A LIBS-MS combined flow cytometry single cell element imaging method and analyzer

Abstract

The present invention discloses a method for elemental imaging of single cells using LIBS-MS combined with flow cytometry, including preparing a single cell suspension labeled with a metal isotope label antibody, delivering the single cell suspension to an erosion area by flow injection, performing high-speed scanning of the erosion area by femtosecond laser ablation, performing LIBS detection and ICP-TOF-MS detection, and comparing the detection signals with each other to form a single cell comprehensive imaging. On the other hand, the present invention discloses an analyzer combined with LIBS-MS, including a single cell sample automatic injection system, a femtosecond laser ablation atomization module, a sample pool, a fiber optic spectrometer, and an inductively coupled plasma time-of-flight mass spectrometer. The LIBS-MS combination of the present invention obtains more comprehensive elemental information and a wider content dynamic range, combining the advantages of high throughput of flow technology, high number of detection channels of mass spectrometry detection, low interference between channels, and providing in-situ information of imaging technology; at the same time, the analyzer can also detect a variety of samples such as aqueous solutions and tissue sections, and is suitable for a variety of different detection scenarios.

Description

Technical Field

[0001] The invention belongs to the technical field of laser ablation mass spectrometry detection, and in particular relates to an element imaging method and an analyzer for a LIBS-MS combined flow cytometry single cell. Background Art

[0002] Flow cytometry (FCM) is a biological technique that performs rapid quantitative analysis and sorting of cells or other biological particles (such as microspheres, bacteria, small model organisms, etc.) arranged in a single file in a liquid flow. Flow cytometry obtains effective information from a large number of individual measurement results with individual differences by performing statistical analysis. It can simultaneously analyze gene expression and protein expression in cell samples at the single-cell level, and can also detect cell functions such as cell activity, cell cycle, cell apoptosis, cell proliferation and cell oxidation. The routine operation process of flow cytometry mainly includes the preparation of single-cell suspension, labeling of fluorescent antibodies, on-machine detection and data processing of flow cytometer, etc. For details, see Figure 1 .

[0003] Traditional flow cytometers (flow cytometry) are fluorescence-based detection systems. After more than 40 years of development, they have become very mature and have been widely used in various fields from basic research to clinical practice, covering fields such as cell biology, immunology, hematology, oncology, pharmacology, genetics, and clinical testing, playing an important role in various disciplines. Flow cytometers use lasers as light sources to generate scattered light and fluorescence signals, and these signals are detected by detectors such as photodiodes or photomultiplier tubes, so as to quickly screen and analyze single cells in a solution or analyze and separate and purify specific cell subpopulations. Flow cytometers refer to Figure 2, including a light source 001, a mobile phase pipeline 002 with a sleeve structure, a forward angle scatter detector 003, a side angle scatter detector 004, multiple fluorescence signal detectors 005 and multiple beam splitters 006. Flow cytometers analyze various parameters of cells through optical signals. Optical signals include scattered light signals and fluorescent signals. Scattered light signals can be used to reflect physical information such as cell size and granularity, including forward angle (i.e. 0 angle) scatter (forward scatter, FSC) and side angle scatter (also known as 90 angle scatter) (sidescatter, SSC). General flow cytometers have these two channels. Fluorescence signals mainly include two parts: ① Autofluorescence, that is, the fluorescence emitted by fluorescent molecules inside cells after being irradiated with light without fluorescent staining; ② Characteristic fluorescence, that is, the fluorescence emitted by the fluorescent dyes bound to the cells after being irradiated with light, and its fluorescence intensity is weak, and the wavelength is different from the irradiation laser. Fluorescence signals can be used to reflect chemical characteristics of cells such as antigen expression. The number of fluorescence channels is determined according to the different flow cytometers.

[0004] However, traditional flow cytometry has encountered bottlenecks in its development process and still has some defects, which are mainly manifested in three aspects. First, due to the limitation of fluorescent dyes, spectral leakage, autofluorescence and other problems cannot be avoided. Second, the overlap of emission spectra of different fluorescent groups will cause signals between channels to interfere with each other. Third, the number of detection channels of traditional flow cytometers (<20) has been difficult to improve qualitatively. All of these have made experimental design and compensation calculations quite complicated, and experimental operations are quite inconvenient.

[0005] In response to the bottlenecks and defects of traditional flow cytometry, scientists have developed mass spectrometry flow cytometry technology. Mass cytometry (CyTOF) uses antibodies with metal isotope labels to label cells, and analyzes the label composition on each cell through time-of-flight mass spectrometry to conduct in-depth research on cell phenotypes and functions. Unlike traditional flow cytometers that use detectors such as photodiodes or photomultiplier tubes for detection, mass spectrometry uses stable heavy metal isotopes (mainly lanthanides) instead of fluorescent groups as signal reporting groups. The cells are then introduced into the CyTOF analyzer and atomized into droplets. The droplets are vaporized, atomized, and ionized, and then brought into the mass spectrometer through potential acceleration. Finally, the filtered ion cloud is analyzed using a TOF-MS detector. For a schematic diagram of the mass cytometry principle, see Figure 3 .

[0006] The droplet atomization process is a very important step in mass spectrometry flow cytometry, which directly affects the sensitivity of detection. Since the general nebulizer only disperses the mobile phase carrying single cells into single-cell droplets, a spray chamber is required between it and the ICP source (ICP torch) to further disintegrate and disperse the cells. The specific process diagram is shown in Figure 4 After the mass spectrometry sample is prepared, it enters the instrument in the form of a cell suspension and is divided into small droplets by a nebulizer, each of which contains a cell. After the droplets containing cells enter the spray chamber, a high flow of argon gas further atomizes and disperses the aerosol droplets (some instruments are also equipped with a heating device, and the heating temperature in this link can reach 200°C), and the atomized aerosol is transmitted to the ICP source for plasma.

[0007] Mass spectrometry flow cytometry has the advantages of high channel number, low inter-channel interference, low background interference and high sensitivity, but it has the following main defects:

[0008] 1. Traditional mass spectrometry usually uses high-flow argon gas to atomize droplets. Some instruments also need to heat the atomization chamber to 200 degrees Celsius for atomization. At present, the atomization efficiency is generally 10-20%, and the transmission efficiency is generally below 10%, and the overall atomization effect is poor;

[0009] 2. Through atomization, cells usually enter the droplet phase as a whole and then form an aerosol. The test result is the overall information of the cells, without the in situ information of the cells.

[0010] With the further development of technology, cytometry has developed imaging technology based on the detection of overall information, such as Imaging Flow Cytometry (IFC). Imaging flow cytometry uses bright-field transmission imaging (Bright-field image) instead of forward angle scattering signal to provide more accurate cell positioning information and cell morphology information, etc.; and the fluorescence signal generated by the cell through excitation is imaged and amplified, and then decomposed according to different fluorescence wavelength ranges and projected onto different CCD array strips, realizing simultaneous imaging of multiple fluorescence channels, that is, Figure 2 The scattered light detector and fluorescence detector in the apparatus are replaced with corresponding imaging detectors.

[0011] In the field of mass spectrometry, there is also an imaging mass cytometry (IMC) with the same name. However, this technology is not based on a flow system, but usually images fixed cell samples (such as tissue sections, etc.). This is due to several factors: on the one hand, the mass spectrometry imaging signal is realized by scanning, that is, the information of each pixel is distinguished by time resolution instead of spatial resolution. Traditional mass spectrometry imaging takes a long time, so if the cells are not fixed (fixation methods include chemical fixation, frozen sections, etc.), the position information will be lost; on the other hand, ICP-MS or ICP-TOF-MS is difficult to accurately measure light elements in cells (including common elements in organic matter and elements corresponding to some common inorganic salts), such as: C, H, O, N, F, Na, etc., so that the cell location / morphology information provided by the non-specific signal of mass spectrometry detection is not as easy to extract as bright field imaging. Compared with imaging flow cytometry, the detection throughput of imaging mass spectrometry cytometry based on fixed tissue sections is more limited.

[0012] In order to further detect light elements in cells, the present invention combines LIBS technology with flow imaging detection. LIBS is the abbreviation of Laser-Induced Breakdown Spectroscopy. This technology uses the plasma generated by a pulsed laser to ablate and excite the material in the sample, and obtains the spectrum emitted by the atoms excited by the plasma through a spectrometer to identify the elemental composition in the sample. LIBS can be used for the detection and analysis of solid, liquid, gaseous samples and slurry substances, and provides spatial resolution analysis for elements that are difficult to detect with other conventional technologies (such as H, Be, B, Li, C, O, N, F, Na and other light elements), and can detect content ranging from ppm to %. However, the existing LIBS imaging technology is also mainly used for fixed tissue slice samples; when it is applied to solution samples, the matrix effect (nonlinear effect in detection) is more serious, which is mainly caused by two reasons. On the one hand, compared with other states of matter, the plasma lifetime in aqueous solution is shorter, and water has a higher ionization potential and high electronegativity. Therefore, compared with air, the contribution of charged particles in laser-induced plasma is minimal, which results in poor plasma generated in water. On the other hand, splashing and shaking caused by thermal effects on the liquid will also bring nonlinearity to the detection results, reducing the accuracy of the results.

[0013] Therefore, how to combine flow cytometry, mass spectrometry imaging and LIBS technology to simultaneously obtain the high throughput of flow cytometry, the high number of detection channels of mass spectrometry detection and the high spatial resolution of imaging technology is an urgent problem to be solved. Summary of the invention

[0014] In view of this, the purpose of the present invention is to provide a LIBS-MS combined flow cytometry single cell element imaging method. On the basis of flow technology, the liquid surface of the erosion port is subjected to high-speed scanning and erosion through a galvanometer mechanism, and the main light element signals are detected by LIBS synchronously during erosion to provide cell positioning / morphology information, and the generated aerosol is subjected to ICP-TOF-MS detection of heavy metal isotope ion signals to provide specific information (such as specific binding of label antibodies, etc.), and the two are referenced to each other to realize single-cell flow mass spectrometry imaging.

[0015] To achieve the above object, the present invention provides a method for elemental imaging of single cells by LIBS-MS combined with flow cytometry, comprising the following steps:

[0016] preparing a single cell suspension labeled with a metal isotope labeling antibody, wherein the metal isotope labeling antibody comprises a labeling element;

[0017] The single-cell suspension was delivered to the denuded area by flow injection;

[0018] Performing high-speed femtosecond laser ablation scanning on the ablation area, detecting LIBS spectrum signals generated during femtosecond laser ablation, and acquiring a LIBS signal sequence;

[0019] The aerosol generated during femtosecond laser ablation is detected by ICP-TOF-MS mass spectrometry to obtain the mass spectrum signal sequence;

[0020] Comparing the LIBS signal sequence with the mass spectrometry signal sequence to form a comprehensive elemental imaging of a single cell;

[0021] The LIBS signal sequence includes LIBS signals of at least one element among C, H, O, N, Na or F; and the mass spectrum signal sequence includes the mass spectrum signal of the tag element.

[0022] Preferably, the laser pulse width of the femtosecond laser ablation is less than 500 fs, and the repetition frequency is 1 kHz to 1 MHz; the spot diameter of the femtosecond laser ablation is 1 to 5 μm, and the flow rate of the flow injection is 0.1 to 10 μL / min.

[0023] Preferably, the LIBS signal sequence is used to provide cell localization information and / or cell morphology information.

[0024] Preferably, the mutual comparison of the LIBS signal sequence and the mass spectrometry signal sequence includes correcting the pipeline transmission time, and the formation of comprehensive elemental imaging of single cells includes restoring the time series of signals to spatial coordinate associations according to the focus position sequence of femtosecond laser ablation high-speed scanning.

[0025] Another aspect of the present invention provides a LIBS-MS combined analyzer, comprising a single cell sample automatic injection system, a femtosecond laser ablation atomization module, a sample pool, a fiber optic spectrometer and an inductively coupled plasma time-of-flight mass spectrometer;

[0026] The single cell sample automatic injection system is used for flow injection;

[0027] The femtosecond laser ablation atomization module includes a femtosecond laser, a galvanometer mechanism and a focusing lens; the femtosecond laser is used to generate ablation laser, the galvanometer mechanism is used to switch the focus position of the ablation laser at a high speed in a preset ablation area, and the focusing lens is used to focus the ablation laser;

[0028] The optical fiber spectrometer includes a spectrometer body, a spectrum collector and a transmission optical fiber; the spectrum collector is connected to the spectrometer body through the transmission optical fiber, and the spectrum collector is used to collect LIBS signals;

[0029] The inductively coupled plasma time-of-flight mass spectrometer comprises an ICP torch and a TOF-MS detector;

[0030] The sample pool comprises a sample pool housing, a multi-purpose analysis cup and a sample holder, and an air inlet is arranged on the sample pool housing;

[0031] The multi-purpose analysis cup is fixedly arranged on the sample pool housing, and is provided with a window glass, a sampling tube and a jack; the sampling tube is used to guide out the aerosol generated during femtosecond laser ablation, and the sampling tube is connected to the gas path of the inductively coupled plasma time-of-flight mass spectrometer; the jack is used to insert a spectrum collector under the condition of a sealed connection; one end of the sampling tube is opened toward the inside of the sample pool, and the other end of the sampling tube is arranged outside the sample pool; one end of the jack is opened toward the inside of the sample pool, and the other end of the jack is opened toward the outside of the sample pool;

[0032] The sample holder is arranged inside the sample pool; the sample holder includes an erosion tube, one end of which is connected to the liquid path of the single-cell sample automatic injection system; the other end of the erosion tube is located on the upper surface of the sample holder, and an optical liquid level sensor is arranged on the erosion tube, and the optical liquid level sensor is connected to the signal of the single-cell sample automatic injection system.

[0033] Preferably, the sample holder further comprises a wafer slot and a support spring; the wafer slot is used to place a sample carrier, and the support spring is used to support and fix the sample carrier so that the upper surface of the sample carrier is at a height corresponding to the upper surface of the sample holder.

[0034] Preferably, the pulse width of the ablation laser is less than 500 fs, and the repetition frequency is 1 kHz to 1 MHz; the scanning speed of the galvanometer mechanism is greater than or equal to 20.0 rad / s, and the positioning speed is greater than or equal to 30.0 rad / s.

[0035] Preferably, the single-cell sample automatic injection system comprises a high-precision injection pump, a multi-way valve and a quantitative ring, and the high-precision injection pump and the quantitative ring are respectively connected to the multi-way valve pipeline.

[0036] Preferably, the sample pool further comprises a moving stage and a transmission illuminator, wherein the transmission illuminator is arranged below the sample holder, and a high-transmittance glass is arranged at the bottom of the sample holder, and evenly distributed fixing holes are arranged on the high-transmittance glass.

[0037] Preferably, the analyzer is used for the aforementioned LIBS-MS coupled flow cytometry single-cell elemental imaging method, and can also be used for other imaging and detection methods such as mass spectrometry imaging of tissue sections or total elemental analysis of aqueous solutions.

[0038] The beneficial effects of the present invention are:

[0039] (1) The element imaging method of the present invention can achieve micro-area ablation of a flowing sample through high-speed laser ablation scanning, so that the speed of laser ablation matches the flow sampling speed, and the spatial coordinate correlation is converted into a time series; through the combination of LIBS-MS detection, the wide detection element range of LIBS detection and the high sensitivity of ICP-TOF-MS detection are matched, and the two detection methods are detected separately during the same laser ablation, so that the non-specific information (such as cell location / morphology, etc.) provided by the major light elements in a single cell and the specific information provided by the immunolabeling of the metal label antibody are compared. The final single-cell full-element imaging combines the advantages of high throughput of flow technology, high number of detection channels of mass spectrometry detection, low interference between channels and providing in situ information of imaging technology;

[0040] (2) The element imaging method and analyzer of the present invention use a combination of femtosecond laser and high-speed three-dimensional galvanometer scanning technology to realize laser ablation atomization technology to replace the traditional mass spectrometry flow atomizer, and the atomization efficiency is increased from 10-20% to nearly 100%, and the transmission efficiency is increased from 10% to more than 90%, which greatly increases the sampling volume of cells and improves the sensitivity and accuracy of instrument analysis;

[0041] (3) The femtosecond laser used in the element imaging method and analyzer of the present invention significantly improves the ionization effect and reduces the thermal effect. Compared with the nanosecond laser, the LIBS spectrum enhancement effect can be achieved by several dozen times, the signal-to-noise ratio of the spectrum line is significantly enhanced, the detection limit is lower, and the enhancement effect is particularly obvious for samples with large matrix effects such as liquid samples;

[0042] (4) The multi-purpose analysis cup of the analyzer of the present invention can not only realize the collection of sample particle aerosol, but also realize the collection of spectral signals, thereby obtaining LIBS spectral signals and ICP-TOF-MS mass spectrometric signals at the same time, providing an improved data multiple detector for mass spectrum interference elements, and further confirming the element concentration and position distribution;

[0043] (5) Compared with the LIBS detection and ICP-TOF-MS detection adopted by the present invention, the former is more suitable for the detection of elements with higher content, while the latter is more suitable for the detection of elements with lower content. When the two are used together, the linear range is matched, and a wide dynamic range detection of element content from ppm to % can be achieved; the detectable element range also covers heavy elements and various light elements commonly found in biological samples, such as C, H, O, N, Na, F, etc.;

[0044] (6) In addition to performing LIBS-MS coupled flow cytometry single-cell element imaging, the analyzer of the present invention can also be used for quantitative detection of element content in aqueous solutions, mass spectrometry imaging of tissue section samples, etc., and is suitable for a variety of different detection application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 A flow chart of flow cytometry in the prior art;

[0047] Figure 2 It is a structural schematic diagram of a flow cytometer in the prior art;

[0048] Figure 3 It is a schematic diagram of the process of mass spectrometry flow cytometry in the prior art;

[0049] Figure 4 A schematic diagram of the nebulization process in mass spectrometry flow cytometry in the prior art;

[0050] Figure 5 A schematic diagram of the structure of a multi-purpose analyzer system according to an embodiment of the present invention;

[0051] Figure 6 It is a structural schematic diagram of a multi-purpose analysis cup according to an embodiment of the present invention;

[0052] Figure 7 A schematic diagram of the structure of a sample holder according to an embodiment of the present invention;

[0053] Figure 8 The present invention is a flowchart of a method for elemental imaging of single cells using LIBS-MS combined with flow cytometry. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Embodiment 1

[0056] like Figure 5 As shown, the multi-purpose analyzer system of this embodiment includes: a single cell sample automatic injection system 100, a femtosecond laser ablation atomization module 200, a sample pool 300 (including a multi-purpose analysis cup 320), a fiber optic spectrometer 400, and an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS mass spectrometer 500);

[0057] The single cell sample automatic injection system 100 includes a sample tube, a working solution tube, a high-precision injection pump 110, a multi-way valve 120 and a quantitative ring 130, and preferably also includes components such as a liquid level controller and a flushing station. The sample tube is used to store a single cell suspension sample with a label; the working solution tube is used to store a carrier liquid and a working liquid; the high-precision injection pump 110 can accurately adjust the flow rate of the mobile phase and can operate at an extremely low and stable flow rate; the high-precision injection pump 110 is connected to the multi-way valve 120 pipeline, and the quantitative ring 130 is also connected to the multi-way valve 120 pipeline when in use.

[0058] The femtosecond laser ablation atomization module 200 includes a femtosecond laser 210, an attenuator 220, a beam expander 230, a (three-axis) galvanometer mechanism 240, an adapter ring 250, an objective lens 260 and a coaxial observation system 270, etc.; the femtosecond laser 210, the attenuator 220, the beam expander 230, the coaxial observation system 270 and the galvanometer mechanism 240 are connected in sequence, and the adapter ring 250 is used to install the focusing lens (i.e., the objective lens 260) on the galvanometer mechanism 240; the coaxial observation system 270 has a camera function to obtain an image of the focusing plane, which is used to select the scanning range of the galvanometer mechanism 240; the femtosecond laser 210 preferably has a wavelength of 515nm or less, a pulse width of less than 500fs, and a repetition frequency of 1kHz to 1MHz; the attenuator 220 can be selected as a transmission type, which can make the laser output energy adjustable from 0 to 100%. The beam expander 230 is used to expand the ablation laser beam to the same aperture as the incident hole of the galvanometer mechanism 240. The galvanometer mechanism 240 uses a reflective lens with a coating consistent with the laser wavelength, a scanning speed of ≥20.0rad / s, and a positioning speed of ≥30.0rad / s, which is used to switch or scan the focus position of the laser at high speed within a certain range; in the multi-purpose analyzer system of this embodiment, the femtosecond laser 210, the galvanometer mechanism 240 and the mobile stage adopt a linkage mode, the large-scale translation of the sample is completed by the mobile stage, and the high-speed switching of the focus position within a small range is achieved by the galvanometer mechanism 240; at the same time, a galvanometer trigger mode is adopted, that is, the galvanometer mechanism 240 sends a trigger signal to the femtosecond laser 210, and at the same time sends a trigger signal to the single cell automatic sampling system 100, the ICP-TOF-MS mass spectrometer 500 and the fiber spectrometer 400 to ensure that the instruments work in coordination. According to the analysis of the background technology section, the key to combining flow technology and mass spectrometry imaging technology is to increase the speed of focused scanning. Since the single cells in the flow system are not fixed but move in real time in the pipeline, the full ablation scanning sequence of a single cell needs to be completed before the relative position information is lost in order to use the detection signal generated by each ablation to perform imaging restoration. The ablation frequency of traditional mass spectrometry imaging or LIBS imaging for fixed slice samples is generally below 10 Hz or 10 to 100 Hz, so it is difficult to complete its combination with flow technology; the combination of the femtosecond laser 210 and the galvanometer mechanism 240 in this embodiment can greatly increase the ablation frequency to 1 kHz to 1 MHz, making it possible to combine the two.

[0059] The sample pool 300 includes a sample pool housing 310, a multi-purpose analysis cup 320, a sample holder 330, a transmission illuminator 340, a (three-axis) precision moving stage 350, etc. The sample pool housing 310 is provided with one or more air inlets. The working carrier gas is fed into the sample pool 300 from the air inlet and is sent out of the sample pool 300 from the sampling tube 322 in the multi-purpose analysis cup 320; the sampling tube 322 is connected to the ICP-TOF-MS mass spectrometer 500 by pipeline. The sample holder 330 is provided on the precision moving stage 350, and the transmission illuminator 340 provides background lighting from below the sample holder 330. Helium is preferably used as the working carrier gas, and those skilled in the art may also select other types of carrier gas according to actual needs.

[0060] Multi-purpose analysis cup 320 Figure 6 As shown, the multi-purpose analysis cup 320 is fixed to the center of the upper part of the sample pool housing 310 by means of fixing screws 324. The main body material is stainless steel or PEEK material, the upper surface is a circular plane, the center of the plane is a circular window glass 321, the axis of the laser beam passes through the window glass 321, and the lower part of the window glass 321 is a cylindrical hollow cavity structure, and the lower end opening is connected to the inner cavity of the sample pool 300; the multi-purpose analysis cup 320 is provided with an inclined stainless steel sampling tube 322, which is at an angle of 30 to 60 degrees with the horizontal plane, and the lower end opening of the sampling tube 322 is a cylindrical hollow cavity structure. The sampling tube 322 is used to collect aerosol samples formed by mixing particles and carrier gas after laser ablation atomization. The other end of the sampling tube 322 is outside the sample pool housing 310 and is connected to the inlet tube of the ICP-TOF-MS mass spectrometer 500 through a PFA tube to transport the aerosol sample to the mass spectrometer for analysis. There are two fiber optic spectrum collector sockets 323a / b on both sides of the sampling tube 322. The axis direction of the socket 323a / b is at an angle of 30 to 60° with the axis direction of the laser beam. The socket 323a / b is a through hole, and the lower end outlet is the inner wall of the cylindrical hollow cavity. The socket 323a / b is provided with a soft sealing structure. After the spectrum collector of the fiber optic spectrometer 400 is inserted, the soft sealing structure is tightly combined with the transmission optical fiber 410 to ensure that there is no air leakage. The fiber optic spectrum collector is inserted into the socket to capture the spectral signal. When in use, two optical fibers can be inserted at the same time, or one optical fiber can be used. When the optical fiber is not inserted or only one optical fiber is used, the jack 323a / b can be blocked with a soft plug to prevent air leakage. Those skilled in the art can adjust the insertion depth of the optical fiber spectrum collector according to the focal length of the focusing lens in the spectrum collector to ensure that the spectrum collector can collect the plasma emission spectrum in the LIBS detection. The radiation signal of the plasma is coupled to the transmission optical fiber 410 through the integrated spectrum collector and sent to the main body of the multi-channel optical fiber spectrometer 400, and then split and detected by a CCD detector or an enhanced charge coupled device (ICCD).

[0061] like Figure 7 As shown, the sample holder 330 is a concave groove structure. The bottom of the sample holder 330 is provided with a high light-transmitting organic glass 333 with evenly distributed small holes (for setting fixing devices such as mushroom nails) to allow the illumination light of the transmission lamp 340 to pass through; an erosion tube is fixed on the sample holder 330, and the erosion tube is a thin right-angle curved tube with openings at both ends. The left side of the horizontal section is connected to the single-cell sample automatic injection system 100, and the vertical section is coaxially arranged with the laser beam (measured at the center position of the scanning range of the galvanometer mechanism 240), and one end of the vertical section is an outlet 331; on the vertical section of the erosion tube, an optical liquid level sensor 332 is arranged near the outlet 331, and the optical liquid level sensor 332 is linked with the high-precision injection pump 110 to control the liquid level and flow rate in the erosion tube.

[0062] like Figure 8 As shown, the element imaging method of single cells using LIBS-MS combined with flow cytometry using the multi-purpose analyzer of this embodiment includes the following steps:

[0063] Preparation of single cell suspension labeled with metal isotope label antibody: culture and process the sample (including non-adherent cells from culture, bacteria, yeast, blood and tissue, etc.) into a single cell suspension, and label the cells with antibodies labeled with metal isotopes. Those skilled in the art can select a variety of different antibodies and corresponding metal isotope labels (usually lanthanide metal isotopes) according to actual needs, respectively, to label various different targets in the cell, and finally form multi-channel mass spectrometry imaging;

[0064] Instrument preparation: introduce carrier gas / protective gas into the sample pool 300; adjust the height of the precision moving stage 350 through the coaxial observation system 270, let the laser focus on the center of the outlet 331 of the erosion tube (i.e., the erosion area), and select the high-speed scanning range of the ablation laser so that it can completely cover the outlet 331 of the erosion tube; adjust the insertion depth of the optical fiber spectrum collector to ensure that the spectrum collector can collect the plasma emission spectrum; set laser ablation atomization parameters such as laser frequency, energy density, spot size and carrier gas flow rate; set the initial flow rate of the single-cell sample automatic injection system 100 (e.g., 0.1-10 μL / min); technicians in this field can adjust the spot diameter of the laser focus (e.g., 1-5 μm) as needed, so that the ratio of the liquid volume ablated by the galvanometer mechanism 240 to the time consumed in a complete horizontal scan (i.e., the speed of laser ablation atomization) matches the flow rate of the single-cell sample automatic injection system 100;

[0065] Injection: The single cell suspension is loaded by the high-precision injection pump 110 and accurately delivered to the detection area at the outlet 331 of the erosion tube of the sample holder 330 at a stable flow rate; during the injection process, the single cell automatic injection system 100 further accurately and automatically adjusts the flow rate through the sensor signal feedback of the optical liquid level sensor 332 to match the speed of laser ablation atomization;

[0066] Laser ablation atomization-LIBS detection: The ablation laser is triggered by the galvanometer mechanism 240, and a trigger signal is sent to the single cell automatic sampling system 100, the ICP-TOF-MS mass spectrometer 500 and the fiber spectrometer 400 at the same time. The entire device starts working according to the pre-set parameters under computer control; the femtosecond laser ablation atomization module 200 generates a femtosecond laser through the optical path system, and is focused on the cell micro-area at high speed through the galvanometer mechanism 240 to perform ablation atomization of the cell micro-area, and at the same time, a high-energy plasma is generated to excite the atoms therein and emit a LIBS signal with elemental characteristics. The optical fiber spectrum collector collects the emitted spectral signal and transmits it to the main body of the fiber spectrometer 400 through the transmission optical fiber 410 for spectral separation, and is detected by a CCD detector or an enhanced charge coupled device (ICCD); LIBS can detect light elements including C, H, O, N, Na and other light elements present in the main amount, as well as heavy metal elements introduced by label antibodies, but due to the charged liquid during the ablation atomization process, The plasma life is short, and the detection sensitivity and linear range are limited. Therefore, the LIBS signal of light elements can be mainly collected to provide non-specific information such as cell location / morphology; through the high-speed scanning and focusing function of the galvanometer mechanism 240, the liquid flowing out of the outlet of the erosion tube 331 can be completely ablated and atomized, and the signal sequence generated by the LIBS detection is subtracted from the background signal of the mobile phase to obtain the location of the area corresponding to the cell; compared with the nanosecond laser commonly used in the LIBS method in the prior art (the wavelength is generally 1064nm, the pulse width is <15ns, and the repetition frequency is generally 10Hz), the femtosecond pulse width ablation laser used in this embodiment (the wavelength is 515nm and below, the pulse width is <500fs, and the repetition frequency is up to 1MHz) has a smaller thermal effect, so it has less impact on the splashing or shaking of the liquid, so that the linearity of the generated signal is better, and the pulse energy concentration can also increase the plasma generation ratio, so that the signal-to-noise ratio of the spectrum line is significantly enhanced, and the detection limit is further reduced;

[0067] ICP-TOF-MS detection: Laser ablation atomizes cell micro-areas to form tiny nanoparticles, which form aerosols with carrier gas. The aerosols are transmitted along the mass spectrometer injection pipeline to the ICP-TOF-MS mass spectrometer 500, ionized by the ICP torch, and then detected by the time-of-flight mass spectrometer detector (TOF-MS) to obtain more accurate and sensitive mass spectrometry signals of heavy metal isotope labels;

[0068] Data processing: The signal sequence collected in the above steps is further processed. By correcting the pipeline transmission time, the mass spectrometry signal sequence and the LIBS signal sequence are compared with each other, and the full element content map of the cell microregion can be obtained; according to the focus position sequence during laser ablation high-speed scanning, the time series of the signal is restored to the spatial position association, and the image is reconstructed in the spatial coordinate system to obtain the comprehensive element imaging of the single cell.

[0069] In some other embodiments, the purpose is not to obtain the in-situ distribution information of the elements of a single cell, but only to obtain the element content map of the entire cell (equivalent to the combination of traditional mass spectrometry flow cytometry CyTOF and LIBS), and the objective lens 260 used for laser focusing can be replaced with a field lens that can form a larger spot size to speed up the scanning speed and thus be suitable for a higher liquid flow rate; after deducting the background signal of the mobile phase, the signal sequence obtained is accumulated for the signals corresponding to the single cell to obtain the overall content of the element to be measured in the cell. In some other embodiments, a similar method can also be used to determine the element content in the solution to be measured, by replacing the objective lens 260 used for laser focusing with a field lens, and replacing the single cell suspension sample with the solution to be measured (it is necessary to filter out particulate matter that may block the pipeline) and loading it into the quantitative ring 130 through the high-precision injection pump 110 and the multi-way valve 120.

[0070] Embodiment 2

[0071] Compared with the first embodiment, the multi-purpose analyzer system of this embodiment is further integrated with a tissue section mass spectrometry imaging function (ie, IMC).

[0072] Specifically, if Figure 7 As shown, in the sample holder 330, slide slots for fixing tissue carriers such as glass slides 334 are also provided on both sides of the denudation tube, and support springs 335 are provided in the slide slots. When in use, the glass slide 334 carrying the tissue slice sample is placed in the slide slot, and the glass slide 334 is fixed by the support of the support spring 335 (and can be further fixed by mushroom nails), and the upper surface of the glass slide 334 and the outlet 331 of the denudation tube are at the same horizontal plane height.

[0073] The method for 2D & 3D elemental imaging of tissue slices using the multi-purpose analyzer of this embodiment comprises the following steps:

[0074] Preparation of tissue section samples labeled with metal isotope label antibodies: After the biological tissue is labeled with the antibody with the metal isotope label, a glass slide 334 or a tissue target plate carrying the tissue section sample is formed by frozen sectioning, fixation and paraffin embedding, etc., and the glass slide 334 or the tissue target plate is fixed in the slide card slot of the sample holder 330;

[0075] Instrument preparation: introduce carrier gas / protective gas into the sample pool 300; adjust the height of the precision moving stage 350 through the coaxial observation system 270 to focus the laser on the center of the tissue slice, and select the imaging area, i.e., the range of laser focus scanning; adjust the insertion depth of the optical fiber spectrum collector to ensure that the spectrum collector can collect the plasma emission spectrum; set the laser ablation atomization parameters such as the laser frequency, energy density, spot size, and carrier gas flow rate; since the sample is fixed, sufficient imaging scanning time is provided, so the spatial resolution can be improved as much as possible, for example, the focus spot size is set to <1μm;

[0076] Laser ablation atomization-LIBS detection: The ablation laser is triggered by the galvanometer mechanism 240, and a trigger signal is sent to the ICP-TOF-MS mass spectrometer 500 and the fiber spectrometer 400 at the same time. The entire device starts working according to the pre-set parameters under computer control; the femtosecond laser ablation atomization module 200 generates a femtosecond laser through the optical path system, which is focused on the tissue micro-region at high speed through the galvanometer mechanism 240 to perform ablation atomization of the tissue micro-region, and at the same time, a high-energy plasma is generated to excite the atoms therein and emit a LIBS signal with elemental characteristics. The optical fiber spectrum collector collects the emitted spectral signal and transmits it to the main body of the optical fiber spectrometer 400 through the transmission optical fiber 410 for spectral separation, and detects it through a CCD detector or an enhanced charge coupled device (ICCD);

[0077] ICP-TOF-MS detection: After laser ablation of the tissue micro-area, tiny nanoparticles are formed, which form aerosols with the carrier gas. The aerosols are transmitted along the mass spectrometer injection pipeline to the ICP-TOF-MS mass spectrometer 500, ionized by the ICP torch, and then detected by the time-of-flight mass spectrometer detector (TOF-MS) to obtain more accurate and sensitive mass spectrometry signals of heavy metal isotope labels; after correcting the pipeline transmission time, the mass spectrometry signal sequence and the LIBS signal sequence are compared with each other to obtain the full element content spectrum of the tissue micro-area;

[0078] Data processing: The signal sequences acquired in the above steps are further processed to obtain 2D or 3D elemental imaging of the tissue slice.

[0079] From the description of the above embodiments, it can be seen that the mass spectrometry imaging method of the present invention has the advantage of more detection channels compared with the fluorescence imaging method, and the interference between each channel is low. The specific signal generated by the heavy metal isotope label does not have problems such as fluorescence cross-talk and signal interference such as tissue autofluorescence. Through the complementary advantages of LIBS detection and ICP-TOF-MS detection, rapid detection of all elements from major elements with a content of 1% to trace elements with a content of ppb, from light elements to heavy metal elements, is achieved in one stripping. By comparing the LIBS detection signal of the non-specific major elements in the cell with the ICP-TOF-MS detection signal of the specific heavy metal isotope label, the element distribution imaging of the flowing single cell is achieved, and not only the average distribution of the elements of the entire cell is detected, but also the in-situ element distribution of the micrometer scale inside the cell is provided. The multi-purpose analyzer system of the present invention realizes the element analysis and imaging functions in multiple types of samples such as single cell suspensions, biological tissue slices and aqueous solution samples through the mutual integration of modules; the laser ablation atomization technology is realized by combining femtosecond laser + high-speed three-dimensional galvanometer scanning technology to replace the traditional mass spectrometry flow atomizer, and the atomization efficiency is increased from 10-20% to nearly 100%, and the transmission efficiency is increased from 10% to more than 90%, which greatly increases the sampling amount of cells and improves the sensitivity and accuracy of instrument analysis; the femtosecond laser light source of the present invention (wavelength of 515nm and below, pulse width <500fs, weight The repetition frequency is as high as 1MHz, which significantly improves the ionization effect and reduces the thermal effect. Compared with nanosecond laser, which can achieve dozens of times the LIBS spectrum enhancement effect, the signal-to-noise ratio of the spectrum line is significantly enhanced, and the detection limit is lower; the high-frequency triggering of the femtosecond laser light source and the high-speed scanning and focusing of the galvanometer mechanism also retain the spatial distribution information of the elements in the flowing sample to a greater extent, while also reducing the influence of factors such as liquid splashing and shaking caused by thermal effects on the spatial distribution information; the multi-purpose analysis cup can also conveniently choose to perform a single ICP-TOF-MS test or LIBS test at the same time as the ablation.

[0080] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0081] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for elemental imaging of single cells using LIBS-MS combined with flow cytometry, characterized in that: The steps include: preparing a single cell suspension labeled with a metal isotope labeling antibody, wherein the metal isotope labeling antibody comprises a labeling element; The single-cell suspension was delivered to the denuded area by flow injection; Performing high-speed femtosecond laser ablation scanning on the ablation area, detecting LIBS spectrum signals generated during femtosecond laser ablation, and acquiring a LIBS signal sequence; The aerosol generated during femtosecond laser ablation is detected by ICP-TOF-MS mass spectrometry to obtain the mass spectrum signal sequence; Comparing the LIBS signal sequence with the mass spectrometry signal sequence to form a comprehensive elemental imaging of a single cell; The LIBS signal sequence includes the LIBS signal of at least one element among C, H, O, N, Na or F; the mass spectrometry signal sequence includes the mass spectrometry signal of the tag element; and the LIBS signal sequence is used to provide cell localization information and / or cell morphology information.

2. The element imaging method according to claim 1, characterized in that: The laser pulse width of the femtosecond laser ablation is less than 500fs, and the repetition frequency is 1kHz-1MHz; the spot diameter of the femtosecond laser ablation is 1-5μm, and the flow rate of the flow injection is 0.1-10μL / min.

3. The element imaging method according to claim 1, characterized in that: Comparing the LIBS signal sequence and the mass spectrometry signal sequence with each other includes correcting the pipeline transmission time, and the formation of comprehensive element imaging of single cells includes restoring the time series of signals to spatial coordinate association according to the focus position sequence of femtosecond laser ablation high-speed scanning.

4. A LIBS-MS combined analyzer, characterized in that: It includes a single-cell sample automatic injection system, a femtosecond laser ablation atomization module, a sample pool, a fiber optic spectrometer, and an inductively coupled plasma time-of-flight mass spectrometer; The single cell sample automatic injection system is used for flow injection; The femtosecond laser ablation atomization module includes a femtosecond laser, a galvanometer mechanism and a focusing lens; the femtosecond laser is used to generate ablation laser, the galvanometer mechanism is used to switch the focus position of the ablation laser at a high speed in a preset ablation area, and the focusing lens is used to focus the ablation laser; The optical fiber spectrometer includes a spectrometer body, a spectrum collector and a transmission optical fiber; the spectrum collector is connected to the spectrometer body through the transmission optical fiber, and the spectrum collector is used to collect LIBS signals; The inductively coupled plasma time-of-flight mass spectrometer comprises an ICP torch and a TOF-MS detector; The sample pool comprises a sample pool housing, a multi-purpose analysis cup and a sample holder, and an air inlet is arranged on the sample pool housing; The multi-purpose analysis cup is fixedly arranged on the sample pool housing, and is provided with a window glass, a sampling tube and a jack; the sampling tube is used to guide out the aerosol generated during femtosecond laser ablation, and the sampling tube is connected to the gas path of the inductively coupled plasma time-of-flight mass spectrometer; the jack is used to insert a spectrum collector under the condition of a sealed connection; one end of the sampling tube is opened toward the inside of the sample pool, and the other end of the sampling tube is arranged outside the sample pool; one end of the jack is opened toward the inside of the sample pool, and the other end of the jack is opened toward the outside of the sample pool; The sample holder is arranged inside the sample pool; the sample holder comprises an erosion tube, one end of which is connected to the liquid path of the single cell sample automatic injection system; the other end of the erosion tube is located on the upper surface of the sample holder, and an optical liquid level sensor is arranged on the erosion tube, and the optical liquid level sensor is connected to the signal of the single cell sample automatic injection system; The analyzer is used for the element imaging method of single cells combined with LIBS-MS flow cytometry as described in any one of claims 1 to 3.

5. The analyzer according to claim 4, characterized in that The sample holder also includes a slide holder slot and a support spring; the slide holder slot is used to place the sample carrier, and the support spring is used to support and fix the sample carrier so that the upper surface of the sample carrier is at a height corresponding to the upper surface of the sample holder.

6. The analyzer according to claim 5, characterized in that The pulse width of the ablation laser is less than 500 fs, and the repetition frequency is 1 kHz to 1 MHz; the scanning speed of the galvanometer mechanism is greater than or equal to 20.0 rad / s, and the positioning speed of the galvanometer mechanism is greater than or equal to 30.0 rad / s.

7. The analyzer according to claim 4, characterized in that The single cell sample automatic injection system comprises a high-precision injection pump, a multi-way valve and a quantitative ring, and the high-precision injection pump and the quantitative ring are respectively connected to the multi-way valve pipeline.

8. The analyzer according to claim 4, characterized in that The sample pool also includes a moving stage and a transmission illuminator. The transmission illuminator is arranged below the sample holder. A high-transmittance glass is arranged at the bottom of the sample holder. The high-transmittance glass is provided with evenly distributed fixing holes.

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