Ion source and mass spectrometry imaging system for high resolution mass spectrometry imaging

Abstract

The application relates to an ion source (00) for high-resolution mass spectrum imaging and a mass spectrum imaging system, the ion source (00) comprising a first carrier table (001), a second carrier table (002), an ionization electrode (003), a high-voltage power supply (004), a scanning probe (005), a micro flow pump (006) and a control device (007); the first carrier table (001) is provided with a first holder (011) for clamping a slide (110), and the tissue slice sample (220) on the front surface of the slide (110) clamped by the first holder (011) faces the sample inlet (330) of a mass spectrometer (33); the second carrier table (002) is provided with a second holder (012) for clamping the scanning probe (005), and the scanning probe (005) is connected with the micro flow pump (006) through a conduit (008); the ionization electrode (003) faces the sample inlet (330) of the mass spectrometer (33) and is located on the back surface of the slide (110) clamped by the first holder (011), and the ionization electrode (003) is electrically connected with the high-voltage power supply (004); and the control device (007) is electrically connected with the first carrier table (001), the second carrier table (002), the high-voltage power supply (004) and the micro flow pump (006) respectively. Thus, a picoliter droplet analytical electrospray ion source (00) with higher sensitivity and spatial resolution in mass spectrum imaging technology is realized.

Description

Ion sources and mass spectrometry imaging systems for high-resolution mass spectrometry imaging Technical Field

[0001] This disclosure relates to the field of mass spectrometry imaging, and more particularly to an ion source and a mass spectrometry imaging system for high-resolution mass spectrometry imaging. Background Technology

[0002] Mass spectrometry imaging (MSI) is a novel molecular imaging technique that can visualize and analyze molecules of a specified mass number in a sample region, obtaining the relative abundance and spatial distribution characteristics of analytes on the sample surface. It has become an important tool and a hot topic in clinical medical research. Its principle involves ionizing substances on the sample surface, sending them into a mass spectrometer, obtaining the ionic intensity of the analytes distributed in space, and then reconstructing the spatial distribution information of the analyte molecules in the sample using imaging software. In recent years, MSI has become an important method for research in oncology, disease, pharmacology, drugs, and neuroscience. Compared with other techniques (such as positive ion tomography and magnetic resonance spectroscopy), MSI can directly detect metabolites and lipids related to the occurrence and development of biomedical research subjects without labeling or molecular imprinting. It can obtain multi-point, multi-dimensional, high-throughput, and visualized molecular spatial information, enabling research on tumor types, biomarkers, drug metabolism, and other aspects.

[0003] Mass ionization (MSI) primarily employs three commonly used ion source technologies: Secondary Ion Mass Spectrometry (SIMS), Matrix-Assisted Laser Desorption Ionization (MALDI), and Desorption Electrospray Ionization (DESI). These three ion source technologies differ in their underlying principles, resulting in varying applicability and performance characteristics.

[0004] SIMS primarily utilizes a primary ion beam to bombard the surface of the analyte, then introduces secondary ions sputtered from the surface into the mass analyzer of the mass spectrometer for mass separation and measurement. Its main advantage within MSI is its high detection resolution, reaching approximately 100 nm. However, because the primary ion beam typically has high energy, it easily generates a large number of fragment ions, failing to retain information about complete molecules, resulting in less than satisfactory detection performance for biomolecules.

[0005] MALDI primarily utilizes matrix absorption of laser energy, which is then transferred to sample molecules, allowing them to desorb, evaporate, and ionize under conditions with minimal fragmentation. This offers significant advantages for the detection of biomolecules such as proteins and lipids. However, the MALDI matrix directly affects ionization efficiency, spatial resolution of MSI, and purity.

[0006] SIMS and MALDI require vacuum conditions, making sample handling inconvenient. In contrast, DESI utilizes the impact of atomized charged droplets on the sample surface to generate sputtering, which is then used for mass spectrometry analysis. This DESI technique operates at atmospheric pressure, requires no sample pretreatment, and is suitable for the analysis of metabolites, lipids, and proteins. Currently, DESI used for MSI has insufficient sensitivity and low spatial resolution (~40 μm). Therefore, there is an urgent need for an ion source with higher sensitivity, higher spatial resolution for mass spectrometry imaging, and without the limitations of vacuum conditions.

[0007] Summary of the Invention

[0008] In view of this, this disclosure proposes an ion source and a mass spectrometry imaging system for high-resolution mass spectrometry imaging, which can effectively improve the spatial resolution and sensitivity of mass spectrometry imaging without the limitations of vacuum conditions and sample conditions.

[0009] According to one aspect of this disclosure, an ion source is provided, comprising: a first stage, a second stage, an ionization electrode, a high-voltage power supply, a scanning probe, a microfluidic pump, and a control device; the first stage is provided with a first holder for holding a glass slide, on the front of which a tissue section sample to be analyzed is placed, with the tissue section sample on the front of the glass slide held by the first holder facing the inlet of the mass spectrometer; the second stage is provided with a second holder for holding the scanning probe, the scanning probe being connected to the microfluidic pump via a conduit, the microfluidic pump being used to deliver an extraction solution to the scanning probe via the conduit; the ionization electrode is directly opposite the inlet of the mass spectrometer and located on the back side of the glass slide held by the first holder, the ionization electrode being electrically connected to the high-voltage power supply; the control device is electrically connected to the first stage, the second stage, the high-voltage power supply, and the microfluidic pump respectively; wherein, the control device... The device is configured to: control the first stage to move along a specified path until the current test point on the tissue section sample on the slide is directly opposite the inlet of the mass spectrometer; control the second stage to move until the distance between the tip of the scanning probe and the current test point reaches a specified distance; control the microfluidic pump to deliver extraction liquid to the scanning probe so that the scanning probe outputs a picolinate-volume droplet of extraction liquid to the current test point, and the extraction of the surface material of the current test point is completed after the extraction droplet contacts the current test point; control the high-voltage power supply to apply a specified voltage to the ionization electrode so that the ionization electrode generates an electric field, and the extraction droplet after the surface material extraction is completed flies into the inlet of the mass spectrometer under the action of the electric field force of the electric field, so that the mass spectrometer outputs the mass spectrum corresponding to the current test point based on the ions ionized from the surface material extracted in the flying extraction droplet.

[0010] In one possible implementation, the ion source further includes a camera and an impedance measurement device. The camera faces the tissue section sample and the scanning probe, and the impedance measurement device is connected to the scanning probe and used to measure the resistance value between the tip of the scanning probe and the surface of the test point. The control device is electrically connected to both the camera and the impedance measurement device. The step of controlling the second stage to move until the distance between the tip of the scanning probe and the current test point reaches a specified distance includes: controlling the camera to acquire a real-world image of the scanning probe and the tissue section sample; and analyzing the real-world image of the tip of the scanning probe and the tissue section... The relative positions between samples are controlled to move the second stage until the tip of the scanning probe is aligned with the current test point on the tissue section sample; the impedance measurement device is controlled to measure the current resistance value between the tip of the scanning probe and the surface of the current test point; according to a preset resistance-distance mapping relationship, the current distance corresponding to the current resistance value is determined, whereby the resistance-distance mapping relationship characterizes the mapping relationship between the resistance value and the distance between the tip of the scanning probe and the surface of the test point; based on the difference between the current distance corresponding to the current resistance value and the specified distance, the second stage is controlled to move until the distance between the tip of the scanning probe and the current test point reaches the specified distance.

[0011] In one possible implementation, controlling the microfluidic pump to deliver extractant to the scanning probe includes: controlling the microfluidic pump to continuously deliver extractant to the scanning probe, or controlling the microfluidic pump to deliver extractant to the scanning probe intermittently; wherein, the impedance measuring device measures the resistance between the tip of the scanning probe and the surface of the test point by applying a voltage to the tip of the scanning probe and measuring the current value between the tip of the scanning probe and the surface of the test point; when controlling the microfluidic pump to continuously deliver extractant to the scanning probe, the control device is further configured to: alternately control the voltage applied by the impedance measuring device and the voltage applied by the high-voltage power supply in a timing sequence.

[0012] In one possible implementation, the microfluidic pump includes a microsyringe and a driving device. The microsyringe includes a needle and a piston, the piston being fixed to the driving device. The needle stores an extractant and is connected to the scanning probe via a conduit. Controlling the microfluidic pump to deliver the extractant to the scanning probe includes controlling the driving device to drive the piston to move at a specified speed for a specified duration, thereby delivering a specified volume of extractant to the scanning probe, the specified volume including a picoliter volume.

[0013] In one possible implementation, the control device is further configured to: acquire a visible light image of the tissue slice sample; in response to marking at least one region of interest on the visible light image, divide each region of interest in the marked at least one region of interest into multiple test points according to a preset resolution; wherein the preset resolution includes subcellular or cellular resolution, and the preset resolution is lower than the movement resolution of the first stage; determine the movement path based on the multiple test points divided from each region of interest in the at least one region of interest; wherein the movement path includes multiple test points in at least one region of interest.

[0014] In one possible implementation, the control device is further configured to: acquire, for any region of interest, the mass spectra of multiple test points within the region of interest output by the mass spectrometer, wherein the mass spectra characterize the relationship between the ion mass-to-charge ratio and ion intensity of the surface material at the test point; and generate a mass spectrum image corresponding to the region of interest based on the mass spectra of the multiple test points within the region of interest, wherein the mass spectrum image characterizes the molecular composition of the surface material at each test point within the region of interest.

[0015] In one possible implementation, the extracted droplets after surface material extraction are introduced into the mass spectrometer in the form of droplets or electrosprays under the influence of the electric field force of the electric field, and the surface material extracted from the extracted droplets in the electric field is ionized during the process of entering the mass spectrometer.

[0016] In one possible implementation, the scanning probe includes a universal capillary for electrospray ionization, the universal capillary being conical with a pointed tip and its tail end being detachably connected to the second holder; the second holder having an internal cavity and a bypass channel on its sidewall, the bypass channel being connected to the conduit for transmitting the extract delivered by the microfluidic pump.

[0017] In one possible implementation, the specified distance includes 5-10 μm, and the specified voltage includes 2-3 kV.

[0018] According to another aspect of this disclosure, a mass spectrometry imaging system is provided, the system comprising: the ion source, and a mass spectrometer.

[0019] According to various aspects of this disclosure, it is possible to extract surface substances from tissue section samples using picoliter-level extraction droplets. The extraction droplets form at the tip of a scanning probe, directly contacting the tissue section sample and forming a limited contact surface. This contact surface has dimensions in both length and width on the micrometer scale. The extracted surface substances are incorporated into the extraction droplets and, under the influence of a high-voltage electric field, enter the mass spectrometer inlet. This allows the mass spectrometer to achieve cellular or subcellular spatial resolution, effectively improving sensitivity. Furthermore, it eliminates the need for vacuum operation and limitations on the analyzed samples, allowing operation in atmospheric pressure environments. Additionally, it enables the direct use of primary ions ionized from the extraction droplets during mass spectrometry imaging analysis, resulting in richer mass spectrometric signals, higher sensitivity, and higher resolution. This achieves a picoliter-level droplet desorbing electrospray ionization source with higher sensitivity and spatial resolution in mass spectrometry imaging technology.

[0020] Other features and aspects of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0021] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this disclosure together with the specification and serve to explain the principles of this disclosure.

[0022] Figure 1 shows a schematic diagram illustrating the basic working principle of an ion source according to an embodiment of the present disclosure.

[0023] Figure 2a shows a schematic diagram of the structure of an ion source 00 according to an embodiment of the present disclosure.

[0024] Figure 2b shows a schematic diagram of a mass spectrometry imaging system according to an embodiment of the present disclosure.

[0025] Figure 3a shows a schematic diagram of the structure of another ion source 00 according to an embodiment of the present disclosure.

[0026] Figure 3b shows a schematic diagram of another mass spectrometry imaging system according to an embodiment of the present disclosure.

[0027] Figure 4 shows a real-world image of a scanning probe 005 and a tissue section sample 220 according to an embodiment of the present disclosure.

[0028] Figure 5 shows a measurement schematic diagram of an impedance measuring device 010 according to an embodiment of the present disclosure.

[0029] Figure 6 shows a schematic diagram of the relationship between displacement and measured resistance according to an embodiment of the present disclosure.

[0030] Figure 7 shows a schematic diagram of a resistance-distance mapping relationship according to an embodiment of the present disclosure.

[0031] Figure 8 shows a schematic diagram of the structure of a microfluidic pump 006 according to an embodiment of the present disclosure.

[0032] Figure 9 shows a schematic diagram of the workflow of a discrete scanning mode according to an embodiment of the present disclosure.

[0033] Figures 10a and 10b show schematic diagrams of mass spectrometry signal scanning results obtained using the ion source 00 of the present disclosure and the commercial DESI ion source, respectively.

[0034] Figure 11 shows a schematic diagram comparing the relative intensities of mass spectrometry signals obtained using the ion source 00 of this disclosure embodiment and a commercial DESI ion source.

[0035] Figure 12 shows a schematic diagram of mass spectrometry imaging results of nine metabolite solutions obtained using ion source 00 according to an embodiment of the present disclosure.

[0036] Figure 13 shows mass spectrometry images obtained by mass spectrometry analysis of 12 typical metabolites with added internal standards using ion source 00 of the present disclosure and commercial DESI ion source, respectively.

[0037] Figure 14 shows a mass spectrum obtained by mass spectrometry analysis of a tissue section sample using the ion source 00 of this embodiment of the present disclosure.

[0038] In the attached figure, 00 is the ion source; 001 is the first stage; 002 is the second stage; 003 is the ionization electrode; 004 is the high-voltage power supply; 005 is the scanning probe; 006 is the microfluidic pump; 007 is the control device; 008 is the catheter; 009 is the camera; 010 is the electrical impedance measurement device; 011 is the first holder; 012 is the second holder; 110 is the glass slide; 220 is the tissue section sample; 33 is the mass spectrometer; 330 is the sample inlet; 061 is the microsyringe; 0611 is the needle; 0612 is the piston; and 062 is the driving device. Detailed Implementation

[0039] Various exemplary embodiments, features, and aspects of this disclosure will now be described in detail with reference to the accompanying drawings. The same reference numerals in the drawings denote elements that have the same or similar functions. Although various aspects of the embodiments are shown in the drawings, they are not necessarily drawn to scale unless specifically indicated otherwise.

[0040] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0041] In this document, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent three cases: A alone, A and B simultaneously, and B alone. Furthermore, the term "at least one" in this document means any combination of at least two of any one or more elements. For example, including at least one of A, B, and C can mean including any one or more elements selected from the set consisting of A, B, and C.

[0042] It should be understood that the terms "first," "second," etc., in the claims, specification, and drawings of this disclosure are used to distinguish different objects, rather than to describe a specific order. The terms "comprising" and "including" as used in the specification and claims of this disclosure indicate the presence of the described features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or collections thereof.

[0043] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In some instances, methods, means, components, and circuits well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0044] To facilitate understanding of the ion source and the mass spectrometry imaging system constructed based on the ion source proposed in this disclosure, the basic working principle of the ion source is first introduced with reference to Figure 1. This mainly includes: a tissue section sample prepared on a glass slide is placed in front of the mass spectrometer's inlet; an ionization electrode is placed on the back of the glass slide. The relative position of the ionization electrode and the mass spectrometer's inlet can be reasonably set according to actual conditions, so that a reasonable electric field distribution can be formed when an electric field is applied between the ionization electrode and the mass spectrometer. A scanning probe is placed on the current analyte point of the tissue section sample, maintaining a certain distance from the tissue section sample. The scanning probe contains an organic solvent, organic acid, or other suitable extraction liquid to achieve selective extraction of the surface substances of the analyte point. At the start of extraction, the scanning probe extrudes a picolinate-volume droplet of extraction liquid from the needle tip. As the droplet grows, it comes into contact with the surface of the analyte point, and extracts the surface substances of the analyte point during the contact process. Then, under the influence of the high-voltage electric field generated by the ionization electrode, the electric force on the extraction droplet overcomes the surface tension between it and the surface of the analyte, causing it to fly into the mass spectrometer inlet. The surface substances extracted from the droplet are either ionized in the air or ionized in the mass spectrometer inlet. These ions are analyzed by the mass spectrometer to obtain the mass spectrum of the current analyte. After completing the mass spectrometry analysis of the current analyte, the next analyte is moved to the ionization electrode, and the above ionization and mass spectrometry analysis process is repeated to obtain the mass spectra of each analyte on the sample. Then, the mass spectrum image of the sample is obtained based on the mass spectra of each analyte using existing mass spectrometry imaging algorithms.

[0045] The ion source proposed in this disclosure includes mechanical, optical, electronic, and hydraulic hardware systems and corresponding software control systems. Specifically, Figure 2a shows a schematic diagram of the structure of an ion source 00 according to an embodiment of this disclosure. Based on the ion source 00 shown in Figure 2a, this disclosure also provides a mass spectrometry imaging system as shown in Figure 2b. The mass spectrometry imaging system shown in Figure 2b includes the ion source 00 shown in Figure 2a and a mass spectrometer 33. As shown in Figures 2a and 2b, the ion source 00 includes:

[0046] The mass spectrometer comprises a first stage 001, a second stage 002, an ionization electrode 003, a high-voltage power supply 004, a scanning probe 005, a microfluidic pump 006, and a control device 007. The first stage 001 is equipped with a first clamp 011 for holding a glass slide 110. A tissue section sample 220 to be analyzed is placed on the front of the glass slide 110, with the tissue section sample 220 facing the inlet 330 of the mass spectrometer 33. The second stage 002 is equipped with a second clamp 021. The holder 012 holds the scanning probe 005, which is connected to the microfluidic pump 006 via the conduit 008. The microfluidic pump 006 is used to deliver the extraction solution to the scanning probe 005 through the conduit 008. The ionization electrode 003 faces the sample inlet 330 of the mass spectrometer 33 and is located on the back of the glass slide 110 held by the first holder 011. The ionization electrode 003 is electrically connected to the high-voltage power supply 004. The control device 007 is electrically connected to the first stage 001, the second stage 002, the high-voltage power supply 004, and the microfluidic pump 006, respectively.

[0047] Among them, control device 007 is configured as follows:

[0048] According to the specified moving path, the first stage 001 is controlled to move to the current test point on the tissue section sample 220 on the slide 110, which is directly opposite the sample inlet 330 of the mass spectrometer 33. The moving path includes multiple test points on the tissue section sample 220.

[0049] Control the second stage 002 to move until the distance between the tip of the scanning probe 005 and the current point to be measured reaches the specified distance;

[0050] The microfluidic pump 006 is controlled to deliver the extractant to the scanning probe 005, so that the scanning probe 005 outputs an extractant droplet of picolinate volume to the current test point. After the extractant droplet contacts the current test point, the surface material of the current test point is extracted.

[0051] The high-voltage power supply 004 applies a specified voltage to the ionization electrode 003 to generate an electric field. After the surface material is extracted, the extract droplets fly into the sample inlet 330 of the mass spectrometer 33 under the action of the electric field force. The mass spectrometer 33 outputs the mass spectrum corresponding to the current test point based on the ions ionized from the surface material extracted in the flying extract droplets.

[0052] In practical applications, both the first stage 001 and the second stage 002 can employ high-precision three-dimensional moving platforms. The glass slide 110 is fixed to the first stage 001 via a first clamp 011 adapted to it, facilitating easy insertion and removal of the slide after use. The scanning probe 005 is fixed to the second stage 002 via a second clamp 012 adapted to it, facilitating easy loading and replacement of the scanning probe 005. A glass slide 110 containing tissue section sample 220 is placed on one side of the first stage 001, facing the inlet 330 of the mass spectrometer 33, so that the extraction droplet can fly into the inlet 330; the scanning probe 005 is located on the same side of the first stage 001 where the glass slide 110 is located, and is used to add a picolinate volume of extraction droplet to the surface of the tissue section sample 220; the ionization electrode 003 is located on the other side of the first stage 001. During the operation of the ion source, the position of the ionization electrode 003 relative to the inlet 330 of the mass spectrometer 33 can remain stationary to achieve a stable electric field distribution between the two.

[0053] In one possible implementation, the scanning probe 005 may include a general-purpose capillary for electrospray ionization (ESI). This capillary is conical with a pointed tip, and its tail end is detachably connected to a second holder. The second holder has an internal cavity and a bypass channel on its sidewall, which is connected to a conduit 008 for transmitting the extractant delivered by the microfluidic pump 006. Optionally, the inner diameter of the tip of the general-purpose capillary may be 2–3 micrometers, and the cone angle may be 12–18°. It should be understood that the scanning probe 005 of this embodiment is not limited to a general-purpose capillary with the above parameters; any capillary capable of achieving the required functions of the scanning probe 005 is acceptable.

[0054] In one possible implementation, the region of interest (ROI) for mass spectrometry imaging can be obtained by pre-staining and optically imaging the tissue section sample on the slide 110. For example, ROI markers can be pre-set on the slide 110 to facilitate accurate selection of these ROIs during visible light imaging. These ROIs can be mapped to the movement path of the first stage 001; that is, the control device 007 can generate a movement path based on the ROIs, convert the movement path into a sequence of control commands, and transmit it to the driver of the first stage 001 to drive the first stage 001 to move according to the movement path. Specifically, the control device 007 is further configured to:

[0055] Obtain visible light images of 220 tissue section samples;

[0056] In response to marking at least one region of interest on a visible light image, each region of interest in the marked at least one region of interest is divided into multiple detection points according to a preset resolution; wherein, the preset resolution includes subcellular or cellular resolution, and the preset resolution is lower than the movement resolution of the first stage 001;

[0057] A movement path is determined based on multiple test points divided within each region of interest (ROI) at least once; wherein the movement path contains multiple test points within at least one ROI.

[0058] In practical applications, the tissue section sample 220 can be a stained tissue section sample. The visible light image of the tissue section sample 220 can be captured by a camera, or the visible light image of the tissue section sample 220 can be transmitted from other electronic devices to the control device 007, etc. The embodiments of this disclosure do not limit the method of acquiring the visible light image of the tissue section sample 220.

[0059] In practical applications, the control device 007 may be equipped with a display device. After acquiring the visible light image of the tissue section sample 220, the display device can show the visible light image of the tissue section sample 220 and provide relevant controls for marking regions of interest (ROIs). This allows the user to mark at least one ROI on the displayed visible light image. The ROI can be understood as the tissue region on the tissue section sample 220 to be analyzed by mass spectrometry. Of course, the visible light image with the marked ROI can also be directly input into the control device 007, and this embodiment of the present disclosure does not limit this approach.

[0060] The aforementioned preset resolution can be understood as the size of each measured point. For example, each region of interest can be divided according to a preset resolution of 5 μm, and the size of each measured point within each region of interest can be 5 μm. It should be understood that the position of the region of interest marked in the visible light imaging image is known, and the position of each measured point within each region of interest is also known. Based on the position of each measured point within each region of interest, a movement path containing multiple measured points within at least one region of interest can be determined. This movement path can be a horizontal movement followed by a vertical movement, or a vertical movement followed by a horizontal movement; this embodiment does not limit this. Thus, during the operation of the ion source 00, the first stage 001 can be controlled to move according to the determined movement path until the current measured point on the tissue section sample 220 on the slide 110 is directly opposite the inlet 330 of the mass spectrometer 33. The current measured point can be any one of the multiple measured points divided from the aforementioned at least one region of interest.

[0061] The motion resolution of the first stage 001 can be understood as the lateral and longitudinal motion accuracy of the first stage 001, or the minimum distance it can move. Setting a preset resolution lower than the motion resolution of the first stage 001 can make the unit movement time of the scanning probe 005 in the same region of interest greater than the mass spectrometry scanning cycle of the mass spectrometer 33, thereby obtaining a higher intensity mass spectrometry signal. This can ensure the stability of the ion source during continuous operation while ensuring the spatial resolution during mass spectrometry imaging, thus achieving a higher resolution mass spectrometry imaging effect.

[0062] In one possible implementation, the specified distance achieved by controlling the second stage 002 to move to the distance between the tip of the scanning probe 005 and the current test point can be a distance on the micrometer scale, for example, the specified distance can include 5-10 μm. To achieve the above-mentioned distance control with micrometer-level precision, a schematic diagram of another ion source 00 is shown in Figure 3a, and Figure 3b shows a mass spectrometry imaging system including the ion source 00 of Figure 3a and the mass spectrometer 33. As shown in Figures 3a and 3b, the ion source 00 may also include: a camera 009 and an impedance measurement device 010. The camera 009 faces the tissue slice sample 220 and the scanning probe 005, and the impedance measurement device 010 is connected to the scanning probe 005. The impedance measurement device 010 is used to measure the resistance value between the tip of the scanning probe 005 and the surface of the test point. That is, the scanning probe 005 is connected to the impedance measurement device 010 to monitor the distance between the tip of the scanning probe 005 and the surface of the test point. Camera 009 can be placed on one side of the tissue slice sample 220 for easy observation, and is used to observe the contact process between the scanning probe 005 and the surface of the test point, and the working status of the plasma source.

[0063] The control device 007 is electrically connected to the camera 009 and the impedance measuring device 010. This allows the control of the second stage 002 to move until the distance between the tip of the scanning probe 005 and the current measurement point reaches a specified distance, including:

[0064] The camera 009 is controlled to acquire real-world images of the scanning probe 005 and the tissue section sample 220;

[0065] Based on the relative position between the tip of the scanning probe 005 and the tissue section sample 220 in the real-world image, the second stage 002 is controlled to move until the tip of the scanning probe 005 is aligned with the current test point on the tissue section sample 220.

[0066] Control the impedance measuring device 010 to measure the current resistance value between the tip of the scanning probe 005 and the surface of the current point to be measured;

[0067] Based on the preset resistance-distance mapping relationship, the current distance corresponding to the current resistance value is determined. The resistance-distance mapping relationship characterizes the mapping relationship between the resistance value and the distance between the tip of the scanning probe 005 and the surface of the point to be measured.

[0068] Based on the difference between the current distance corresponding to the current resistance value and the specified distance, the second stage 002 is controlled to move until the distance between the tip of the scanning probe 005 and the current test point reaches the specified distance.

[0069] It should be understood that by acquiring a real-world image containing the scanning probe 005 and the tissue section sample 220 through the camera 009, the relative position between the tip of the scanning probe 005 and the tissue section sample 220 can be determined from the real-world image. Then, by combining the camera imaging principle, the relative spatial position between the tip of the scanning probe 005 and the tissue section sample 220 can be determined. Based on the relative spatial position between the tip of the scanning probe 005 and the tissue section sample 220, the second stage 002 can be controlled to move until the tip of the scanning probe 005 is aligned with the current test point on the tissue section sample 220. This is equivalent to initially adjusting the relative spatial position between the scanning probe 005 and the tissue section sample 220 until the tip of the scanning probe 005 is aligned with the current test point on the tissue section sample 220. For example, Figure 4 shows a real-world image of a scanning probe 005 and a tissue section sample 220. The scanning probe 005 can move in the scanning direction (i.e., the moving path) shown in the figure to extract and perform mass spectrometry analysis on the surface material of multiple test points on the tissue section sample 220.

[0070] Once the tip of the scanning probe 005 is aligned with the test point, the impedance measurement device 010 can be activated to measure the resistance between the internal electrode of the scanning probe 005 and the surface of the test point, thereby determining the distance between the scanning probe 005 and the test point surface. The measurement accuracy can reach the micrometer level. Specifically, the impedance measurement device 010 can measure the resistance between the scanning probe 005 tip and the test point surface by applying a voltage to the tip of the scanning probe 005 and measuring the current between them. As shown in Figure 5, the droplet generated by the tip of the scanning probe 005 has a certain impedance after contacting the test point surface. Applying a voltage to the tip of the scanning probe 005 and the test point surface creates a path between them, allowing for the measurement of the current. Using Ohm's law, the resistance between the scanning probe 005 tip and the test point surface can be calculated. In Figure 5, h represents the distance (tip height) between the scanning probe 005 tip and the test point surface.

[0071] In practical applications, the impedance measuring device 010 can be implemented, for example, by using a series low-voltage power supply, an ammeter, and a switch. The control device 007 can control the start and stop of the impedance measuring device 010 by controlling the opening and closing of the switch. The low-voltage power supply can apply a low-voltage voltage to the tip of the scanning probe 005 and the surface of the point to be measured. The ammeter can measure the current value between the two, thereby obtaining the resistance value between the tip of the scanning probe 005 and the surface of the point to be measured. It should be understood that the embodiments of this disclosure do not limit the specific circuit structure of the impedance measuring device 010, as long as it can achieve the required function.

[0072] As described above, the moving path contains multiple test points. If the scanning probe 005 is moved in the scanning direction shown in Figure 4, the relationship between the moving displacement and the measured resistance shown in Figure 6 can be measured by the impedance measuring device 010, that is, the resistance value in the scanning direction can be measured.

[0073] In practical applications, the resistance-distance mapping relationship can be pre-calibrated through experimental testing. For example, the second stage 002 can control the tip of the scanning probe 005 to reach multiple calibration distances with the sample surface, and the impedance measurement device 010 can apply voltage to measure the corresponding resistance value in each calibration interval. Then, the resistance-distance mapping relationship can be constructed or fitted by the calibration distance and the corresponding resistance value. This resistance-distance mapping relationship can be expressed as an empirical formula or a graph. For example, Figure 7 shows a schematic diagram of a resistance-distance mapping relationship, where the tip height is the distance between the tip of the scanning probe 005 and the sample surface. As shown in Figure 7, when the scanning probe 005 is farther from the sample surface (i.e., the larger the tip height), the generated droplet is larger and the resistance is greater; when the scanning probe 005 is closer to the sample surface (i.e., the smaller the tip height), the generated droplet is smaller and the resistance is smaller.

[0074] Based on the aforementioned resistance-distance mapping relationship, after the impedance measuring device 010 measures the current resistance value between the tip of the scanning probe 005 and the surface of the current test point, the current distance corresponding to the current resistance value can be determined based on this resistance-distance mapping relationship. This is equivalent to converting the measured current value and calculated resistance value into a distance value (i.e., the current distance), and accurately controlling the distance between the scanning probe 005 and the sample surface to reach the specified distance based on the difference between the converted distance value and the specified distance. This method enables micron-level distance control and compensates for potential unevenness on the sample surface, ensuring the safety and scanning stability of the scanning probe 005 during the extraction operation and preventing physical damage caused by the scanning probe 005 scratching the sample surface.

[0075] In practical applications, the aforementioned control of the microfluidic pump 006 to deliver the extraction solution to the scanning probe 005 includes: controlling the microfluidic pump 006 to continuously deliver the extraction solution to the scanning probe 005, or controlling the microfluidic pump 006 to intermittently deliver the extraction solution to the scanning probe 005. Continuous delivery of the extraction solution to the scanning probe 005 can be understood as a continuous operating mode, in which the microfluidic pump 006 can remain continuously on, allowing the scanning probe 005 to continuously add extraction solution drops to multiple test points on the tissue section sample 220. Intermittent delivery of the extraction solution to the scanning probe 005 can be understood as a discrete operating mode, in which the microfluidic pump 006 can be turned off after delivering the extraction solution once, allowing the scanning probe 005 to add extraction solution drops to one test point, and then turned on again after the scanning probe 005 moves to the next test point. This allows the scanning probe 005 to intermittently add extraction solution to multiple test points on the tissue section sample 220. It should be understood that those skilled in the art can select the operating mode of the microfluidic pump 006 according to actual needs, and this disclosure does not limit this.

[0076] Considering that there may be mutual interference between the voltage applied by the impedance measuring device 010 and the specified voltage applied by the high-voltage power supply 004 when the microfluidic pump 006 continuously delivers the extractant to the scanning probe 005, the control device 007 can be configured to alternately control the voltage applied by the impedance measuring device 010 and the specified voltage applied by the high-voltage power supply 004 in a time sequence. For example, this can be achieved by designing pulse control signals corresponding to the impedance measuring device 010 and the high-voltage power supply 004, respectively. This embodiment of the present disclosure does not limit this approach. In this way, mutual interference between the impedance measuring device 010 and the high-voltage power supply 004 is prevented, while ensuring that the mass spectrometer 33 generates an effective mass spectrometry signal in the time domain.

[0077] To achieve precise control of the microfluidic pump 006 delivering the extractant to the scanning probe 005, thereby enabling the scanning probe 005 to output a picolinate-volume droplet of extractant to the current test point, in one possible implementation, Figure 8 shows a schematic diagram of the microfluidic pump 006. As shown in Figure 8, the microfluidic pump 006 includes a microsyringe 061 and a driving device 062. The microsyringe 061 includes a needle tube 0611 and a piston 0612. The piston 0612 is fixed to the driving device 062, and its moving distance and speed can be precisely controlled. The driving device 062 can be any high-precision driving device known in the art, and this embodiment does not limit it. The needle tube 0611 stores the extractant and is connected to the scanning probe 005 through a conduit 008 to achieve the delivery of the extractant.

[0078] Based on the aforementioned microfluidic pump 006, controlling the microfluidic pump 006 to deliver the extractant to the scanning probe 005 includes: driving the piston 0612 to move at a specified speed for a specified duration via a control drive device 062, thereby delivering a specified volume of extractant to the scanning probe 005, the specified volume including a picolinate volume. By using this microfluidic pump 006, precise control and delivery of the extractant volume can be achieved.

[0079] It should be understood that the speed and duration of the drive device 062 can be adjusted according to actual needs, that is, the speed and duration of the piston 0612 movement can be controlled, thereby controlling the flow rate and volume of the liquid in the syringe 0611, and thus achieving precise control of the volume of the extract. Specifically, let the speed of the piston 0612 in Figure 8 be v, the density of the extract be ρ, and the atmospheric pressure be P0, then according to Bernoulli's equation, the fluid pressure P at the tip of the scanning probe 005 is given by formula (1):

[0080] For a single particle in the extract droplet at the tip of the scanning probe 005, with the tip of the scanning probe 005 as the origin, its position coordinates are represented as shown in Figure 8. Then, according to Newton's second law, the above formula (1) can be transformed into formula (2):

[0081] Where m represents the mass of the particle. Let A represent the acceleration of the particle, and let A represent the cross-sectional area at the tip of the scanning probe 005. Then, by dividing both sides of equation (2) by ρ and integrating, we can obtain the relationship between the volume V of the extract, the piston velocity v, and the time t shown in equation (3):

[0082] In practical applications, the specified speed and duration required for a specified volume can be calibrated based on the above formula (3), and the calibrated specified duration and speed can be used to control the drive device 062 to drive the piston 0612 to move, so as to deliver a specified volume of extract to the scanning probe 005, thereby enabling the scanning probe 005 to output a picolinate volume of extract droplets to the current test point.

[0083] In this process, after a picoliter-volume extraction droplet comes into contact with the current test point, the extraction of the surface material at the current test point can be completed in a short time (e.g., within 100ms). After that, the high-voltage power supply 004 can be controlled to apply a specified voltage value to the ionization electrode 003 so that the ionization electrode 003 generates a high-voltage electric field.

[0084] In practical applications, the distance between the ionization electrode 003 and the first stage 001 can be on the order of millimeters, that is, the distance between the ionization electrode 003 and the glass slide 110 held by the first clamp 011 on the first stage 001 can be on the order of millimeters. The ionization electrode 003 and the high-voltage power supply 004 can be connected by wires. The high-voltage power supply 004 can generally generate a high-voltage level of -5 to 5kV. The voltage value of the specified voltage applied by the high-voltage power supply 004 to the ionization electrode 003 can be adjusted according to actual needs. The specified voltage can be a high-voltage DC voltage; for example, the specified voltage when the ionization electrode 003 is working can include 2 to 3kV, that is, the amplitude of the high-voltage DC voltage when the ionization electrode 003 is working can be set to 2 to 3kV, either positive or negative.

[0085] In this process, the extracted droplets, after surface material extraction, can enter the inlet 330 of the mass spectrometer 33 in the form of droplets or electrospray under the influence of the electric field. The surface material extracted from the droplets in the electric field ionizes during its entry into the mass spectrometer 33. Based on the ionized ions from the extracted surface material in the droplets, the mass spectrometer 33 outputs a mass spectrum corresponding to the current analyte. It should be understood that those skilled in the art can use any existing mass spectrometer to perform mass spectrometry analysis on the ionized ions from the extracted droplets to obtain a mass spectrum of the surface material at the current analyte. The mass spectrum can be understood as a graphical mass spectrometry signal. It should be understood that this embodiment does not limit the mass spectrometry analysis process of the mass spectrometer 33.

[0086] As described above, the moving path may include multiple test points within at least one region of interest. It should be understood that for each test point, ionization and mass spectrometry analysis can be performed using the aforementioned ion source 00 combined with the mass spectrometer 33 to obtain a mass spectrum for each test point within each region of interest. The mass spectrum characterizes the relationship between the ion mass-to-charge ratio (m / z value) and ion intensity of the surface material at the test point. Furthermore, based on existing mass spectrometry imaging algorithms and the mass spectra of multiple test points within each region of interest, a mass spectrometry image of each region of interest is generated. The mass spectrometry image characterizes the molecular composition of the surface material at each test point within the region of interest. Therefore, the control device 007 can also be configured to: acquire the mass spectra of each of the multiple test points within the region of interest output by the mass spectrometer 33 for any region of interest; and generate a mass spectrometry image corresponding to the region of interest based on the mass spectra of each of the multiple test points within the region of interest. It should be understood that those skilled in the art can use known mass spectrometry imaging algorithms, data processing software, etc., to generate a mass spectrometry image corresponding to the region of interest based on the mass spectra of each of the multiple test points within the region of interest, i.e., obtain the mass spectrometry imaging result of the region of interest. This embodiment of the present disclosure does not limit this.

[0087] As shown in Figures 2b and 3b above, the core hardware system of the ion source mainly includes a first stage 001 and a first holder 011 for controlling the glass slide, a second stage 002 and a second holder 012 for controlling the scanning probe 005, a microfluidic pump 006, a scanning probe 005, an impedance measurement device 010, an ionization electrode 003, a high-voltage power supply 004, a camera 009, a control device 007, and corresponding drivers and controllers for these hardware components or subsystems. These hardware systems can be integrated into a compact, plug-and-play ion source device that is compatible with the inlet mechanisms of various mass spectrometers through optimized design and customization. Furthermore, during the operation of the ion source 00, precise control can be achieved through design, including controlling the volume of droplets generated by the scanning probe, controlling the distance between the scanning probe and the sample surface, controlling the timing of the second stage, and the switching sequence of the ionization electrode, to coordinate the overall working pace of the various components of the ion source.

[0088] Based on the ion sources shown in Figures 2b and 3b above, this disclosure also proposes a procedure for mass spectrometry imaging analysis of tissue section samples using ion source 00. Specifically, the tissue to be analyzed is first processed into a section sample and placed on a glass slide. During mass spectrometry imaging, the glass slide 110 containing the tissue section sample 220 is placed on the first stage 001 and clamped and fixed by the first clamp 011. Then, the relative position of the tissue section sample 220 and the scanning probe 005 is observed through the camera 009. The scanning probe 005 is moved by adjusting the second stage 002 so that the scanning probe 005 is close to the surface of the tissue section sample 220. The electrical impedance measurement device 010 is used to make the distance between the scanning probe 005 and the surface of the current test point of the tissue section sample 220 reach 5-10 μm. Then, the first stage 011 can be controlled to translate along a predetermined moving path (such as translating with a unit movement of 5 μm as described above). For each test point, the distance between the scanning probe 005 and the tissue slice sample 220 is controlled to reach 5-10 μm. Then, the extraction droplets are generated by controlling the microfluidic pump 006 for extraction and the high voltage power supply 004 is controlled to ionize the extracted substances in the extraction droplets to obtain the mass spectrometry signal of the test point. That is, the piloliter-level extraction droplets are added to the sample surface by the scanning probe 005, and the extraction droplets can cover the subcellular area. After the extractant droplets extract substances from the tissue section sample, they are pushed away from the sample surface by the strong electric field generated by the ionization electrode 003. The droplets then enter the mass spectrometer inlet 33 as a whole droplet or electrospray for subsequent mass spectrometry analysis, obtaining the mass spectrometry signal of the current analyte point in the sample. This process is repeated, scanning each analyte point on the tissue section sample to obtain a series of mass spectrometry signals. A mass spectrometry image can then be reconstructed based on this information. Specifically, by controlling the first stage 001 to move the tissue section sample, surface substances at multiple analyte points within the region of interest are extracted, and the mass spectrometry signals of these multiple analyte points within the region of interest are obtained and fused into a two-dimensional mass spectrum. Each pixel in the mass spectrum represents a mass-to-charge ratio and ion intensity containing a large amount of biomolecular information. The distribution of the mass spectrometry signal intensity of a specific ion in the mass spectrum reflects its relative abundance distribution at different locations in the sample. This relative abundance distribution can be represented by the intensity of a certain color, obtaining the mass spectrometry image of that substance. Alternatively, images of different colors (i.e., different ions) can be superimposed to obtain a complete mass spectrometry image.

[0089] In practical applications, the ion source 00 proposed in this embodiment can employ a discrete scanning mode in the high-resolution mass spectrometry imaging process. Figure 9 shows a schematic diagram of the workflow of a discrete scanning mode according to an embodiment of this disclosure. The movement path during mass spectrometry imaging can be pre-determined using alignment marks on the slide 110 with the aid of visible light imaging of the tissue section sample. Before scanning a specific measurement point on the sample, the microfluidic pump 006, high-voltage power supply 004, and impedance measurement device 010 are all in a turned-off state. They are turned on as needed during the scanning process and turned off after use. As shown in Figure 9, the workflow of this discrete scanning mode can include: fixing the tissue section sample 220 on the first stage 001, i.e., fixing the tissue section sample 220; acquiring a real-world image of the scanning probe 005 and the tissue section sample 220 through the camera 009, and initially adjusting the spatial relative position of the scanning probe 005 and the tissue section sample 220, i.e., positioning the scanning probe 005; moving the scanning probe 005 close to the surface of the tissue section sample 220, turning on the impedance measurement device 010, and using the tip height feedback control of the scanning probe 005 to make the distance between the scanning probe 005 and the surface of the tissue section sample 220 reach about 5-10 μm, and turning off the impedance measurement device 010; turning on the microfluidic pump 006 to add the extraction liquid, and after the droplet increases in size, it contacts the surface of the tissue section sample 220. The extraction of the surface material of the current test point on the tissue section sample 220 can be completed in a short time (<100ms), i.e., achieving millisecond-level micro-extraction of the piezometric extraction droplet, and turning off the microfluidic pump 006; turning on the high-voltage power supply 004, and in the electric... A specified voltage of 2-3 kV is applied to the electrode 003, causing the extraction droplets on the sample surface to fly off and enter the injection port of the mass spectrometer 33, obtaining a mass spectrometry signal and a mass spectrum of a single test point, thus realizing the electrospray injection mass spectrometer 33. Then, it can be determined whether the scan is complete, i.e., whether all test points along the moving path have been scanned. If not, the first stage 001 is moved approximately 5 μm (this value can be smaller, its setting can match the required imaging resolution) along the preset moving path to the next test point, and the impedance detection is activated. The device 010 performs tip height feedback control to control the surface distance between the scanning probe 005 and the next test point on the tissue section sample 220 to reach 5-10 μm; repeating the above process of "liquid addition-extraction-detection-movement positioning" to complete the mass spectrometry imaging analysis of all test points on the moving path and obtain the mass spectrum of all test points on the moving path; then, the mass spectrum of all test points can be reconstructed into a mass spectrometry image (i.e., mass spectrometry imaging image) through data processing software, which is to obtain the mass spectrometry imaging result of the tissue section sample.

[0090] In practical applications, the ion source 00 proposed in this embodiment can also employ a continuous scanning mode during high-resolution mass spectrometry imaging. The movement path in continuous scanning mode can be pre-determined using the visible light image of the tissue section sample with the aid of alignment marks on the slide 110. Before scanning a specific point on the sample, the microfluidic pump 006, high-voltage power supply 004, and impedance measurement device 010 are all in a turned-off state. They are turned on as needed during the scanning process and turned off after use. The workflow of this continuous scanning mode may include: fixing the tissue section sample 220 on the first stage 001; acquiring a real-world image of the scanning probe 005 and the tissue section sample 220 through the camera 009; initially adjusting the relative spatial position of the scanning probe 005 and the tissue section sample 220, and aligning the scanning probe 005 with the first test point in the scanning path on the horizontal plane; moving the scanning probe 005 close to the surface of the tissue section sample 220; activating the impedance measurement device and using tip height feedback control to ensure that the distance between the scanning probe 005 and the surface of the first test point on the tissue section sample 220 is approximately 5-10 μm; and activating the microfluidic flow meter. Pump 006 continuously adds extraction solution at a stable rate. Simultaneously, the slide sample 220 is moved stably along a specified path (i.e., the first stage 001 is moved). The scanning probe 005 is kept at a distance of approximately 5-10 μm from the surface of the tissue slide sample 220 by the impedance detection device 010 and the tip height feedback control. Next, the high-voltage power supply 004 is kept on, and a specified voltage of 2-3 kV is applied to the ionization electrode 003, causing the extraction solution droplets from multiple test points on the sample surface to continuously fly off and enter the injection port of the mass spectrometer 33. The mass spectrometer 33 records the mass spectrum of each test point, including the m / z value and the corresponding ion intensity I of each test point. As the scan progresses, the mass spectra collected point by point are aggregated into a quaternary dataset containing spatial information (x-axis, y-axis, mass-to-charge ratio m / z, ion intensity I), where each measurement point corresponds to a mass spectrum. During this continuous scanning process, the voltage applied by the impedance detection device 010 and the voltage applied by the high-voltage power supply 004 to the ionization electrode 003 cannot and do not need to be turned on simultaneously. That is, the voltage applied by the impedance detection device 010 and the voltage applied by the high-voltage power supply 004 to the ionization electrode 003 can be turned on alternately. This prevents mutual interference and ensures that the mass spectrometer generates an effective signal in the time domain. After scanning the specified movement path, the microfluidic pump 006, the high-voltage power supply 004, the impedance measurement device 010, etc., are turned off. Then, the mass spectra of all measurement points on the movement path are reconstructed into mass spectrometry images by the data processing software, which yields the mass spectrometry imaging results of the tissue slice sample 220.

[0091] It should be understood that the ion source 00 of this disclosure embodiment can provide the above-mentioned discrete scanning mode and continuous scanning mode, so that the user can select any desired scanning mode to realize mass spectrometry analysis of any tissue slice sample, and this disclosure embodiment does not limit this.

[0092] According to the ion source 00 of this disclosure, surface substances of tissue section samples can be extracted using picoliter-level extraction droplets. The extraction droplets form at the tip of the scanning probe, directly contacting the tissue section sample and forming a limited contact surface. This contact surface has dimensions in both length and width at the micrometer level. The extracted surface substances are incorporated into the extraction droplets and, under the influence of a high-voltage electric field, enter the mass spectrometer inlet. This allows the mass spectrometer to achieve cellular or subcellular spatial resolution, effectively improving sensitivity. Furthermore, it eliminates the need for vacuum operation and limitations on the analyzed samples, allowing operation in atmospheric pressure environments. Additionally, it enables the direct use of primary ions ionized from the extraction droplets during mass spectrometry imaging analysis, resulting in richer mass spectrometry signals, higher sensitivity, and higher resolution. In other words, it realizes a picoliter-level droplet desorbing electrospray ion source with higher sensitivity and spatial resolution in mass spectrometry imaging technology.

[0093] Based on the ion source proposed in the above embodiments of this disclosure, this disclosure also proposes a mass spectrometry imaging system, including: the aforementioned ion source 00 and a mass spectrometer 33. The novel mass spectrometry imaging system proposed in this disclosure, using the ion source 00 to establish a sample tissue section, facilitates the implementation of highly sensitive metabolomics analysis methods, achieving label-free, highly sensitive, and high-coverage mass spectrometry imaging technology, providing innovative tools for metabolomics analysis in life sciences, clinical medicine, metabolomics, and molecular histology.

[0094] The ion source 00 proposed in this disclosure has the following advantages over existing ion source technologies:

[0095] (1) It has fewer restrictions on samples and operating environment, and possesses most of the advantages of DESI, such as the ability to obtain richer molecular information. At the same time, it does not require chemical modification of the sample or the addition of additional markers, nor does it require complex surface pretreatment, such as chemical fixation or metal coating. The preparation process is relatively simple, reducing sample damage and avoiding deviations in analytical results caused by the introduction of other chemical substances. In addition, the imaging process does not require a vacuum environment, making it widely applicable.

[0096] (2) The spatial resolution is high. Compared with the mainstream DESI technology, the ion source 00 proposed in this embodiment is used for mass spectrometry imaging with extraction sites as small as subcellular level. The spatial resolution of mass spectrometry imaging is improved by about one order of magnitude, which can more accurately reveal the spatial distribution of molecules in biological tissues.

[0097] (3) Higher sensitivity: Compared to mainstream DESI technology, the ion source 00 proposed in this embodiment uses picoliter droplets to extract the analyte (i.e., the surface material of the test point) and directly sends the extracted droplets into the mass spectrometer for analysis, avoiding the use of secondary ions and improving sensitivity. Compared to MALDI technology, the ion source 00 proposed in this embodiment does not introduce matrix interference; compared to SIMS technology, it can preserve complete molecular information and meet the needs of accurate identification of biomolecules.

[0098] (4) Multiple extractions increase analyte coverage. The ion source 00 proposed in this embodiment uses picoliter droplet contact extraction to extract analytes without causing physical impact damage to tissue section samples. Therefore, different analytes can be extracted from the same tissue section sample using different extraction solvents, which has a significant advantage in increasing the coverage of analytes.

[0099] To better understand the advantages of the ion source 00 proposed in the above embodiments of this disclosure compared to existing ion source technologies, the resolution of the picoliter droplet desorbing electrospray ion source can be characterized by a rhodamine grid deposited on a glass substrate. The average width of the rhodamine grid lines is 10 μm, and the spacing between the grid lines is 10 μm. Using the ion source 00 in continuous scanning mode to scan the rhodamine grid at a constant speed yields the mass spectrometry signal scanning results shown in Figure 10a. Simultaneously, using a commercially available DESI ion source operating in continuous scanning mode to scan the rhodamine grid at a constant speed yields the mass spectrometry signal scanning results shown in Figure 10b. As shown in Figures 10a and 10b, the grid line widths measured by the ion source 00 proposed in this embodiment and the commercial DESI ion source are 14.1 μm and 37.9 μm, respectively. It can be understood that the measured grid line width can be obtained based on the product of the duration of the peak signal and the scanning speed. Using the full width at half maximum (FWHM) definition of resolution, the resolution of the ion source 00 in this embodiment is 4.1 μm, while the resolution of the commercial DESI ion source is 27.9 μm. This indicates that the spatial resolution of mass spectrometry imaging using the ion source 00 in this embodiment is approximately seven times that of the commercial DESI ion source.

[0100] By using the ion source 00 of this disclosure and a commercial DESI ion source to perform mass spectrometry analysis on six standard metabolite solutions with added internal standards (i.e., glucose Glu, reserpine, phosphatidylcholine PC (34:1), glutathione GSH, glutamine Gln, and acetylcholine), the relative intensity (i.e., ionic intensity) of the mass spectrometry signals shown in Figure 11 can be obtained. This indicates that the sensitivity of the ion source 00 of this disclosure is improved by about 40% compared with the commercial DESI ion source.

[0101] For the biological tissue homogenate shown in Figure 12(a), the ion source of this embodiment can be used to obtain the mass spectrometry imaging results of the biological tissue homogenate with nine metabolite solutions (spermine, taurine, xanthine, glucose phosphate, acetylcholine, glutamine, phosphatidylcholine PC (34:1), fatty acid FA-C18:1, and L-carnitine) shown in Figures 12(b) to 12(j) respectively. As can be seen from the mass spectrometry imaging results, the distribution of different molecules has obvious spatial specificity, reflecting the biochemical characteristics of different types of cells in the biological tissue and revealing the spatial chemical heterogeneity of the biological tissue. Furthermore, the ion source 00 of this embodiment can obtain more than 1200 metabolites by mass spectrometry analysis, which is better than the less than 900 metabolites of the commercial DESI ion source. This demonstrates the high coverage of the ion source 00 of this embodiment in complex biological samples.

[0102] Figure 13 shows mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing 12 typical metabolites using ion source 00 of the present invention and commercial DESI ion source, respectively. Figures 13(a1) and 13(a2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite Spermine using ion source 00 of the present invention and commercial DESI ion source, respectively. Figures 13(b1) and 13(b2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite Taurine using ion source 00 of the present invention and commercial DESI ion source, respectively. Figures 13(c1) and 13(c2) are... The following are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite L-Carnitine using ion source 00 of the present invention and a commercial DESI ion source. Figures 13(d1) and 13(d2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite Acetylcholine using ion source 00 of the present invention and a commercial DESI ion source, respectively. Figures 13(e1) and 13(e2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite phosphatidylethanolamine PE (P38:1) using ion source 00 of the present invention and a commercial DESI ion source, respectively. Figures 13(f1) and 13(f2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite glucose using the ion source 00 of this embodiment and the commercial DESI ion source, respectively. Figures 13(g1) and 13(g2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite FA-C18:1 with internal standard using the ion source 00 of this embodiment and the commercial DESI ion source, respectively. Figures 13(h1) and 13(h2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite glutamine using the ion source 00 of this embodiment and the commercial DESI ion source, respectively. Figures i1 and i2 are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite phosphatidylserine PS (38:5) using ion source 00 of the present invention and commercial DESI ion source, respectively. Figures j1 and j2 are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite Xanthine using ion source 00 of the present invention and commercial DESI ion source, respectively. Figures k1 and k2 are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite PC (34:1) using ion source 00 of the present invention and commercial DESI ion source, respectively.Figures 13(l1) and 13(l2) are mass spectrometry images obtained by mass spectrometry analysis of biological tissue homogenates containing the typical metabolite phosphatidylglycerol PG (36:1) using the ion source 00 of this disclosure and the commercial DESI ion source, respectively. In the above mass spectrometry images, the darker the color, the greater the ion intensity. Therefore, the mass spectrometry image obtained using the ion source 00 of this disclosure shows stronger detail and contrast than that obtained using the commercial DESI ion source. These results demonstrate that the picoliter-level droplet desorbed electrospray ion source proposed in this disclosure has significant advantages in terms of resolution, sensitivity, and coverage in mass spectrometry imaging.

[0103] Figure 14 shows the mass spectrum obtained by mass spectrometry analysis of tissue section samples using the ion source 00 of this embodiment. The ion source 00 can use an acetonitrile aqueous solution ACN / H2O (8:2) extractant and a continuous scan mode. As shown in Figure 14, substances with a mass-to-charge ratio of approximately 180 to 560 are metabolites, and substances with a mass-to-charge ratio of approximately 620 to 1120 are lipids. Figure 14 also shows mass spectrum images with mass-to-charge ratios m / z = 251.4, 343.7, 734.4, and 895.1. As shown in Figure 14, the mass spectrum generated using the ion source 00 of this embodiment shows a strong peak signal (i.e., intensity peak), which proves that the ion source 00 proposed in this embodiment can effectively ionize the surface substances of the tissue section samples, that is, it has good usability.

[0104] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of this disclosure. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of an instruction, which contains one or more executable instructions for implementing a specified logical function. In some alternative implementations, the functions marked in the blocks may occur in a different order than those marked in the drawings. For example, two consecutive blocks may actually be executed substantially in parallel, and they may sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each block in the block diagrams and / or flowcharts, and combinations of blocks in the block diagrams and / or flowcharts, can be implemented using a dedicated hardware-based system that performs the specified function or action, or using a combination of dedicated hardware and computer instructions.

[0105] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. An ion source, characterized by, The ion source comprises: a first carrier, a second carrier, an ionization electrode, a high-voltage power supply, a scanning probe, a micro flow pump, and a control device; the first carrier is provided with a first holder, the first holder is used for clamping a glass slide, the glass slide has a tissue section sample to be analyzed on the front surface, and the tissue section sample on the front surface of the glass slide clamped by the first holder faces the sample inlet of a mass spectrometer; the second carrier is provided with a second holder, the second holder clamps the scanning probe, the scanning probe is connected to the micro flow pump through a conduit, and the micro flow pump is used for conveying an extraction solution to the scanning probe through the conduit; the ionization electrode faces the sample inlet of the mass spectrometer and is located on the back surface of the glass slide clamped by the first holder, and the ionization electrode is electrically connected to the high-voltage power supply; the control device is electrically connected to the first carrier, the second carrier, the high-voltage power supply, and the micro flow pump, respectively; The control device is configured to: control the first carrier to move to a current test point on the tissue section sample on the glass slide to face the sample inlet of the mass spectrometer according to a specified movement path, the movement path including a plurality of test points on the tissue section sample; control the second carrier to move to a distance between the needle tip of the scanning probe and the current test point to reach a specified distance; control the micro flow pump to convey the extraction solution to the scanning probe, so that the scanning probe outputs a picoliter volume of extraction solution droplets to the current test point, and the extraction solution droplets complete the extraction of surface substances of the current test point after contacting the current test point; control the high-voltage power supply to apply a specified voltage to the ionization electrode to generate an electric field, and the extraction solution droplets after the extraction of surface substances fly into the sample inlet of the mass spectrometer under the action of the electric field force of the electric field, so as to output a mass spectrum corresponding to the current test point based on the ions ionized by the surface substances extracted from the flying extraction solution droplets by the mass spectrometer.

2. The ion source of claim 1, wherein, The ion source further comprises a camera and an electrical impedance measurement device, the camera faces the tissue section sample and the scanning probe, the electrical impedance measurement device is connected to the scanning probe, and the electrical impedance measurement device is used for measuring the electrical resistance value between the needle tip of the scanning probe and the surface of the test point; the control device is electrically connected to the camera and the electrical impedance measurement device, respectively; The control of the second carrier to move to a distance between the needle tip of the scanning probe and the current test point to reach a specified distance comprises: controlling the camera to capture a real scene image of the scanning probe and the tissue section sample; controlling the second carrier to move to the current test point on the tissue section sample according to the relative position between the needle tip of the scanning probe and the tissue section sample in the real scene image; controlling the electrical impedance measurement device to measure the current electrical resistance value between the needle tip of the scanning probe and the surface of the current test point; According to a preset resistance-distance mapping relationship, a current distance corresponding to the current resistance value is determined, the resistance-distance mapping relationship representing a mapping relationship between resistance values and distances between a tip of the scanning probe and a surface of the to-be-measured point; According to a difference between the current distance corresponding to the current resistance value and the specified distance, the second object table is controlled to move to a distance between the tip of the scanning probe and a current to-be-measured point reaching the specified distance.

3. The ion source of claim 2, wherein, The control of the micro flow pump to deliver the extraction liquid to the scanning probe includes: controlling the micro flow pump to continuously deliver the extraction liquid to the scanning probe, or controlling the micro flow pump to deliver the extraction liquid to the scanning probe at intervals. The electrical impedance measurement device measures the resistance value between the tip of the scanning probe and the surface of the to-be-measured point by applying a voltage to the tip of the scanning probe and the surface of the to-be-measured point and measuring a current value between the tip of the scanning probe and the surface of the to-be-measured point. In the case of continuously controlling the micro flow pump to deliver the extraction liquid to the scanning probe, the control device is further configured to: alternately control the electrical impedance measurement device to apply a voltage and the high-voltage power supply to apply a specified voltage in time sequence.

4. The ion source of claim 1 or 3, wherein The micro flow pump includes a microsyringe and a driving device, the microsyringe includes a needle tube and a piston, the piston is fixed to the driving device, the needle tube stores the extraction liquid and is connected to the scanning probe through a catheter. The control of the micro flow pump to deliver the extraction liquid to the scanning probe includes: By controlling the driving device to drive the piston to move at a specified speed for a specified time length, a specified volume of the extraction liquid is delivered to the scanning probe, and the specified volume includes a picoliter volume.

5. The ion source of claim 1, wherein, The control device is further configured to: Obtain a visible light imaging image of the tissue section sample; In response to marking at least one region of interest on the visible light imaging image, divide each region of interest in the marked at least one region of interest into a plurality of to-be-detected points according to a preset resolution, wherein the preset resolution includes a subcellular level or a cellular level resolution, and the preset resolution is lower than a movement resolution of the first object table; According to the plurality of to-be-detected points divided in each region of interest in the at least one region of interest, determine the movement path, wherein the movement path contains the plurality of to-be-measured points in the at least one region of interest.

6. The ion source of claim 5, wherein, The control device is further configured to: For any region of interest, obtain a mass spectrum image of each to-be-detected point in the region of interest output by the mass spectrometer, the mass spectrum image representing a relationship between ion mass-to-charge ratio and ion intensity of surface material of the to-be-detected point; According to the mass spectrum image of each to-be-detected point in the region of interest, generate a mass spectrum image corresponding to the region of interest, the mass spectrum image representing molecular composition of surface material of each to-be-detected point in the region of interest. After the surface material extraction is completed, the extraction liquid droplet flies into the sample inlet of the mass spectrometer in the form of a droplet or an electrospray under the action of the electric field force of the electric field, and the extracted surface material in the extraction liquid droplet in the electric field ionizes in the process of flying into the mass spectrometer.

7. The ion source of claim 1, wherein, ​ 8. The ion source of claim 1, wherein, The scanning probe comprises a general capillary for the field of electrospray ionization, the general capillary is conical, the tip is pointed, and the tail end is detachably connected with the second holder; The second holder has a cavity inside and a bypass channel is arranged on the side wall, the bypass channel is connected with the conduit and is used for transmitting the extraction solution delivered by the micro flow pump.

9. The ion source of claim 1, wherein, The specified distance includes 5-10 μm, and the specified voltage includes 2-3 kV.

10. A mass spectrometry imaging system, characterized by, The system comprises the ion source according to any one of claims 1 to 9, and a mass spectrometer.

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