Desorption electrospray and secondary photoionization mass spectrometry imaging device combined with ion funnel and working method of desorption electrospray and secondary photoionization mass spectrometry imaging device

Abstract

The invention discloses a desorption electrospray and secondary photoionization mass spectrometry imaging device combined with an ion funnel, which comprises a double-light-source secondary ionization component, a two-stage ion funnel, a desorption electrospray ionization source and a three-degree-of-freedom displacement imaging platform for carrying a glass slide of a measured object, the desorption electrospray ionization source is arranged above the three-degree-of-freedom displacement imaging platform and located on one side of the glass slide, an electrospray sampling pipe for sampling molecule-ion droplet clusters is arranged at an inlet of the double-light-source secondary ionization assembly, and the electrospray sampling pipe is arranged above the three-degree-of-freedom displacement imaging platform and located on the other side of the glass slide; an outlet of the double-light-source secondary ionization assembly is connected with an inlet of the two-stage ion funnel, and an outlet of the two-stage ion funnel is externally connected with mass spectrometry equipment. Through the integrated design of double-light-source same-cavity secondary ionization, gas phase doping and the two-stage ion funnel, the ionization efficiency and the ion transmission efficiency of the to-be-analyzed object can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry analysis technology, and in particular to a desorption electrospray and secondary photoionization mass spectrometry imaging device and its working method that incorporates an ion funnel. Background Technology

[0002] Traditional mass spectrometry imaging, which uses charged droplets or lasers to directly desorb and ionize molecules from the surface of biological tissues, suffers from low ionization efficiency, with less than one-thousandth of molecules being ionized. This severely hinders the improvement of mass spectrometry sensitivity and image quality. Secondary ionization techniques can significantly improve ionization efficiency without affecting the primary desorption / ionization mechanism, increasing ion signal intensity by 2-3 orders of magnitude. Furthermore, the existence of secondary ionization can greatly enhance instrument sensitivity, compensating for the low ionization efficiency caused by sacrificing desorption area in pursuit of high spatial resolution, thus resolving the contradiction between high spatial resolution and high sensitivity in single imaging methods. Moreover, secondary ionization can overcome the polarity discrimination effect of primary ionization (except for ESI as a secondary ionization method). Therefore, this method has become a mainstream approach in emerging imaging technologies and is attracting increasing attention from researchers in mass spectrometry imaging methodology.

[0003] In recent years, with the development and advancement of more efficient ionization methods, ambient ionization mass spectrometry (AIMS), which simplifies sample pretreatment, operates at room temperature and pressure, and enables rapid detection and quantification of target analytes in samples, has become a highly anticipated research area in mass spectrometry. AIMS is a "soft ionization" technique that ionizes neutral particles through photoradiation. It features fast analysis speed, low sample consumption, high sensitivity, no need for sample pretreatment, high ionization efficiency, and few fragment ions. Compared to desorption electrospray ionization (DESI), it also offers advantages such as no ionization polarity discrimination and weak matrix effects. Therefore, AIMS has been commercially available as an ion source coupled with liquid chromatography or gas chromatography.

[0004] However, in the high background gas pressure environment of DESI / PI, collisions between ions and gas molecules dominate the ion trajectory, forcing the beam radius to continuously expand and resulting in ion loss. On the other hand, during the transmission of desorbed and ionized ions to the high-vacuum mass analyzer for detection and analysis in low-vacuum or atmospheric pressure environments, beam expansion occurs due to rapid changes in gas pressure. Therefore, it is necessary to utilize ion guiding devices to efficiently control the ion trajectory in different vacuum regions, reduce ion loss, and thus improve ion transmission efficiency and instrument sensitivity. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one objective of this invention is to propose a desorption electrospray and secondary photoionization mass spectrometry imaging device and its operating method that combines an ion funnel. By leveraging the high spatial resolution of desorption electrospray ionization, and improving ion ionization efficiency through post-photoionization technology and low-vacuum transmission efficiency through the ion funnel, the invention enhances instrument sensitivity, thereby exploring higher imaging spatial resolution and achieving superior imaging quality.

[0006] In a first aspect, the present invention proposes a desorption electrospray and secondary photoionization mass spectrometry imaging device combining an ion funnel, comprising a dual-source secondary ionization component, a dual-stage ion funnel, a desorption electrospray ionization source, and a three-degree-of-freedom displacement imaging platform for mounting a glass slide containing the analyte. The desorption electrospray ionization source is located above the three-degree-of-freedom displacement imaging platform on one side of the glass slide. The desorption electrospray ionization source is used to perform desorption electrospray ionization on the surface of the analyte within the glass slide above the three-degree-of-freedom displacement imaging platform, generating molecular-ion droplet clusters containing ions and neutral molecules. An electrospray sampling tube for sampling the molecular-ion droplet clusters is provided at the inlet of the secondary ionization component, and the electrospray sampling tube is located above the three-degree-of-freedom displacement imaging platform on the other side of the glass slide. The outlet of the dual-source secondary ionization component is connected to the inlet of the dual-stage ion funnel, and a mass spectrometry analysis device is externally connected to the outlet of the dual-stage ion funnel.

[0007] Preferably, the dual-source secondary ionization assembly includes a photoionization tube, the outlet of which is connected to the inlet of the bi-stage ion funnel. A synchrotron radiation light transmission tube is provided on the side of the photoionization tube away from the bi-stage ion funnel. A vacuum window for maintaining the vacuum level of the synchrotron radiation light transmission tube is provided between the synchrotron radiation light transmission tube and the photoionization tube. A synchrotron radiation light interface is provided at the end of the synchrotron radiation light transmission tube away from the photoionization tube. An electrospray sampling tube is located on the side wall of the photoionization tube near the end of the synchrotron radiation light transmission tube. A vacuum ultraviolet lamp is provided on the opposite side of the photoionization tube. A dopant introduction tube is also provided near the electrospray sampling tube of the photoionization tube.

[0008] Preferably, the dual-stage ion funnel includes a high-pressure ion funnel and a low-pressure ion funnel. The outlet of the high-pressure ion funnel is connected to the inlet of the low-pressure ion funnel. The outlet of the low-pressure ion funnel is externally connected to the mass spectrometry analysis device. A sample inlet capillary is provided on the side wall of the high-pressure ion funnel away from the low-pressure ion funnel. The inlet of the sample inlet capillary is connected to the outlet of the dual-stage ion funnel. The outlet of the sample inlet capillary is connected to the interior of the high-pressure ion funnel. A vacuum port for connecting an external vacuum device is provided on the side wall of the high-pressure ion funnel away from the sample inlet capillary.

[0009] Preferably, a sample inlet capillary heating cone is provided on the outside of the sample inlet capillary, and a sample inlet heating assembly is provided inside the sample inlet capillary heating cone near the sample inlet capillary.

[0010] Preferably, the three-degree-of-freedom displacement imaging platform includes a first stepper motor for driving the glass slide carrying the object to be measured to move along the X direction, a second stepper motor for driving the glass slide carrying the object to be measured to move along the Y direction, and a manual adjustment mechanism for adjusting the height of the glass slide carrying the object to be measured in the Z direction. The first stepper motor and the second stepper motor are controlled by a control program to realize mass spectrometry imaging sampling of the object to be measured.

[0011] Preferably, the desorption electrospray ionization source includes an inner layer of a liquid-conducting capillary and an outer layer of a spray needle. The liquid-conducting capillary and the spray needle are coaxially mounted. The inlet of the liquid-conducting capillary is used to introduce the electrospray solution and apply a high voltage. The outlet of the inner liquid-conducting capillary is located near the outlet of the outer spray needle. High-pressure gas is introduced into the interior of the outer spray needle. Under the action of the high-pressure gas, the liquid flowing out of the inner liquid-conducting capillary is atomized into charged droplets and sprayed onto the surface of the object to be tested.

[0012] Preferably, the outlet of the dual-stage ion funnel is provided with a standardized interface for mass spectrometry instruments.

[0013] Preferably, a water-cooling assembly is provided on the outside of the synchrotron radiation optical transmission tube near the vacuum window. The water-cooling assembly has a water-cooling cavity inside. The outside of the water-cooling assembly is provided with a cooling water inlet pipe for introducing cooling water into the water-cooling cavity and a cooling water outlet pipe for leading out the cooling water after it has absorbed heat from the water-cooling cavity.

[0014] Secondly, the present invention proposes a method for operating a desorption electrospray and secondary photoionization mass spectrometry imaging device combining an ion funnel, comprising any one of the above-mentioned schemes of the desorption electrospray and secondary photoionization mass spectrometry imaging device combining an ion funnel, and the steps of the method are as follows:

[0015] S1: Fix the glass slide carrying the test object onto the three-degree-of-freedom displacement imaging platform, and adjust the height in the Z direction so that the sample surface is in the optimal spray position of the desorption electrospray ionization source;

[0016] S2: Electrospray solution is introduced into the inner liquid-conducting capillary of the desorption electrospray ionization source and a high voltage is applied. At the same time, high-pressure gas is introduced into the outer spray needle to generate desorption electrospray ionization on the surface of the test object and form molecular-ion droplet clusters.

[0017] S3: Molecular-ion droplet clusters are introduced into the photoionization tube through an electrospray sampling tube, and gaseous dopant is introduced into the photoionization tube through a dopant introduction tube;

[0018] S4: Select the working mode of the dual-source secondary ionization component. When working mode one is selected, execute step S5; when working mode two is selected, execute step S6; when working mode three is selected, execute step S7.

[0019] S5: Turn on only the vacuum ultraviolet lamp and turn off the synchrotron radiation light, so that the molecular-ion droplet clusters can complete the secondary ionization under the vacuum ultraviolet lamp, and proceed to step S8;

[0020] S6: Introduce only synchrotron radiation light, turn off the vacuum ultraviolet lamp, so that the molecular-ion droplet clusters can complete secondary ionization under synchrotron radiation light, and proceed to step S8;

[0021] S7: Turn on the vacuum ultraviolet lamp and introduce synchrotron radiation light at the same time, so that the molecular-ion droplet clusters can complete the secondary ionization, and proceed to step S8;

[0022] S8: The ion beam generated by the secondary photoionization is focused sequentially through a high-pressure ion funnel and a low-pressure ion funnel and then introduced into the mass spectrometry analysis device for mass spectrometry analysis.

[0023] S9: Under the drive of the three-degree-of-freedom displacement imaging platform, the analyte is scanned at a predetermined step distance, and imaging reconstruction is performed based on the mass spectrometry data at different scanning positions to obtain the spatial distribution of the target analyte.

[0024] The beneficial effects of this invention are:

[0025] (1) It can achieve rapid switching or simultaneous use of two photoionization sources: synchrotron radiation light and vacuum ultraviolet lamp. Synchrotron radiation photoionization sources have advantages such as high luminous flux, adjustable photon energy, and high collimation, but the facilities are fixed and the experimental flexibility is limited; vacuum ultraviolet lamps have advantages such as simple operation and flexibility. Therefore: (1) This invention can meet the needs of different experiments by rapidly switching the secondary ionization source, and the switching process will not produce unknown operational variables; (2) When vacuum ultraviolet lamps and synchrotron radiation are used simultaneously, a larger photoionization area can be obtained, further improving the photoionization efficiency;

[0026] (2) Compared with photoionization mass spectrometry imaging devices that combine desorption electrospray ionization: (1) Synchrotron radiation has higher brightness and higher flux, up to a maximum of 10 16 (1) Photon / s significantly improves the photoionization efficiency of the analyte; (2) Synchrotron radiation energy is continuously adjustable, allowing for the selection of different energies for different compounds, achieving near-threshold ionization of the analyte and avoiding the generation of a large number of fragment ions due to high photon energy; (3) Synchrotron radiation has a wide spectral range and continuously adjustable energy, enabling selective ionization of the analyte, thus achieving imaging of a specific substance; (4) For compounds with the same mass-to-charge ratio, the photoionization efficiency (PIE) spectrum can be scanned, and isomers can be identified and complex components can be analyzed by fitting the photoionization cross-section (PICS) curve;

[0027] (3) The dual-stage ion funnel used has the following advantages: (1) The dual-stage funnel can sample at higher pressures and significantly reduce ion loss, achieving efficient ion transport and focusing under rough vacuum conditions, improving ion transport efficiency and instrument sensitivity. (2) Sample molecule droplet clusters enter the high-pressure ion funnel in an orthogonal direction with the airflow, which can prevent neutral gas molecules from entering the subsequent vacuum chamber and reduce the gas pressure accumulation at the funnel outlet. The eddy current generated at the electrode edge is conducive to the desolvation of the sample droplets and improves instrument sensitivity. (3) The high-pressure ion funnel has a large inner diameter electrode, which can accommodate more ions and reduce ion loss caused by space charge effect. The low-pressure ion funnel has a smaller inner diameter and outlet size, which can modulate ions into a more concentrated ion beam, which is conducive to improving instrument resolution. (4) The PCB material ion funnel electrode has a lower cost and is easy to disassemble and assemble, which can reduce the overall cost of the equipment and simplify the assembly and operation process.

[0028] (4) Combining secondary ionization technology, which improves ionization efficiency, with ion funnel technology with high ion transport efficiency, improves imaging sensitivity from two directions: increasing ion yield and reducing ion loss. This solves the problem of signal intensity decline caused by the improvement of imaging spatial resolution and optimizes instrument stability, reliability and imaging quality.

[0029] (5) Compared with the solution dopant introduction method, the introduction of gas phase dopant can: (1) make the dopant mix more uniformly with the sample gas molecules, improve the ionization efficiency and the molecular ion reaction efficiency between the two; (2) avoid the competitive ionization of the analyte by the solvent introduced by the droplet dopant, and reduce the cluster ion signal peak formed by the dopant and the sample molecules, thereby improving the signal-to-noise ratio; (3) avoid the contamination of the photoionization tube by the dopant droplets, thereby improving the stability of the instrument; (4) the present invention is usually loaded into a commercial high-resolution orbital trap mass spectrometer for use in conjunction with the gas phase dopant, which has a lower mass-to-charge ratio and reacts fully with the analyte, avoiding the generation of a strong space charge effect in the orbital trap, which affects the volume of the trap. Traditional dopant such as acetone and toluene are more likely to fill the trap, affecting the sensitivity of trace detection. Attached Figure Description

[0030] In the attached diagram:

[0031] Figure 1 This is a schematic diagram of the structure of the desorption electrospray and secondary photoionization mass spectrometry imaging device combining an ion funnel proposed in this invention.

[0032] Figure 2 The present invention proposes Figure 1 A sectional view;

[0033] Figure 3 This is a partially enlarged view of the dual-source secondary ionization assembly proposed in this invention;

[0034] Figure 4 The present invention proposes Figure 3 A sectional view;

[0035] Figure 5 The images show positive ion mode mass spectra obtained from desorption electrospray ionization (DESI) and desorption electrospray ionization-secondary ionization (DESI / PI) mass spectrometry analyses proposed in this invention.

[0036] in, Figure 5 (a) indicates using only DESI mode. Figure 5 (b) in the diagram represents the DESI / PI mode using toluene as a solution dopant. Figure 5 (c) in the figure represents the DESI / PI mode using propylene as a vapor phase dopant;

[0037] Figure 6 The distribution diagram of choline (m / z 104) and cholesterol (m / z 369) in mouse brain homogenate obtained by desorption electrospray ionization (DESI) and desorption electrospray ionization-secondary ionization (DESI / PI) mass spectrometry analysis proposed in this invention is shown.

[0038] in, Figure 6(a), (c), and (e) in the image show mass spectrometry images of choline at m / z 104.107;

[0039] Figure 6 The cholesterol mass spectrometry images at m / z 369.36 shown in (b), (d), and (f) are as follows;

[0040] Figure 6 (a) and (b) in the examples only use DESI mode. Figure 6 (c) and (d) in the diagram use the DESI / PI mode with toluene as the solution dopant. Figure 6 (e) and (f) in the diagram use the DESI / PI mode with propylene as the gas phase dopant.

[0041] In the figure: 1-Dual-source secondary ionization assembly, 2-Sample injection capillary heating cone, 3-Instrument standardization interface, 4-Dual-stage ion funnel, 5-Desorption electrospray ionization source, 6-Three-degree-of-freedom displacement imaging platform;

[0042] 11-Vacuum UV lamp, 12-Dopant introduction tube, 13-External hexagonal cap, 14-External hexagonal boss, 15-Electrospray sampling tube, 16-Water cooling assembly, 17-Cooling water inlet pipe, 18-Wrench slot, 19-Synchrotron radiation optical interface;

[0043] 41-Vacuum extraction port, 42-High-pressure ion funnel, 43-Low-pressure ion funnel, 44-Sample inlet heating assembly, 45-Sample inlet capillary;

[0044] 110-Cooling water outlet pipe, 111-Vacuum window, 112-Sample injection sealing graphite, 113-UV lamp sealing graphite, 114-Synchrotron radiation light transmission tube, 115-Photoionization tube. Detailed Implementation

[0045] Reference Figure 1 and Figure 3A desorption electrospray and secondary photoionization mass spectrometry imaging device combining an ion funnel includes a dual-source secondary ionization component 1, a dual-stage ion funnel 4, a desorption electrospray ionization source 5, and a three-degree-of-freedom displacement imaging platform 6 for mounting a glass slide containing the analyte. The desorption electrospray ionization source 5 is located above the three-degree-of-freedom displacement imaging platform 6 on one side of the glass slide. The desorption electrospray ionization source 5 is used to perform desorption electrospray ionization on the surface of the analyte within the glass slide above the three-degree-of-freedom displacement imaging platform 6, generating molecular-ion droplet clusters containing ions and neutral molecules. An electrospray sampling tube 15 for sampling the molecular-ion droplet clusters is provided at the inlet of the secondary ionization component 1. The electrospray sampling tube 15 is located above the three-degree-of-freedom displacement imaging platform 6 on the other side of the glass slide. The outlet of the dual-source secondary ionization component 1 is connected to the inlet of the dual-stage ion funnel 4, and a mass spectrometry analysis device is externally connected to the outlet of the dual-stage ion funnel 4.

[0046] Obviously, based on the above: by placing the glass slide on the three-degree-of-freedom displacement imaging platform 6 and adjusting the platform position, the desorption electrospray ionization source 5 is arranged on one side of the glass slide to perform desorption electrospray ionization on the surface of the analyte, forming molecular-ion droplet clusters containing ions and neutral molecules on the surface of the analyte; the electrospray sampling tube 15 located on the other side of the glass slide samples the above molecular-ion droplet clusters and introduces them into the dual-source secondary ionization component 1. In this component, the sampled molecular-ion clusters are further ionized and transported. Then, the ions obtained are sent from the outlet of the dual-source secondary ionization component 1 into the dual-stage ion funnel 4. After being guided, focused and transported step by step by the dual-stage ion funnel 4, they are output to the external mass spectrometry analysis equipment to complete the mass analysis. The above arrangement allows for two advantages. First, the desorption electrospray ionization source 5 and the electrospray sampling tube 15 are positioned opposite each other on the glass slide, which facilitates the formation of stable and repeatable molecular-ion droplet clusters on the surface of the analyte and timely collection of sample information. Second, the series connection between the dual-source secondary ionization component 1 and the dual-stage ion funnel 4 enables ions generated from the sample surface and entering the system through sampling to be effectively collected and transported to the mass spectrometry analysis equipment within a large pressure gradient range. This improves the ionization utilization rate and ion transport efficiency of the analyte, providing a better signal foundation and spatial resolution conditions for subsequent mass spectrometry imaging.

[0047] In this embodiment, refer to Figure 3 and Figure 4The dual-source secondary ionization assembly 1 includes a photoionization tube 115. The outlet of the photoionization tube 115 is connected to the inlet of the dual-stage ion funnel 4. A synchrotron radiation light transmission tube 114 is provided on the side of the photoionization tube 115 away from the dual-stage ion funnel 4. A vacuum window 111 is provided between the synchrotron radiation light transmission tube 114 and the photoionization tube 115 to maintain the vacuum level of the synchrotron radiation light transmission tube 114. A synchrotron radiation light interface 19 is provided at the end of the synchrotron radiation light transmission tube 114 away from the photoionization tube 115. An electrospray sampling tube 15 is provided on the side wall of the photoionization tube 115 near the end of the synchrotron radiation light transmission tube 114. A vacuum ultraviolet lamp 11 is provided on the opposite side of the photoionization tube 115. A dopant introduction tube 12 is also provided near the electrospray sampling tube 15 of the photoionization tube 115.

[0048] Specifically, the molecular-ion droplet clusters generated by the desorption electrospray ionization source 5 enter the photoionization tube 115 with an inner diameter of 7 mm through the electrospray sampling tube 15 with an inner diameter of 2 mm. The gaseous dopant enters the photoionization tube 115 through the dopant introduction tube 12 with an inner diameter of 1 mm and mixes evenly with the droplet clusters. The dopant is one of propylene or 1,3-butadiene. An electric heating wire is wound around the outside of the photoionization tube 115 to heat the internal gas to approximately 300°C, preventing condensation of the gaseous sample and dopant on the wall surface and enhancing the reactivity of the molecular ions. A vacuum ultraviolet lamp 11 emits photons at the sample entry point, serving as one of the light sources for secondary ionization. It is sealed to the photoionization tube 115 via an ultraviolet lamp sealing graphite 113. A synchrotron radiation light source is connected via a KF 25 standard flange synchrotron radiation light interface 19, transmitting the synchrotron radiation light axially into the synchrotron radiation light transmission tube 114, serving as another light source for secondary ionization. The synchrotron radiation light transmission tube 114 and the photoionization tube 115 are connected by threads and tightened with a wrench at the external hexagonal boss 14 and the wrench slot 18. A 1mm thick vacuum window 111 is installed between the synchrotron radiation light transmission tube 114 and the photoionization tube 115 to maintain the 10mm thickness of the synchrotron radiation light transmission tube 114. -3 A vacuum on the order of Pa is achieved, enabling high-efficiency synchrotron radiation light transmission. The window 111 is made of magnesium fluoride (Mg2F) or lithium fluoride (LiF). The window 111 is vacuum-sealed with the synchrotron radiation light transmission tube 114 and the photoionization tube 115 by extruding a silicone gasket. The dual-source secondary ionization device 1 is connected to the sample introduction capillary heating cone 2 of the bi-stage ion funnel 4 via a sample introduction sealing graphite 112.

[0049] Clearly, based on the above, the above structural arrangement achieves thorough mixing and secondary ionization of the gaseous dopant and droplet clusters, which is beneficial to improving the ionization efficiency of the analyte and enhancing signal stability. On the other hand, the coaxial integration of a vacuum ultraviolet lamp 11 and a synchrotron radiation light source within the same photoionization tube 115 while maintaining the vacuum environment of the synchrotron radiation light transmission tube 114 facilitates flexible implementation of secondary ionization under different light source modes or in a combined mode. It also takes into account high light flux, good vacuum performance, and convenient mechanical connections for disassembly and assembly, thereby improving the overall adaptability and reliability of the mass spectrometry imaging device.

[0050] In this embodiment, refer to Figure 2 The dual-stage ion funnel 4 includes a high-pressure ion funnel 42 and a low-pressure ion funnel 43. The outlet of the high-pressure ion funnel 42 is connected to the inlet of the low-pressure ion funnel 43. The outlet of the low-pressure ion funnel 43 is connected to an external mass spectrometry analysis device. A sample inlet capillary 45 is provided on the side wall of the high-pressure ion funnel 42 away from the low-pressure ion funnel 43. The inlet of the sample inlet capillary 45 is connected to the outlet of the dual-stage ion funnel 4, and the outlet of the sample inlet capillary 45 is connected to the interior of the high-pressure ion funnel 42. A vacuum evacuation port 41 for connecting an external vacuum evacuation device is provided on the side wall of the high-pressure ion funnel 42 away from the sample inlet capillary 45.

[0051] Specifically, ions generated in the photoionization tube 115 are introduced into the vacuum environment of the two-stage ion funnel 4 via the sample introduction capillary 45. The main structure of the ion funnel consists of a high-pressure ion funnel 42 and a low-pressure ion funnel 43. During operation, the radio frequency parameters of the two-stage ion funnel are 0.74MHz and 100Vpp. An external vacuum pump is connected to the vacuum extraction port 41 to maintain a vacuum level of 0.7~22 Torr inside the high-pressure ion funnel 42. The low-pressure ion funnel 43 is partially installed inside the vacuum chamber of the commercial instrument, where the vacuum level is maintained at 1~2 mbar. The ions entering the ion funnel are affected by the combined action of the airflow and the electric field. The gaseous sample is ejected radially from the end of the sample introduction capillary 45. Due to the pressure difference, a supersonic jet is generated. Since the molecular part is uncharged, it is swept radially by the fluid to the opposite pump port and then removed. Furthermore, the fluid is disturbed near the electrode edge, generating vortices, which enhances the desolvation effect. The ions are deflected by the electric field and continue to be focused and transported downstream along the axial direction. The high-pressure ion funnel 42 has a larger electrode inner diameter, which can accommodate more ions and reduce the negative impact of space charge effect on ion transport. The larger diameter of the funnel's conical outlet reduces the radial potential well depth, allowing more ions to pass through and widening the lower limit of permissible transport quality, resulting in higher ion transport efficiency. In the low-pressure ion funnel 43, the electrode inner diameter is reduced and the number of electrodes with equal inner diameters is increased, thus maintaining high collimation of the ion beam. The smaller outlet diameter further focuses the ion beam to a smaller size, resulting in less spatial and energy dispersion, improving the transport efficiency of the subsequent ion optical system and increasing instrument resolution.

[0052] In this embodiment, refer to Figure 1 and Figure 2 A sample inlet capillary heating cone 2 is provided on the outside of the sample inlet capillary 45, and a sample inlet heating assembly 44 is provided inside the sample inlet capillary heating cone 2 near the sample inlet capillary 45.

[0053] Specifically, the injection tube heating assembly 44 is used to heat the injection tube, thereby preventing ion clusters from condensing and adsorbing on the metal tube wall and achieving the effect of desolventizing.

[0054] In this embodiment, the three-degree-of-freedom displacement imaging platform 6 includes a first stepper motor for driving the glass slide carrying the object to be measured to move along the X direction, a second stepper motor for driving the glass slide carrying the object to be measured to move along the Y direction, and a manual adjustment mechanism for adjusting the height of the glass slide carrying the object to be measured in the Z direction. The first stepper motor and the second stepper motor are controlled by a control program to realize mass spectrometry imaging sampling of the object to be measured.

[0055] Specifically, during operation, the glass slide carrying the sample is placed on the stage. The vertical height is adjusted by manually rotating the Z-axis adjustment screw, controlling the distance between the sample and the electrospray sampling tube 15 to optimize ion sampling efficiency. A control program is written in LabVIEW to realize the manual adjustment of the X and Y axis stepper motors and the spectral acquisition logic control, thereby performing mass spectrometry imaging scanning sampling.

[0056] In this embodiment, refer to Figure 1 The desorption electrospray ionization source 5 includes an inner layer of a liquid-conducting capillary and an outer layer of a spray needle. The liquid-conducting capillary and the spray needle are coaxially mounted. The inlet of the liquid-conducting capillary is used to introduce the electrospray solution and apply a high voltage. The outlet of the inner liquid-conducting capillary is located near the outlet of the outer spray needle. High-pressure gas is introduced into the interior of the outer spray needle. Under the action of the high-pressure gas, the liquid flowing out of the inner liquid-conducting capillary is atomized into charged droplets and sprayed onto the surface of the object to be tested.

[0057] Specifically, an electrospray solution is introduced into the inner capillary at a flow rate of 0.18 L / min, and a voltage of +3500V positive ion mode or -5000V negative ion mode is applied to charge the solution. High-pressure nitrogen gas of 0.4 MPa is introduced into the nozzle on the outside of the capillary. During operation, the high-pressure nitrogen gas is ejected through the nozzle outlet, and the charged liquid flows out through the front end of the capillary and is atomized under the action of the high-pressure nitrogen gas.

[0058] In this embodiment, refer to Figure 1 The outlet of the dual-stage ion funnel 4 is equipped with a standardized interface 3 for mass spectrometry instruments.

[0059] Clearly, based on the above, the interface compatibility and electrical connection issues between the modular two-stage ion funnel device and commercial instruments can be resolved through standardized interfaces, thus enabling it to work in conjunction with commercial instruments.

[0060] In this embodiment, refer to Figure 4 A water-cooling assembly 16 is provided on the outside of the synchrotron radiation optical transmission tube 114 near the vacuum window 111. A water-cooling cavity is provided inside the water-cooling assembly 16. A cooling water inlet pipe 17 for introducing cooling water into the water-cooling cavity and a cooling water outlet pipe 110 for leading out the cooling water after it has absorbed heat from the water-cooling cavity are provided on the outside of the water-cooling assembly 16.

[0061] Specifically, the vacuum window 111 is not resistant to high temperatures, and the vacuum window 111 is cooled by a cooling water circulation formed by the water cooling component 16.

[0062] As another embodiment of this application, this embodiment proposes a working method of a desorption electrospray and secondary photoionization mass spectrometry imaging device that combines an ion funnel, including any one of the above-mentioned desorption electrospray and secondary photoionization mass spectrometry imaging devices that combine an ion funnel.

[0063] The working method and steps are as follows:

[0064] S1: Fix the glass slide carrying the test object onto the three-degree-of-freedom displacement imaging platform 6, and adjust the height in the Z direction so that the sample surface is in the optimal spray position of the desorption electrospray ionization source 5;

[0065] S2: Electrospray solution is introduced into the inner liquid-conducting capillary of the desorption electrospray ionization source 5 and a high voltage is applied. At the same time, high-pressure gas is introduced into the outer spray needle to generate desorption electrospray ionization on the surface of the analyte and form molecular-ion droplet clusters.

[0066] S3: Molecular-ion droplet clusters are introduced into photoionization tube 115 through electrospray sampling tube 15, and gaseous dopant is introduced into photoionization tube 115 through dopant introduction tube 12.

[0067] S4: Select the working mode of the dual-light source secondary ionization component 1. When working mode one is selected, execute step S5; when working mode two is selected, execute step S6; when working mode three is selected, execute step S7.

[0068] S5: Turn on only the vacuum ultraviolet lamp 11 and turn off the synchrotron radiation light, so that the molecular-ion droplet clusters can complete the secondary ionization under the vacuum ultraviolet lamp, and proceed to step S8;

[0069] S6: Introduce only synchrotron radiation light, turn off vacuum ultraviolet lamp 11, so that the molecular-ion droplet clusters can complete secondary ionization under synchrotron radiation light, and proceed to step S8;

[0070] S7: Turn on the vacuum ultraviolet lamp 11 and introduce synchrotron radiation light to make the molecular-ion droplet clusters complete the secondary ionization, and proceed to step S8;

[0071] S8: The ion beam generated by the secondary photoionization is focused sequentially through the high-pressure ion funnel 42 and the low-pressure ion funnel 43 and then introduced into the mass spectrometry analysis equipment for mass spectrometry analysis.

[0072] S9: The analyte is scanned at a predetermined step distance under the drive of the three-degree-of-freedom displacement imaging platform 6, and imaging reconstruction is performed based on the mass spectrometry data at different scanning positions to obtain the spatial distribution of the target analyte.

[0073] Clearly, based on the above, this method, while maintaining the high spatial resolution advantage of desorption electrospray ionization, introduces switchable dual-source secondary ionization and gas-phase doping, combined with the efficient ion transport of the two-stage ion funnel, to achieve sensitive detection of non-polar and weakly polar components and high ion utilization, which is beneficial for obtaining mass spectrometry imaging results with good signal-to-noise ratio and rich imaging details.

[0074] To more clearly illustrate the implementation plan and its effects, we will use the attached diagram as an example:

[0075] See attached document Figure 5 This invention compares mass spectra under three different mass spectrometry modes:

[0076] (1) Use only DESI mode ( Figure 5 (a));

[0077] (2) DESI / PI mode using toluene as a solution dopant ( Figure 5 (b));

[0078] (3) The DESI / PI mode proposed in this invention using propylene as a gas-phase dopant ( Figure 5 (c)).

[0079] Experimental results show that, compared with the traditional DESI mode, the DESI / PI mode with added dopants (including solution doping and gas-phase doping) significantly improves the richness of mass spectrometry information and significantly enhances the signal intensity of target substances such as lipids and sterols. Secondly, compared with the traditional toluene solution dopant, the use of propylene as a gas-phase dopant in this invention further significantly improves the signal intensity of lipid substances. For example, the PC(34:1) signal intensity at m / z 760.58 is increased from approximately... Upgraded to more than This demonstrates that the gas-phase introduction method enables more uniform mixing of the dopant with the sample and a more complete reaction, thereby achieving higher ionization efficiency and signal intensity.

[0080] See attached document Figure 6 This invention further compares mass spectrometry images in three modes:

[0081] (1) Use only DESI mode ( Figure 6 (a), (b));

[0082] (2) DESI / PI mode using toluene as a solution dopant ( Figure 6 (c), (d));

[0083] (3) The DESI / PI mode proposed in this invention using propylene as a gas-phase dopant ( Figure 6 (e), (f)).

[0084] Specifically: Figure 6 Images (a), (c), and (e) show mass spectrometry images of choline at m / z 104.107. Due to the high ionization efficiency of DESI for strongly polar compounds, the choline signal is already quite strong in DESI mode. Figure 6The cholesterol mass spectrometry images at m / z 369.36 shown in (b), (d), and (f) demonstrate that cholesterol, being a weakly polar compound, suffers from low ionization efficiency in the traditional DESI mode, resulting in weak signal intensity. However, the DESI / PI mode, by introducing secondary photoionization, achieves efficient ionization of both polar and non-polar substances, thus significantly enhancing the cholesterol signal. Furthermore, compared to using the solution dopant toluene, the use of the gaseous dopant propylene in this invention further increases the cholesterol signal intensity by approximately two times. This again verifies the significant advantages of gaseous dopant in improving substance ionization efficiency and imaging signal intensity.

Claims

1. A desorption electrospray and secondary photoionization mass spectrometry imaging device coupled with an ion funnel, characterized by: The device comprises a double light source secondary ionization component (1), a double-stage ion funnel (4), a desorption electrospray ionization source (5) and a three-degree-of-freedom displacement imaging platform (6) for carrying the sample slide to be measured, the desorption electrospray ionization source (5) is arranged above the three-degree-of-freedom displacement imaging platform (6) on one side of the sample slide, the desorption electrospray ionization source (5) is used for desorption electrospray ionization of the surface of the sample in the sample slide above the three-degree-of-freedom displacement imaging platform (6), to generate molecular-ion droplet clusters containing ions and neutral molecules, an electrospray sampling tube (15) for sampling the molecular-ion droplet clusters is arranged at the inlet of the light source secondary ionization component (1), the electrospray sampling tube (15) is arranged above the three-degree-of-freedom displacement imaging platform (6) on the other side of the sample slide, the outlet of the double light source secondary ionization component (1) is connected with the inlet of the double-stage ion funnel (4), and the outlet of the double-stage ion funnel (4) is externally connected with a mass spectrometry device.

2. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 1, wherein: The double light source secondary ionization component (1) comprises a photoionization tube (115), the outlet of the photoionization tube (115) is connected with the inlet of the double-stage ion funnel (4), one side of the photoionization tube (115) away from the double-stage ion funnel (4) is provided with a synchrotron radiation light transmission tube (114), a vacuum window (111) for maintaining the vacuum degree of the synchrotron radiation light transmission tube (114) is arranged between the synchrotron radiation light transmission tube (114) and the photoionization tube (115), one end of the synchrotron radiation light transmission tube (114) away from the photoionization tube (115) is provided with a synchrotron radiation light interface (19), the electrospray sampling tube (15) is arranged at the side wall of the photoionization tube (115) close to one end of the synchrotron radiation light transmission tube (114), the photoionization tube (115) is provided with a vacuum ultraviolet lamp (11) on the side opposite to the electrospray sampling tube (15), and the photoionization tube (115) is also provided with a dopant introduction tube (12) close to the electrospray sampling tube (15).

3. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 1, wherein: The double-stage ion funnel (4) comprises a high-pressure ion funnel (42) and a low-pressure ion funnel (43), the outlet of the high-pressure ion funnel (42) is connected with the inlet of the low-pressure ion funnel (43), the outlet of the low-pressure ion funnel (43) is externally connected with the mass spectrometry device, one end of the high-pressure ion funnel (42) away from the low-pressure ion funnel (43) is provided with a sampling capillary (45), the inlet of the sampling capillary (45) is in communication with the outlet of the double-stage ion funnel (4), the outlet of the sampling capillary (45) is in communication with the inside of the high-pressure ion funnel (42), and a vacuum air outlet (41) for externally connecting a vacuum pumping device is arranged on the side wall of the high-pressure ion funnel (42) away from the sampling capillary (45).

4. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 3, wherein: The outside of the sample injection capillary (45) is provided with a sample injection capillary heating cone (2), and the inside of the sample injection capillary heating cone (2) is provided with a sample injection capillary heating assembly (44) near the position of the sample injection capillary (45).

5. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 1, wherein: The three-degree-of-freedom displacement imaging platform (6) comprises a first stepper motor for driving the measured object slide to move in the X direction, a second stepper motor for driving the measured object slide to move in the Y direction, and a manual adjusting mechanism for adjusting the height of the measured object slide in the Z direction, and the first stepper motor and the second stepper motor are controlled by a control program to realize mass spectrum imaging sampling of the measured object.

6. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 1, wherein: The desorption electrospray ionization source (5) comprises an inner liquid guide capillary and an outer spray needle, the liquid guide capillary and the spray needle are coaxially installed, the inlet of the liquid guide capillary is used for introducing electrospray solution and applying high voltage, the outlet of the inner liquid guide capillary is close to the outlet position of the outer spray needle, and high-pressure gas is introduced into the inner part of the outer spray needle, so that the liquid flowing out of the inner liquid guide capillary is atomized into charged droplets under the action of the high-pressure gas and sprayed onto the surface of the measured object.

7. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 1, wherein: The outlet of the double-stage ion funnel (4) is provided with a mass spectrometer standard interface (3).

8. The desorption electrospray and secondary photoionization mass spectrometry imaging device incorporating an ion funnel according to claim 2, wherein: The outside of the synchrotron radiation light transmission tube (114) is provided with a water cooling assembly (16) near the position of the vacuum window sheet (111), the inside of the water cooling assembly (16) is provided with a water cooling cavity, and the outside of the water cooling assembly (16) is provided with a cooling water inlet pipe (17) for introducing cooling water into the water cooling cavity and a cooling water outlet pipe (110) for leading out the cooling water after absorbing heat in the water cooling cavity.

9. A method of operation of a desorption electrospray and secondary photoionization mass spectrometry imaging apparatus in combination with an ion funnel, characterized by: The working method of the desorption electrospray and secondary photoionization mass spectrum imaging device comprising the combined ion funnel according to any one of claims 1-8 comprises the following steps: S1: fixing the measured object slide on the three-degree-of-freedom displacement imaging platform (6) and adjusting the Z direction height to make the sample surface be at the best spraying position of the desorption electrospray ionization source (5); S2: introducing electrospray solution into the inner liquid guide capillary of the desorption electrospray ionization source (5) and applying high voltage, and introducing high-pressure gas into the outer spray needle, so as to produce desorption electrospray ionization on the surface of the measured object and form molecular-ion droplet clusters; S3: introducing the molecular-ion droplet clusters into the photoionization tube (115) through the electrospray sampling tube (15) and introducing gas-phase dopant into the photoionization tube (115) through the dopant introduction tube (12); S4: selecting the working mode of the double-light-source secondary ionization assembly (1), when mode one is selected, step S5 is executed, when mode two is selected, step S6 is executed, and when mode three is selected, step S7 is executed; S5: only turning on the vacuum ultraviolet lamp (11) and turning off the synchrotron radiation light, so that the molecular-ion droplet clusters complete secondary ionization under the vacuum ultraviolet light, and step S8 is executed; S6: only introducing synchrotron radiation light and turning off the vacuum ultraviolet lamp (11), so that the molecular-ion droplet clusters complete secondary ionization under the synchrotron radiation light, and step S8 is executed; S7: turn on the vacuum ultraviolet lamp (11) and introduce the synchrotron radiation at the same time, so that the molecule-ion droplet cluster completes secondary ionization, and step S8 is performed; S8: the ion beam generated by the secondary photoionization is sequentially focused by the high-pressure ion funnel (42) and the low-pressure ion funnel (43) and then introduced into the mass spectrometry device for mass spectrometry analysis; S9: under the driving of the three-degree-of-freedom displacement imaging platform (6), the measured object is scanned at a predetermined step distance, and imaging reconstruction is performed based on the mass spectrometry data of different scanning positions to obtain the spatial distribution of the target analyte.

Images