum chamber 19 has six openings: a first window 20, a second window 21, a mechanical pump interface 22, a vacuum pump interface 23, a sample inlet interface 24, and an ionization light introduction interface 25. The first window 20 and the second window 21 are both sealed with ultraviolet-fused planar quartz glass; the mechanical pump interface 22 and the vacuum pump interface 23 are both equipped with gate valves.

[0045] like Figure 1 As shown, the fluorescence imaging section includes an excitation light source 1, a CMOS imaging unit 2, an excitation filter 3, an emission filter 4, a reflector 5, a dichroic mirror 6, and an imaging lens 7. The excitation filter 3 filters out stray light outside the excitation wavelength range; the emission filter 4 filters out background noise light; and the dichroic mirror 6 reflects the excitation light and transmits the emitted fluorescence light. The excitation light emitted by the excitation light source 1 is filtered by the excitation filter 3, then reflected sequentially by the reflector 5 and the dichroic mirror 6, and enters through the second window 21 of the vacuum cavity 19. After being focused by the imaging lens 7, it illuminates the sample under test. The fluorescence emitted by the sample under test passes sequentially through the imaging lens 7, the dichroic mirror 6, and the emission filter 4 before being acquired by the CMOS imaging unit 2, obtaining a high-resolution fluorescence image.

[0046] The desorption and ionization of the sample are performed by two independent laser beams. The desorption light is generated by a high-power pulsed laser source 8, with a selectable operating wavelength of 1064 nm or 532 nm, a pulse width of 10 ns, and a pulse frequency of 10–100 Hz. The desorption light generated by this high-power pulsed laser source 8 enters the vacuum cavity 19 through the first window 20, and is then focused by the focusing lens 9 to perpendicularly illuminate the sample plate surface. The focusing lens 9 is mounted on a linear displacement stage, and the focusing position can be adjusted by the displacement stage to achieve an adjustable spot size within the range of 5–10 μm.

[0047] The ionization light and desorption light are arranged perpendicularly and orthogonally. The ionization light generating device is as follows: Figure 2As shown, the ionization light inlet 25 of the vacuum chamber 19 is sealed with a magnesium fluoride lens 27, which is connected to a four-wave mixer 26. One end of the four-wave mixer 26 is fitted with an ultraviolet fused silica lens 28, and the interior is filled with a mixture of xenon and argon gases. The ionization laser source 29 operates at a wavelength of 355 nm, with a pulse width of 10 ns and a pulse frequency of 10–100 Hz. The 355 nm laser generated by this source is focused by the silica lens to produce 118 nm vacuum ultraviolet ionization light in the four-wave mixer. This vacuum ultraviolet ionization light is then focused above the sample plate 30 by the magnesium fluoride lens 27, with the focal point located on the central axis of the electrode, ensuring that the ionization region is at the center of the ion acceleration region.

[0048] like Figure 2 As shown, the sample displacement stage includes a sample plate 30, a three-dimensional displacement stage 31, and a sample feed rod 32. The sample to be tested is placed on the sample plate 30, with the sample-carrying surface of the sample plate horizontally positioned. The sample plate 30 is fixed to the sample feed rod 32, which is mounted on the three-dimensional displacement stage. The sample plate 30 is made of a transparent insulating material to avoid interference with the electric field between the accelerating electrodes; optical glass is preferred. In this embodiment, optical glass with a thickness of 3 mm and a transmittance greater than 95% is used. The distance between the sample-carrying surface of the sample plate and the axis of the electrode can be adjusted via the three-dimensional displacement stage, with an adjustment range of 0–5 mm, to adjust the optimal ion extraction efficiency.

[0049] The mass spectrometry signal acquisition process is as follows: The timing of desorption light, ionization light, and mass spectrometry acquisition is uniformly controlled by a timing controller: The timing controller first outputs a pulse to trigger the high-power pulsed laser source 8 to emit a desorption laser, irradiating the sample surface and causing gentle desorption of the sample in the spot area, forming neutral molecular clusters. After a delay of 2–20 μs, the timing controller outputs a second pulse, which is split by the signal line to simultaneously trigger the ionization laser source and the data acquisition card, starting the 118 nm vacuum ultraviolet ionization light and beginning the acquisition of mass spectrometry signals. The 118 nm ionization light irradiates the neutral molecular clusters, ionizing the neutral molecules into charged ions; the ions gain kinetic energy under the action of the electrode electric field and fly along the axis, are filtered by the ion grid 16, and have their direction corrected by the deflection electrode 17 before entering the field-free flight region and finally reaching the MCP ion detector; the MCP ion detector converts the ion signal into a voltage signal, which is acquired by the data acquisition card; the mass-to-charge ratio (m / z) of the corresponding ion is calculated based on the ion flight time to obtain the mass spectrometry data.

[0050] The image acquisition and control section uses self-developed LabVIEW control software to process the data from the data acquisition card, generate two-dimensional mass spectrometry images, and fuse fluorescence images with mass spectrometry images.

[0051] This invention employs laser desorption / ionization combined with other techniques, significantly enhancing its performance in tracking drug metabolism in biological tissues and visualizing metabolites. This improvement effectively addresses the fragility of secondary ion mass spectrometry in organic molecule analysis, enabling the acquisition of a large amount of molecular information in a single pass, thus providing more comprehensive and high-throughput molecular spatial distribution information and offering a more powerful analytical tool for life sciences and drug development. The system provided by this invention only requires a 5 × 10⁻⁶ cycle. -5 The vacuum environment of Pa is two orders of magnitude lower than that required by secondary ion mass spectrometry, which reduces instrument complexity, cost and maintenance difficulty.

[0052] The method also provides a vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging method, which includes: Step 1, fixing the fluorescently labeled biological tissue sample onto the sample plate 30, sending it into the vacuum chamber 19, and sequentially evacuating the vacuum to a working pressure of 5×10⁻ using a mechanical pump and a molecular pump. 5 Below Pa.

[0053] Step 2: Use a fluorescence imaging module to excite and acquire fluorescence images of biological tissue samples to obtain high-resolution fluorescence images with a resolution ≤1 μm; define the region of interest on the fluorescence image to obtain the target fluorescence image, and generate a two-dimensional mass spectrometry scanning coordinate array based on the target fluorescence image.

[0054] Step 3: Based on the two-dimensional scanning coordinate matrix of the mass spectrometer, the three-dimensional displacement stage 31, controlled by the timing controller, moves the sample to the positions of each point in the preset scanning matrix. The step size of the scanning matrix is ​​5μm to 10μm. The following operations are performed sequentially: the desorption laser module emits a desorption pulsed laser with a working wavelength of 1064nm or 532nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. The laser is incident through the first window 20 and focused on the sample surface with a focused spot diameter of 5μm to 10μm, causing desorption of the sample to form neutral molecular clusters. After a delay of 2μs to 20μs, the ionization laser source 29 generates a pulsed laser with a working wavelength of 355nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. The pulsed laser, after being mixed with four waves, generates 118nm vacuum ultraviolet ionization light, which is incident on the sample through the ionization light introduction interface 25 and focused above the sample, ionizing neutral molecules into ions. After the timing controller controls the desorption laser module to output the desorption pulsed laser, it triggers the ionization laser source and data acquisition card through the delay synchronization to achieve time synchronization of desorption, ionization and data acquisition. After being accelerated by the electrode group, filtered by the ion grid 16 and oriented by the deflection electrode plate 17, the ions enter the field-free flight region, are detected by the MCP ion detector 18 and converted into voltage signals, which are recorded by the data acquisition card to obtain mass spectrometry data at each scanning point.

[0055] Step 4: Based on the mass spectrometry data of each scanning point, stitch the data according to the spatial position of the scanning point array to generate a two-dimensional mass spectrometry image corresponding to each mass-charge ratio.

[0056] Step 5: Map the two-dimensional mass spectrometry image onto the target fluorescence image according to coordinates to achieve pixel-level registration error, and simultaneously obtain the morphological information of the sample, the spatial distribution information of fluorescently labeled molecules, and the spatial distribution information of each mass spectrometry-detected molecule; wherein, the target fluorescence image contains the biological tissue morphological information and fluorescently labeled molecule distribution information of the sample, and the two-dimensional mass spectrometry image contains the distribution information of mass spectrometry-detected molecules.

[0057] The specific steps are as follows: S0, Sample preparation: Biological tissue sections with a thickness of 5-10 μm are fluorescently labeled and fixed onto a transparent insulating sample plate.

[0058] S1. Vacuum Preparation: The sample is placed into the vacuum chamber; the mechanical pump is used to evacuate to 1×10⁻⁶. -1 Below Pa; the molecular pump was pumped to 5 × 10 -5 Once the pressure is below a certain level and the pressure stabilizes, close the gate valve.

[0059] S2. Vacuum in situ fluorescence imaging: The fluorescence imaging part acquires fluorescence images of the sample with a resolution of ≤1μm, showing tissue morphology and the distribution of labeled molecules.

[0060] S3. Select the mass spectrometry analysis area: Select the region of interest on the fluorescence image to obtain the target fluorescence image.

[0061] S4. Mass spectrometry point-by-point scanning: The control software generates a two-dimensional scanning point array with a step size of 5-10 μm; the three-dimensional displacement stage moves the sample to the scanning point; 1064 nm laser desorption is performed, followed by 118 nm vacuum ultraviolet ionization after a delay of 2-20 μs; the MCP detector collects ion signals and generates single-point mass spectrometry data.

[0062] S5. Mass Spectrometry Image Reconstruction: The mass spectrometry data is stitched together according to the scanning matrix to generate two-dimensional mass spectrometry images corresponding to each mass-charge ratio.

[0063] S6. Image Registration and Fusion: The target fluorescence image is fused with the two-dimensional mass spectrometry image. The spatial position of the mass spectrometry image is corrected based on the high-resolution fluorescence image, improving resolution. The target fluorescence image contains information on the biological tissue morphology of the sample and the distribution information of fluorescently labeled molecules, while the two-dimensional mass spectrometry image contains the distribution information of molecules detected by mass spectrometry. Fusion achieves pixel-level registration error correction and simultaneously obtains the morphological information of the sample, the spatial distribution information of fluorescently labeled molecules, and the spatial distribution information of each molecule detected by mass spectrometry.

[0064] S7. Output Results: Generates high-resolution fused images of biological tissue morphology and drug and metabolite distribution, enabling precise localization and qualitative and quantitative analysis.

[0065] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented, for example, in orders other than those illustrated or described herein. Furthermore, the terms “comprising” and “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatuses.

[0066] It should be understood that in this application, "at least one (item)" means one or more, and "more than" means two or more. "And / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can represent three cases: only A exists, only B exists, and both A and B exist simultaneously, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one (item) of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one (item) of a, b, or c can represent: a, b, c, "a and b", "a and c", "b and c", or "a and b and c", where a, b, and c can be single or multiple.

[0067] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be indirect coupling or communication connection through some interfaces, apparatuses, or units, and may be electrical, mechanical, or other forms.

[0068] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0069] Furthermore, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0070] If the integrated unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this application. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0071] Although the description of this application has been quite detailed and particularly focused on several of the described embodiments, it is not intended to limit itself to any of these details or embodiments or any particular embodiment. Rather, it should be considered as effectively covering the intended scope of this application by referring to the appended claims and taking into account the prior art, which provides for a broad possible interpretation of these claims. Furthermore, the foregoing description of this application with respect to embodiments foreseeable by the inventors is intended to provide a useful description, and non-substantial modifications to this application that have not yet been foreseen may still represent equivalent modifications.

[0072] It should be noted that in all specific embodiments of this application, when processing data related to user identity or characteristics, such as user information, user behavior data, user historical data, and user location information, user permission or consent is obtained first. Furthermore, the collection, use, and processing of this data comply with relevant laws, regulations, and standards. In addition, when embodiments of this application require access to sensitive personal information of users, separate permission or consent from the user is obtained through pop-ups or redirection to confirmation pages. Only after obtaining the user's separate permission or consent is the necessary user-related data required for the proper functioning of these embodiments acquired.

Claims

1. A vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device, characterized in that, include: Vacuum chamber (19), electrode assembly, ion grid (16), deflection electrode plate (17), MCP ion detector (18), fluorescence imaging module, desorption laser module, ionization laser module, sample displacement stage and timing controller; The vacuum chamber (19) is provided with: a first window (20), a second window (21), a mechanical pump interface (22), a vacuum pump interface (23), a sample inlet interface (24), and an ionization light introduction interface (25). The electrode assembly is used to provide an accelerating electric field. The ion grid (16) is located at the rear end of the ion emission direction of the electrode assembly and is used to filter neutral particles and particulate impurities. The deflection electrode plate (17) is located at the rear end of the ion grid (16) and is used to adjust the ion flight direction. The MCP ion detector (18) is located at the rear end of the deflection electrode plate (17) and is connected to an external data acquisition card to convert ion signals into voltage signals. The fluorescence imaging module includes: an excitation light source (1), a CMOS imaging unit (2), an excitation filter (3), an emission filter (4), a reflector (5), a dichroic mirror (6), and an imaging lens (7). The fluorescence imaging module is optically connected to the vacuum cavity (19) through the second window (21) and is used to excite and image the sample. The desorption laser module includes a high-power pulsed laser source (8) and a focusing lens (9). The desorption laser module is optically connected to the vacuum cavity (19) through the first window (20). The high-power pulsed laser source (8) is used to generate desorption laser. The desorption laser passes through the first window (20) and the focusing lens (9) in sequence and is focused on the sample surface. The ionization laser module includes: an ionization laser source (29) and a four-wave mixer (26). The four-wave mixer (26) is connected to the vacuum cavity (19) via the ionization light inlet interface (25). A magnesium fluoride lens (27) is sealed at the ionization light inlet interface (25). The ionization laser source (29) is used to generate a 355nm pulsed laser. The pulsed laser generates 118nm vacuum ultraviolet ionization light after passing through the four-wave mixer (26). The vacuum ultraviolet ionization light is focused above the sample through the ionization light inlet interface (25). The sample displacement stage includes: a sample plate (30), a three-dimensional displacement stage (31), and a sample feed rod (32). The sample plate (30) is fixed on the sample feed rod (32). The sample feed rod (32) is installed on the three-dimensional displacement stage (31) to carry the sample and achieve three-dimensional spatial positioning. The timing controller is electrically connected to the desorption laser module, the ionization laser module, and the data acquisition card, respectively, and is used to control the timing synchronization of desorption light emission, ionization light emission, and mass spectrometry data acquisition.

2. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, In the electrode assembly, the distance between the first electrode and the second electrode is 15 mm, and the distance between the remaining adjacent electrode plates is 8 mm; the diameter of the through hole of the second electrode is 2 mm, and the diameter of the through hole of each of the remaining electrode plates is 5 mm.

3. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, The deflection electrode plate (17) consists of two horizontally symmetrically arranged electrode plates; The deflection electrode plate (17) is used to reduce the velocity of ions in the vertical direction by applying an adjustable DC voltage from 0V to 500V; a field-free flight zone is provided between the deflection electrode plate (17) and the MCP ion detector (18), and the length of the field-free flight zone is 0.8m to 1.5m.

4. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, Both the first window (20) and the second window (21) are sealed with ultraviolet-fused planar quartz glass.

5. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, Both the mechanical pump interface (22) and the vacuum pump interface (23) are equipped with gate valves.

6. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, The ionization light inlet (25) is sealed with a magnesium fluoride lens (27), which is used to transmit vacuum ultraviolet ionization light and maintain a vacuum seal.

7. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, The sample plate (30) has a horizontally mounted sample surface, and the distance between the sample surface and the axis of the electrode assembly is adjustable from 0 mm to 5 mm by the three-dimensional displacement stage (31).

8. The vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to claim 1, characterized in that, The sample plate (30) is a transparent insulating material component with a thickness of 3 mm and a light transmittance of more than 95%.

9. A method for dual-mode imaging of ionization mass spectrometry and fluorescence after vacuum in-situ laser desorption / ionization, characterized in that, Applied to the vacuum in-situ laser desorption / ionization mass spectrometry and fluorescence dual-mode imaging device according to any one of claims 1 to 8; the method includes: Step 1: Fix the fluorescently labeled biological tissue sample onto the sample plate (30) and send it into the vacuum chamber (19). Then, use a mechanical pump and a molecular pump to evacuate the vacuum to a working pressure of 5 × 10⁻⁶. -5 Below Pa; Step 2: Use a fluorescence imaging module to excite and acquire fluorescence images of biological tissue samples to obtain high-resolution fluorescence images with a resolution ≤1μm; define the region of interest on the fluorescence image to obtain the target fluorescence image; and generate a two-dimensional mass spectrometry scanning coordinate array based on the target fluorescence image. Step 3: Based on the two-dimensional scanning coordinate array of the mass spectrometer, control the three-dimensional displacement stage (31) to move the sample to the positions of each point in the preset scanning array. The step size of the scanning array is 5μm to 10μm. The following operations are performed sequentially: the desorption laser module emits a desorption pulse laser with a working wavelength of 1064nm or 532nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. The laser is incident through the first window (20) and focused on the sample surface with a focused spot diameter of 5μm to 10μm, causing the sample to desorb and form neutral molecular clusters. After a delay of 2μs to 20μs, the ionization laser source (29) generates a pulse laser with a working wavelength of 355nm, a pulse width of 10ns, and a pulse frequency of 10Hz to 100Hz. Hz; The pulsed laser generates 118nm vacuum ultraviolet ionization light through four-wave mixing, which is incident and focused on the sample through the ionization light introduction interface (25), so that the neutral molecules are ionized into ions; After the timing controller controls the desorption laser module to output the desorption pulsed laser, the ionization laser source and data acquisition card are triggered by the delay synchronization to realize the timing synchronization of desorption, ionization and data acquisition; After the ions are accelerated by the electrode group, filtered by the ion grid (16) and the direction of the deflection electrode plate (17) is corrected, they enter the fieldless flight region, are detected by the MCP ion detector (18) and converted into voltage signals, and are recorded by the data acquisition card to obtain the mass spectrometry data of each scanning point; Step 4: Based on the mass spectrometry data of each scanning point, stitch the data according to the spatial position of the scanning point array to generate a two-dimensional mass spectrometry image corresponding to each mass-charge ratio. Step 5: Map the two-dimensional mass spectrometry image onto the target fluorescence image according to coordinates to achieve pixel-level registration error, and simultaneously obtain the morphological information of the sample, the spatial distribution information of fluorescently labeled molecules, and the spatial distribution information of each mass spectrometry-detected molecule; wherein, the target fluorescence image contains the biological tissue morphological information and fluorescently labeled molecule distribution information of the sample, and the two-dimensional mass spectrometry image contains the distribution information of mass spectrometry-detected molecules.

10. The method for dual-mode imaging of ionization mass spectrometry and fluorescence after vacuum in-situ laser desorption / resorption spectroscopy according to claim 9, characterized in that, The thickness of the biological tissue sample slices is 5 μm to 10 μm.

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