Cryogenic mass spectrometry imaging ion source and mass spectrometer

Abstract

The application discloses a frozen mass spectrum imaging ion source and a mass spectrometer, and relates to the technical field of mass spectrum imaging. The mass spectrum imaging ion source comprises a freezing table and a laser source. The freezing table is used for freezing a sample to be measured in situ, so as to keep the real topographic features of the sample to be measured. The laser source is used for generating laser. The laser is incident on the sample to be measured and interacts with the sample to generate ions to be analyzed. The laser source selects a vacuum ultraviolet to extreme ultraviolet wave band. The laser of the wave band can be strongly absorbed by endogenous water molecules / solution of the frozen biological tissue. On one hand, local micro area thermal desorption generates neutral molecules / ions. On the other hand, the endogenous water molecules / solution releases enough H + and Na + / K + , so that the ionization efficiency of the neutral molecules is greatly improved. Since the laser of the vacuum ultraviolet to extreme ultraviolet wave band can obtain a nanometer scale focusing size, the mass spectrum imaging ion source can be used to obtain mass spectrum imaging information with a submicron to nanometer spatial resolution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry imaging technology, in particular to a frozen mass spectrometry imaging ion source and a mass spectrometer. BACKGROUND

[0002] Mass spectrometry analysis is an analysis method that ionizes a substance first, separates ions according to mass-to-charge ratio, and then measures the intensity of various ion spectral peaks to achieve the purpose of analysis. Molecular mass spectrometry imaging technology is an advanced analysis technology that combines the high sensitivity and spatial resolution of mass spectrometry analysis, and can locate and quantify the distribution of specific molecules on a two-dimensional or three-dimensional sample surface. This technology allows researchers to directly observe the distribution pattern of molecules in the sample, rather than just obtaining average concentration data, which is crucial for various applications in biology, medicine, pharmacology, pathology and other life science fields.

[0003] Currently, the mainstream molecular mass spectrometry imaging technology is matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), which first freezes and slices the tissue sample (generally 10-30 μm thick), transfers the slice to an indium tin oxide glass slide (ITO), and then sprays an organic matrix. The slice after spraying the matrix is placed in a mass spectrometer to perform laser point-by-point scanning to obtain the mass spectrum of each sampling point / pixel, and the two-dimensional coordinates are reconstructed to obtain the mass spectrometry imaging graph. However, the introduction of the organic matrix will cause serious background spectral peak interference in the spectrum, especially for ions with m / z < 500 (metabolites of tissues / cells appear in this mass range); and the matrix sprayed onto the sample surface needs to form a co-crystal with the tissue sample to obtain better signal enhancement, but the traditional matrix-sample co-crystal particles are usually several μm to several tens of μm, so they can only be used for low spatial resolution tissue mass spectrometry imaging analysis, and cannot be used for single-cell mass spectrometry imaging analysis; the highest spatial resolution of the current mainstream MALDI-MSI technology is generally only 5 μm, which also makes it difficult to use for single-cell mass spectrometry imaging analysis. SUMMARY

[0004] The main purpose of the present application is to provide a frozen mass spectrometry imaging ion source, a mass spectrometer and a mass spectrometry imaging method, which aims to maintain the true topography of the sample to be tested while improving the mass spectrometry imaging resolution of the sample to be tested. The strong interaction of laser with endogenous water molecules / solution in frozen biological tissues is used to significantly improve the ionization efficiency of the sample to be tested without applying any matrix, thereby breaking through the bottleneck problem of mutual restriction between spatial resolution and ionization efficiency.

[0005] To achieve the above object, the application provides a frozen mass spectrometry imaging ion source, which comprises:

[0006] A freezing table is arranged for placing and freezing a sample to be tested.

[0007] A laser source is arranged for generating laser light, and the laser light is incident on the sample to be tested and interacts with the sample to generate ions to be analyzed.

[0008] In an embodiment, the refrigeration mode of the freezing table comprises at least one of liquid nitrogen circulation refrigeration and semiconductor refrigeration.

[0009] In an embodiment, the laser source is a pulsed light source.

[0010] In an embodiment, the pulsed light source has a wavelength range of 0.1-192 nm, a beam diameter range of 10 nm-10 mm, a pulse width range of 1 as-1 ms, an instantaneous pulse energy range of 1 nJ-100 mJ, and a pulse frequency range of 0.05 Hz-100 MHz.

[0011] In an embodiment, the frozen mass spectrometry imaging ion source further comprises:

[0012] A two-dimensional moving platform is connected to the freezing table, and the two-dimensional moving platform is arranged for controlling the freezing table to move in two-dimensional directions.

[0013] In an embodiment, the frozen mass spectrometry imaging ion source further comprises:

[0014] A post-ionization source is arranged for ionizing the sample to be tested to increase the ions to be analyzed.

[0015] In an embodiment, the post-ionization source comprises at least one of a laser post-ionization source, an electrospray post-ionization source, and a field-induced post-ionization source.

[0016] In an embodiment, the laser post-ionization source has a wavelength range of 0.1-2940 nm, a beam diameter range of 10 nm-10 mm, a pulse width range of 1 as-1 ms, an instantaneous pulse energy range of 1 nJ-100 mJ, and a pulse frequency range of 0.05 Hz-100 MHz.

[0017] The application further provides a mass spectrometer, which comprises the frozen mass spectrometry imaging ion source described above, and:

[0018] An ion guiding device is arranged for introducing the ions to be analyzed into a mass analyzer.

[0019] The mass analyzer is arranged for separating the ions to be analyzed according to mass-to-charge ratios.

[0020] An ion detector is connected to the mass analyzer for detecting the separated ions to be analyzed and generating detection data.

[0021] In one embodiment, the mass analyzer comprises at least one of a magnetic field analyzer, a quadrupole rod analyzer, an ion trap analyzer, a time of flight analyzer, and a Fourier transform analyzer.

[0022] The technical solution of the present application can keep the real topographic features of the sample to be measured, especially biological tissue, in a high vacuum environment by using a freezing stage to freeze the sample to be measured in situ. Meanwhile, the technical solution of the present application uses a vacuum ultraviolet to extreme ultraviolet band laser to irradiate the sample to be measured, and the vacuum ultraviolet to extreme ultraviolet band laser can be strongly absorbed by endogenous water molecules / solution in the frozen biological tissue, which can produce neutral molecules / ions through local micro-area thermal desorption on one hand, and release enough H + and Na + / K + on the other hand, so that the neutral molecules are subjected to proton attachment ionization and alkali metal ion addition ionization, thereby greatly improving the ionization efficiency of the neutral molecules and realizing mass spectrum imaging. Since the vacuum ultraviolet to extreme ultraviolet band laser can obtain a nanoscale focusing size, the technical solution of the present application can obtain mass spectrum imaging information with a nanoscale spatial resolution. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to the structures shown in the drawings without creative labor for those skilled in the art.

[0024] Figure 1 Structure and principle schematic diagram of an embodiment of the freezing mass spectrum imaging ion source provided by the present application;

[0025] Figure 2 Principle schematic diagram of the existing MALDI-MS technology;

[0026] Figure 3 Principle schematic diagram of the existing SIMS technology.

[0027] Explanation of reference numerals:

[0028] 1, freezing stage; 2, sample to be measured; 3, laser source; 4, two-dimensional moving platform; 5, temperature insulation column.

[0029] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0031] It should be noted that if the embodiments of the present application involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement condition, etc. between components in a certain posture, and if the certain posture changes, the directional indications also change accordingly.

[0032] In addition, if the embodiments of the present application involve descriptions such as “first”, “second”, etc., the descriptions of “first”, “second”, etc. are only for description purposes, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by “first”, “second” can explicitly or implicitly include at least one of the features. In addition, “and / or” or “and / or” appearing throughout the text means that the three parallel schemes include A scheme, or B scheme, or A and B schemes are satisfied at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize it, and when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is also not within the protection scope of the present application.

[0033] The mainstream molecular mass spectrometry imaging technology adopts matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), such as Figure 2As shown, the introduction of the organic matrix in the technology will cause serious background spectrum peak interference in the spectrum, and the highest spatial resolution can only be 5 mu m, which is difficult to be used for single cell mass spectrum imaging analysis. In addition, other traditional mass spectrum imaging such as chemical fixation method, freeze cracking method and freeze drying method are all carried out in a high vacuum environment, and cannot be used for in vivo cell imaging or direct imaging under the condition of keeping the cell complete morphology. Only dry cells or embedded cell slices can be used, but the cell surface often collapses and shrinks during vacuum drying or freeze drying process, so it is difficult to truly reflect the original physiological state. The secondary ion mass spectrometry (SIMS) technology can adopt the freeze mass spectrum imaging, such as Figure 3 As shown, it is used for tissue or biological membrane sample analysis, but it has defects such as surface damage, difficulty in obtaining uniform information of large area sample, high cost, complex operation and maintenance. In addition, some researches such as near-field optical technology, microlens fiber technology, transmission sampling technology and extreme ultraviolet laser ionization technology can improve the spatial resolution to sub-micron level, but they all use direct laser desorption / ionization or laser ionization method, which is easy to cause molecular fragmentation and difficult to obtain complete endogenous molecular information. How to break through the bottleneck of mutual restriction between spatial resolution and ionization efficiency of complete molecules is still a technical problem.

[0034] To solve the above problems, the present application provides a freeze mass spectrum imaging ion source.

[0035] Please refer to Figure 1 In an embodiment of the present application, the freeze mass spectrum imaging ion source comprises:

[0036] A freezing table 1 is arranged in the vacuum cavity and used for placing and freezing a sample 2 to be tested.

[0037] A laser source 3 is used for generating laser; the laser is incident on the sample 2 to be tested and interacts with the sample 2 to be tested to generate ions to be analyzed.

[0038] In this embodiment, the sample 2 to be tested can be a biological sample or a non-biological sample; the biological sample includes animal tissue, plant tissue, insect tissue, human tissue, single cell, 3D cell ball, organoid and other biological tissues or cells.

[0039] In this embodiment, the refrigeration mode of the freezing table 1 can be liquid nitrogen circulation freezing or semiconductor refrigeration sheet refrigeration, or other devices that can obtain subzero temperature.

[0040] In this embodiment, the laser wavelength generated by the laser source 3 is located in the range of vacuum ultraviolet to extreme ultraviolet.

[0041] In this embodiment, the frozen mass spectrometry imaging ion source works under vacuum condition, i.e. the sample 2 to be tested is placed in a vacuum environment, and the laser is transmitted in the vacuum environment and is incident on the surface of the sample to be tested.

[0042] In an optional embodiment, the frozen mass spectrometry imaging ion source comprises a vacuum chamber for providing a vacuum environment with a certain vacuum degree, and the frozen stage is arranged in the vacuum chamber.

[0043] The specific implementation principle of generating the ions to be analyzed using this embodiment is as follows:

[0044] The sample 2 to be tested is placed on the frozen stage 1, and the surface temperature of the platform is rapidly reduced by the frozen stage 1, so that the sample to be tested rapidly enters and remains in a frozen state, realizing in-situ freezing. Compared with the traditional vacuum drying sample or freeze-dried sample, the in-situ freezing method can maintain the in-situ morphology of the sample to be tested (especially the in-situ morphology of biological tissues or cells), and does not cause collapse, shrinkage and molecular degradation. Moreover, since the endogenous solution such as biological tissues or cell liquid in the sample to be tested is frozen, a large amount of volatile substances will not be generated, and therefore the biological tissue or cell sample can be used for mass spectrometry imaging analysis in a high vacuum environment. Compared with the prior art, the present application can perform high-sensitivity detection and imaging analysis on volatile substances in tissues or cells.

[0045] After the vacuum chamber is pumped to a certain vacuum degree, the laser source 3 is started to generate vacuum ultraviolet to extreme ultraviolet band laser, which is incident on the surface of the frozen sample 2 to be tested. The laser in the vacuum ultraviolet to extreme ultraviolet band range can be strongly absorbed by the endogenous water molecules / solution of the frozen biological tissues or cells. On the one hand, the frozen sample to be tested can be locally micro-desorbed to generate neutral molecules; on the other hand, the endogenous water molecules / solution will release a large amount of H + , Na + and K + ions under the action of the laser in the vacuum ultraviolet to extreme ultraviolet band range; wherein, the water molecules are broken under the irradiation of the vacuum ultraviolet or extreme ultraviolet laser to form a large amount of H + , and free radicals and other active molecules such as hydrated electrons and hydroxyl radicals, which further interact with the ions such as Na + and K + surrounded by water molecules or some organic molecules in the solution to strip them from the solvent molecules to form free ions. These ions cause the neutral molecules to undergo proton attachment ionization and alkali metal ion addition ionization to generate target molecules such as [M+H] + , [M+Na] + and [M+K] + (M represents a neutral molecule) to form charged ions, i.e. the neutral molecules are ionized to obtain the ions to be analyzed required for mass spectrometry analysis.

[0046] The ions to be analyzed are used for mass spectrometry analysis. The ions to be analyzed escape into the vacuum chamber and are guided by the ion guide device into the mass analyzer for separation according to the difference in mass-to-charge ratio; further analyzed and detected by the ion detector to obtain mass spectrum data. By scanning the sample to be tested by the laser and collecting mass spectrum data (mass-to-charge ratio and ion intensity) of different regions, a mass spectrum image can be generated on the host computer.

[0047] In this embodiment, the environment in the vacuum chamber can be low vacuum or high vacuum, or other gases (such as one or a mixture of nitrogen, argon, helium, and hydrogen) can be filled as background gas, and the gas pressure range is 1x10 -7 Pa-1x10 5 Pa. The specific vacuum value and the type of background gas are set according to the requirements of the laser source and the sample to be tested.

[0048] In this embodiment, the neutral molecules can be molecules of exogenous drugs and endogenous metabolites of biological tissue or cell samples, and the chemical composition information thereof is analyzed by mass spectrometry to elucidate the disease mechanism and develop new drugs.

[0049] The technical solution of the present application can in-situ freeze the sample to be tested, especially biological tissue, by using a freezing stage, so that the real topographic features of the sample to be tested can be maintained in a high vacuum environment to obtain an in-situ distribution image of the chemical composition of the sample to be tested. At the same time, the technical solution of the present application uses a vacuum ultraviolet to extreme ultraviolet band laser to irradiate the sample to be tested. The vacuum ultraviolet to extreme ultraviolet band laser can be strongly absorbed by endogenous water molecules / solution in the frozen biological tissue. On the one hand, local micro-area thermal desorption can occur to generate neutral molecules / ions, and on the other hand, the endogenous water molecules / solution can release enough H + and Na + / K + ions under the action of the laser, so that the neutral molecules are subjected to proton attachment ionization and alkali metal ion addition ionization, thereby greatly improving the ionization efficiency of the neutral molecules and realizing efficient ionization, which can be used for mass spectrometry imaging. Since the vacuum ultraviolet to extreme ultraviolet band laser can obtain a focusing size of sub-micron to nanometer, the technical solution of the present application can obtain mass spectrometry imaging information with a spatial resolution of sub-micron to nanometer.

[0050] In an embodiment, the laser source 3 is a pulsed light source.

[0051] In the embodiments of the present application, the pulsed light source refers to a light source capable of rapidly emitting high-intensity light pulses in a short time, each pulse lasting from picoseconds (ps) to milliseconds (ms), and the pulse interval being long. The pulsed laser can provide high energy in a short time, effectively ionizing macromolecules in the sample to be tested without excessive decomposition, thereby improving the sensitivity and signal-to-noise ratio of mass spectrometry. Moreover, the pulsed laser helps to improve the spatial resolution, so that the sample components in different regions can be accurately distinguished, thereby realizing the visualization of molecular distribution at the tissue or cell level.

[0052] In this embodiment, the wavelength of the light beam of the pulsed light source ranges from 0.1 to 192 nm, the diameter of the light beam ranges from 10 nm to 10 mm, the pulse width ranges from 1 as to 1 ms, the instantaneous pulse energy ranges from 1 nJ to 100 mJ, and the pulse frequency ranges from 0.05 Hz to 100 MHz.

[0053] According to the actual use, the pulsed light source can be selected to be a vacuum ultraviolet waveband between 120 and 130 nm to effectively ionize the sample without causing a large amount of thermal effect; in order to further improve the resolution, an extreme ultraviolet pulsed light source with a shorter wavelength, for example, 50-120 nm, can also be selected; the diameter of the light beam is selected to be 0.2-5 microns; the pulse width can be selected to be femtosecond-nanosecond level, for example, 1-10 6 femtoseconds to reduce the influence of thermal diffusion and other scattering effects and improve the spatial resolution; the selection of the pulse energy takes into account the ablation depth and ionization efficiency of the sample, for example, the pulse energy can be selected to be in the range of several tens of μJ to several hundred μJ; the selection of the pulse frequency is related to the sampling speed and the prevention of overheating of the sample; a lower pulse frequency, for example, several tens of Hz, allows the sample to have more time to cool, thereby reducing the thermal effect and mass diffusion and improving the quality of the image; a higher pulse frequency is beneficial to improve the acquisition speed, especially for imaging tasks of large area or multiple point sampling, such as a value in the range of 1 kHz-1 MHz. The above values of the wavelength of the light beam, the diameter of the light beam, the pulse width, the instantaneous pulse energy, and the pulse frequency are exemplary descriptions, and the specific values can be determined according to the actual application environment and the parameter indexes of the laser source.

[0054] In an embodiment, the laser source 3 can be selected to be a continuous light source.

[0055] The continuous light source refers to a light source capable of continuously and uniformly emitting laser light in a relatively wide wavelength range. The continuous light source can continuously adjust the output power according to actual needs, so that more accurate energy control can be obtained, which is beneficial to optimize the ionization process and reduce sample damage; in addition, due to the stable emission spectrum property of the continuous light source, the energy output can be maintained stable for a long time, which is beneficial to improve the repeatability and accuracy of the experiment, especially in the case of needing to collect data for a long time or compare multiple experiments.

[0056] In this embodiment, the continuous light source has a wavelength range of 0.1-192 nm, a beam diameter range of 10 nm-10 mm, and an average power greater than 0.1 mW.

[0057] According to actual use, the continuous light source can use a vacuum ultraviolet waveband between 120-130 nm, which can effectively ionize the sample without causing a large amount of heat effect; in order to achieve high spatial resolution, the beam diameter is controlled to be sub-micron to micron, for example, 0.2-5 microns. In order to effectively ionize the sample, the average power of the continuous light source is greater than 0.1 mW; according to the vacuum degree, the refrigeration temperature, and different samples to be measured, the average power can be adjusted to 0.1 mW-1 mW, for example, 0.5 mW can be selected. In order to further improve the resolution, an extreme ultraviolet continuous light source with a shorter wavelength can also be selected, for example, 50-120 nm; the photon energy of the extreme ultraviolet light source is higher, and the desorption / ionization reaction of the sample to be measured is more intense. The above-mentioned values of the beam wavelength, the beam diameter, and the average power are exemplary descriptions, and the specific values can be determined according to the actual application environment and the parameter index of the laser source.

[0058] Referring to Figure 1 In an embodiment, the refrigerated mass spectrometry ion source further comprises:

[0059] A two-dimensional moving platform 4 is connected with the refrigerated table 1; the two-dimensional moving platform 4 is used to control the refrigerated table 1 to move in two-dimensional directions.

[0060] In this embodiment, the two-dimensional moving platform 4 can move in the X-axis and Y-axis directions, thereby driving the refrigerated table 1 to move in the X-axis and Y-axis directions.

[0061] In this embodiment, the mass spectrometry imaging mode is a scanning mode mass spectrometry imaging. The position of the laser source and the incident angle of the laser remain unchanged, and the refrigerated table 1 and the sample to be measured 2 fixed thereon can be moved by controlling the two-dimensional moving platform 4, so as to realize point-by-point scanning of the sample to be measured by the laser. Recording all the ion information to be analyzed of each region of interest on the surface of the sample to be measured can obtain a two-dimensional mass spectrum in the XY direction.

[0062] In this embodiment, the two-dimensional moving platform 4 can be manually controlled, or can be controlled by any one of motor driving, piezoelectric ceramic driving, and piezoelectric motor driving.

[0063] In this embodiment, the two-dimensional moving platform 4 can use a direct reading method to feed back position information, or can use a grating ruler, a capacitive grating ruler, or other feedback forms; the two-dimensional moving platform is controlled according to the feedback position information so that the laser is accurately incident to the target position of the sample to be measured.

[0064] In another embodiment, the mass spectrometry imaging mode can also be selected as a micro-mode mass spectrometry imaging. The micro-mode mass spectrometry imaging combines the spatial resolution advantage of traditional mass spectrometry imaging and the microscopic resolving power of an optical microscope. By superimposing the optical microscope image and the mass spectrometry imaging image, more accurate positioning can be achieved, and higher precision analysis of the molecular distribution on the sample surface can be realized.

[0065] Referring to Figure 1 In an embodiment, the frozen mass spectrometry imaging ion source further comprises:

[0066] The temperature-insulated column 5 is arranged between the two-dimensional moving platform 4 and the freezing table 1, and the two-dimensional moving platform 4 is connected with the freezing table 1 through the temperature-insulated column 5.

[0067] The freezing table 1 and the two-dimensional moving platform 4 are connected through the temperature-insulated column 5, so that heat transfer between the freezing table and the two-dimensional moving platform is avoided, and the refrigeration effect of the freezing table is ensured to be good.

[0068] In an embodiment, the frozen mass spectrometry imaging ion source further comprises:

[0069] The post-ionization source is used to ionize the sample to be tested to increase the ions to be analyzed.

[0070] In this embodiment, in order to further enhance the ionization efficiency of neutral molecules of the sample to be tested, in addition to the desorption / ionization by the first laser (i.e., the laser source described in the above embodiment), a post-ionization source is arranged to assist and / or enhance the ionization of neutral molecules.

[0071] In an embodiment, the post-ionization source comprises at least one of a laser post-ionization source, an electrospray post-ionization source, and a field-induced post-ionization source.

[0072] The laser post-ionization source is another laser source arranged in addition to the above-described laser source, which assists the desorption / ionization of the target region by generating laser of certain wavelength and energy, such as single-photon ionization (SPI), resonance multiphoton ionization (REMPI), and non-resonance multiphoton ionization (NRMPI), to further improve the ionization efficiency of neutral molecules.

[0073] The electrospray post-ionization source generates ions by electrospray. The electrospray is generated by applying high voltage to the liquid to be analyzed, and the remaining ions to be analyzed are left after the droplets are completely evaporated. The electrospray post-ionization source has good applicability to many thermally unstable, fragile macromolecules (such as biological molecules such as proteins, polypeptides, nucleic acids, etc.), which can reduce the fragmentation of the sample, thereby retaining more original molecular information. In addition, it can also be applied to qualitative and quantitative analysis of various compounds such as small molecule compounds, drug metabolites, lipids, etc.

[0074] Field-induced after ionization source uses extremely strong local electric field to extract and ionize electron in gas molecule, and then generates ion, which can be used for mass spectrum analysis of gaseous atom and small molecule.

[0075] In this embodiment, for different laser desorption / ionization states of the to-be-tested sample (such as generating thermally unstable macromolecules, generating gas, and low ionization efficiency), a suitable after ionization source can be selected to assist or enhance ionization of neutral molecules. The after ionization source can be selected as one, or any two or more appropriate combinations.

[0076] In an embodiment, the after ionization source is a laser after ionization source.

[0077] The wavelength range of the light beam of the laser after ionization source is 0.1-2940 nm, the beam diameter range is 10 nm-10 mm, the pulse width range is 1 as-1 ms, the instantaneous pulse energy range is 1 nJ-100 mJ, and the pulse frequency range is 0.05 Hz-100 MHz.

[0078] According to actual use, the laser after ionization source can select an extreme ultraviolet to vacuum ultraviolet band of 50-150 nm to effectively ionize the sample; the beam diameter range is matched with the beam diameter of a continuous light source or a pulsed light source, and can be selected as 10 nm-10 μm; the pulse width can be selected as femtosecond to nanosecond level, such as 1-10 6 femtosecond, to reduce the influence of thermal diffusion and other scattering effects; the instantaneous pulse energy range can be selected as several tens of μJ to several hundreds of μJ, and the pulse frequency can be selected as a lower pulse frequency such as several tens of Hz, to reduce thermal effects and avoid the after ionization source causing the thermal desorption area of the to-be-tested sample to expand.

[0079] In summary, in the embodiments of the present application, the to-be-tested sample, especially biological tissue or cells, can be in-situ frozen by using a freezing stage, so that the real topographic features of the to-be-tested sample can be maintained in a high vacuum environment. At the same time, the technical solution of the present application uses a vacuum ultraviolet to extreme ultraviolet band laser to irradiate the to-be-tested sample, and the vacuum ultraviolet to extreme ultraviolet band laser can be strongly absorbed by endogenous water molecules / solution in the frozen biological tissue, which can generate neutral molecules / ions through local micro-area thermal desorption on one hand, and release enough H + and Na + / K +The neutral molecules are subjected to proton attachment ionization and alkali metal ion addition ionization, so that the ionization efficiency of the neutral molecules is greatly improved, and the required ions to be analyzed are provided for mass spectrum imaging. Compared with the mainstream MALDI-MS technology, the interference of the organic matrix on the background spectrum peak is avoided, and compared with other mass spectrum imaging technologies using dry samples, the real topographic features of the samples are maintained. Moreover, since the vacuum ultraviolet to extreme ultraviolet band laser can obtain a focusing size of submicron to nanometer, the cold-frozen mass spectrum imaging ion source provided by the application can be used to obtain mass spectrum imaging information with a spatial resolution of submicron to nanometer, thereby breaking through the bottleneck problem that the high spatial resolution and the high ionization efficiency are mutually restricted.

[0080] The application further provides a mass spectrometer, which comprises the cold-frozen mass spectrum imaging ion source described above, and the mass spectrometer comprises:

[0081] An ion guiding device is used to introduce the ions to be analyzed into a mass analyzer.

[0082] The mass analyzer is used to separate the ions to be analyzed according to the mass-to-charge ratio.

[0083] An ion detector is connected to the mass analyzer and is used to detect the separated ions to be analyzed and generate detection data.

[0084] In an optional embodiment, the ion guiding device is designed as a series of electrodes to form an electric field for guiding ion transmission. The main function of the ion guiding device is to transmit the ions generated in the ion source to the mass analyzer in an orderly manner, to ensure that the ions are lost as little as possible during the transmission process, and to ensure the purity and focusing degree of the ion beam, so as to effectively perform subsequent mass separation and detection.

[0085] The mass analyzer separates and detects the ions generated by the ion source according to the mass-to-charge ratio (m / z, i.e., the ratio of mass to charge).

[0086] The ion detector is used to detect and quantify the separated ions, to convert the charge ratio and intensity signal of the ions into an electric signal (i.e., to generate detection data), and the electric signal is transmitted to an upper computer to generate a related mass spectrum image.

[0087] In an embodiment, the mass analyzer comprises at least one of a magnetic field analyzer, a quadrupole rod analyzer, an ion trap analyzer, a time-of-flight analyzer and a Fourier transform analyzer.

[0088] The magnetic field analyzer separates ions with different mass-to-charge ratios by magnetic field. When the ions move in the magnetic field, they will be subjected to a force perpendicular to the direction of the ion velocity. The force is proportional to the charge amount of the ion, the velocity and the magnetic field strength, and is related to the mass of the ion and the angle between the velocity direction and the magnetic field direction. Because the Lorentz force received by ions with different masses is different under the same conditions, the trajectories of the ions will be different after passing through the magnetic field, so that the ions can be separated according to the mass-to-charge ratio (m / z).

[0089] In the quadrupole analyzer, the ions vibrate in the quadrupole field formed by four mutually perpendicular electrodes according to a specific stable condition. Only ions with a specific mass-to-charge ratio can pass through the quadrupole region to reach the detector.

[0090] In the ion trap analyzer, the ions are trapped in a triple quadrupole rod or a linear ion trap. By adjusting the electric field strength, ions within a specific mass range are allowed to exit the trap in sequence and pass through the detector. The ion trap includes a linear ion trap, an orbital ion trap, a rectangular ion trap, etc.

[0091] In the time-of-flight analyzer, the ions are accelerated under the action of an electric field and pass through a constant-length flight tube. The smaller the ion mass, the faster the flight speed, and the shorter the time required to reach the detector. Therefore, different masses of ions can be distinguished according to the time difference of reaching the detector.

[0092] In the Fourier transform analyzer, in some multi-dimensional mass spectrometry techniques (such as Fourier transform ion cyclotron resonance mass spectrometry, FT-ICR MS), Fourier transform is used to analyze the signals of ions revolving in a magnetic field, thereby achieving high-precision measurement of ion mass.

[0093] In this embodiment, the mass analyzer can select any one of the analyzers for separating and detecting ions with different mass-to-charge ratios. The mass analyzer can select one, or any two or more for appropriate combination.

[0094] The mass spectrometer proposed by the present application adopts all the technical solutions of the above-mentioned embodiments, and therefore at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0095] The above only describes exemplary embodiments of the present application, and does not limit the patent scope of the present application. Any equivalent structural transformation made by using the content of the present application specification and drawings, or direct / indirect application in other related technical fields is included in the patent protection scope of the present application.

Claims

1. A cryo-mass spectrometry imaging ion source, characterized in that, include: A freezing stage is used to place and freeze biological tissues to be tested in situ without any matrix applied. A laser source is used to generate laser light in the vacuum ultraviolet to extreme ultraviolet band; the laser light is incident on the biological tissue to be tested and interacts with the biological tissue to generate ions to be analyzed.

2. The cryo-mass spectrometry imaging ion source as described in claim 1, characterized in that, The refrigeration method of the freezing stage includes at least one of liquid nitrogen circulating freezing and semiconductor refrigeration.

3. The cryo-mass spectrometry imaging ion source as described in claim 1, characterized in that, The laser source is a pulsed light source.

4. The cryo-mass spectrometry imaging ion source as described in claim 3, characterized in that, The pulsed light source has a beam wavelength range of 0.1-192nm, a beam diameter range of 10nm-10mm, a pulse width range of 1as-1ms, an instantaneous pulse energy range of 1nJ-100mJ, and a pulse frequency range of 0.05Hz-100MHz.

5. The cryo-mass spectrometry imaging ion source as described in claim 1, characterized in that, The mass spectrometry imaging ion source also includes: A two-dimensional moving platform is connected to the freezing stage; the two-dimensional moving platform is used to control the movement of the freezing stage in a two-dimensional direction.

6. The cryo-mass spectrometry imaging ion source as described in claim 1, characterized in that, The cryo-mass spectrometry imaging ion source also includes: The post-ionization source is used to ionize the sample to increase the number of ions to be analyzed.

7. The cryo-mass spectrometry imaging ion source as described in claim 6, characterized in that, The post-ionization source includes at least one of a laser post-ionization source, an electrospray post-ionization source, and a field-induced post-ionization source.

8. The cryo-mass spectrometry imaging ion source as described in claim 7, characterized in that, The laser post-ionization source has a beam wavelength range of 0.1-2940nm, a beam diameter range of 10nm-10mm, a pulse width range of 1as-1ms, an instantaneous pulse energy range of 1nJ-100mJ, and a pulse frequency range of 0.05Hz-100MHz.

9. A mass spectrometer, characterized in that, Includes the cryo-mass spectrometry imaging ion source according to any one of claims 1 to 8, and: An ion guiding device is used to introduce the ions to be analyzed into a mass analyzer. A mass analyzer is used to separate the ions to be analyzed according to their mass-to-charge ratio; as well as, An ion detector is connected to the mass analyzer. Used to detect the separated ions to be analyzed and generate detection data.

10. The mass spectrometer as described in claim 9, characterized in that, The mass analyzer includes at least one of a magnetic field analyzer, a quadrupole analyzer, an ion trap analyzer, a time-of-flight analyzer, and a Fourier transform analyzer.

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