CN117110175A - Femtosecond laser ablation mass spectrum flow type all-in-one machine and application method thereof - Google Patents
Femtosecond laser ablation mass spectrum flow type all-in-one machine and application method thereof Download PDFInfo
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Abstract
The invention discloses a femtosecond laser ablation mass spectrum flow type integrated machine and a use method thereof, wherein the femtosecond laser ablation mass spectrum flow type integrated machine comprises: the device comprises a liquid path sample injection module, a laser ablation module, a mass spectrum detection module and a sample cell; the liquid path sample injection module is used for acquiring single-cell suspension with a mass spectrum detection tag and sending the single-cell suspension into the sample cell; the laser ablation module is used for generating ablation laser to perform laser ablation on the sample in the sample cell; the mass spectrum detection module is used for generating a mass spectrum detection signal. According to the invention, the femto-second laser ablation is adopted to replace the current mass flow type argon high-speed atomization technology, the atomization efficiency is close to 100%, and the sensitivity and reliability of mass flow cytometry are improved; on the other hand, the full coverage of a single cell sampling area is realized by applying the femtosecond laser and the triaxial high-speed galvanometer, so that no omission is caused basically; and the second laser ablation efficiency is extremely high, so that the sampling amount and the analysis speed of cells are greatly improved, and the transmission efficiency can reach more than 90%.
Description
Technical Field
The invention belongs to the technical field of laser ablation, and particularly relates to a femtosecond laser ablation mass spectrometry flow type all-in-one machine and a use method thereof.
Background
Flow Cytometry (FCM) is a biological technique that rapidly quantitatively analyzes and sorts cells or other biological particles (e.g., microspheres, bacteria, small model organisms, etc.) in a single row in a fluid stream one by one. Flow cytometry can analyze gene expression and protein expression in a cell sample from a single cell level simultaneously, and can detect cell activity, cell cycle, apoptosis, cell proliferation, cell oxidation and other cell functions. The conventional flow cytometry operation flow mainly comprises the steps of preparing single-cell suspension, labeling fluorescent antibody, on-line detection of a flow cytometer, data processing and the like, and is shown in fig. 1 in detail.
Traditional flow cytometry
The conventional flow cytometry (flow cytometry) is a fluorescence-based detection system, has been developed for 40 years, is well-established, is widely applied to various aspects from basic research to clinical practice, covers the fields of cell biology, immunology, hematology, oncology, pharmacology, genetics, clinical examination and the like, and plays an important role in various subjects.
The flow cytometer is an instrument that uses laser as a light source to generate scattered light and fluorescence signals, and detects these signals by a detector such as a photodiode or photomultiplier, thereby rapidly screening and analyzing individual cells in a solution or analyzing a specific cell subset and separating and purifying the same. See FIG. 2 for a specific structure of the flow cytometer.
The flow cytometer analyses various parameters of cells by optical signals. The 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 Scatter (FSC) and Side Scatter (SSC), both of which are common flow cytometry. The fluorescent signal mainly comprises two parts: (1) autofluorescence, i.e., fluorescence emitted by fluorescent molecules inside cells after irradiation with light, without fluorescent staining; (2) the characteristic fluorescence, namely the fluorescence emitted by the cells after being irradiated by the fluorescent dye on the dyed combination, has weaker fluorescence intensity and different wavelength from the irradiation laser. The fluorescent signal may be used to reflect the chemical properties of the cell, such as antigen expression. The number of fluorescence channels is determined according to the difference of the flow cytometry.
The unique single cell analysis technique of Flow Cytometry (FCM), the ability to analyze multiple fluorescent parameters on millions or more cells, enables Flow to help us understand complex biological processes. However, the traditional flow technique encounters a bottleneck in the development process, and has some defects, namely, the problems of spectrum leakage, autofluorescence and the like cannot be avoided due to the limitation of fluorescent dye on three aspects, and signals among channels can be mutually interfered due to the overlapping of emission spectrums of different fluorescent groups; thirdly, the number of detection channels (< 20) of the traditional flow cytometry is difficult to be improved; all of these have made experimental design and compensation calculations quite complex and experimental operations quite inconvenient.
Mass flow cytometry: in view of the bottlenecks and defects of traditional flow cytometry, scientists have studied mass spectrometry flow cytometry. Mass Cytometry (CyTOF) uses antibodies with metal isotope tags to label cells, and the tag composition on each cell is analyzed by time-of-flight Mass spectrometry to conduct intensive study on cell phenotype and function.
Unlike conventional flow cytometry, which uses a detector such as a photodiode or photomultiplier for detection, mass spectrometry uses time-of-flight mass spectrometry (TOF-MS, time of Flight Mass Spectrometer). The TOF-MS collects instantaneous full spectrum information, greatly improves the analysis speed and sensitivity of the instrument, ensures that any important information cannot be lost, and is an analysis instrument with ultrahigh mass resolution and high accurate mass.
Mass flow cytometry refers to the labelling of antibodies with stable heavy metal isotopes (mainly lanthanoids) instead of fluorophores. The cells are then introduced into a CyTOF analyser and atomised into droplets which are vaporised, atomised, ionised and then brought into a mass spectrometer by potential acceleration. Finally, the ion cloud after filtration is analyzed with a TOF detector. A mass flow cytometry schematic is shown in fig. 3.
The droplet atomization link is an important link in mass spectrometry flow cytometry, and directly affects the sensitivity of detection, and a specific flow chart is shown in fig. 4. After the mass flow sample is prepared, the sample enters the instrument in the form of a cell suspension and is divided into small droplets by an atomizer (Nebulizer), each droplet containing one cell. The liquid drops containing the cells then enter an atomizing chamber (Spray chamber), high-flow argon in the atomizing chamber atomizes aerosol liquid drops (a part of instruments are also provided with a heating device outside the atomizing chamber, the temperature can reach 200 ℃), and the atomized aerosol is transmitted to an ICP source for plasma treatment.
Mass flow cytometry has the following advantages:
1. ultra-high flux; the mass spectrum flow type has a very wide atomic weight detection range, and can theoretically detect 135 channels simultaneously;
2. the adjacent channels are free from interference, and calculation compensation is not needed; the metal label replaces the fluorescent label, the cross color interference is avoided, the TOF mass spectrum has ultrahigh resolution capability, and various elements for marking can be completely distinguished. Experimental data shows that the interference between adjacent channels is <0.3%, which is basically negligible, without calculation of the compensation. Thus not only simplifying the experimental process, but also saving the sample and reagent
3. Rare earth elements are used as labels, so that the background interference is small, and the signal-to-noise ratio is extremely high; the mass spectrum flow type uses rare earth elements which are not existed in a living body as detection labels, the covalent coupling of the metal labels and the antibodies is realized through a polymeric chelate, and the nonspecific binding of the metal chelate and cell components is extremely low, so that the ultra-high detection signal-to-noise ratio can be realized.
4. High sensitivity; the mass spectrometer has the dual advantages of a mass spectrometer and a polymer-metal chelating technology.
Meanwhile, the prior art also has the following drawbacks:
1. high-flow argon is adopted for atomization, part of company equipment also needs to heat an atomization chamber to 200 ℃ for atomization, and the current atomization efficiency is generally between 10 and 20 percent;
2. the transmission efficiency is low, so that the working efficiency is very low, and the conventional transmission efficiency is below 10%;
and 3, influencing the sensitivity and accuracy of detection.
Disclosure of Invention
Therefore, one of the purposes of the present invention is to provide a femtosecond laser ablation mass spectrometry integrated machine and a use method thereof, which can greatly improve the atomization efficiency through laser ablation, improve the sensitivity and reliability of mass spectrometry, and greatly improve the sampling amount and analysis speed of cells while realizing full coverage of a single cell sampling area.
To achieve the above object, a first aspect of the present invention provides a femtosecond laser ablation mass spectrometry-flow-type integrated machine, including: the device comprises a liquid path sample injection module, a laser ablation module, a mass spectrum detection module and a sample cell;
the liquid path sample injection module is used for acquiring single-cell suspension with a mass spectrum detection tag and sending the single-cell suspension into the sample cell;
the laser ablation module is used for generating ablation laser to perform laser ablation on a sample in the sample cell and sending the obtained aerosol into the mass spectrum detection module, and comprises a coaxial observation module and a three-dimensional galvanometer module, wherein the coaxial observation module is used for observing a focusing position; the three-dimensional galvanometer module is used for adjusting the focusing position of the ablation laser at a high speed within a preset ablation range; the size of the ablation range is at least one single cell volume; the laser ablation module is a femtosecond laser ablation module;
the mass spectrum detection module is used for generating a mass spectrum detection signal;
the sample cell comprises a sample support, and the sample support is of a concave structure; the sample support comprises an ablation tube and a liquid level sensor, wherein the ablation tube is a right-angle bent tube, one end of the ablation tube is connected with the liquid path sampling module, and the center of the other end of the ablation tube is coaxial with the ablation laser and is provided with a single-cell ablation port; the liquid level sensor is arranged at a position adjacent to the single-cell ablation opening and is used for feeding back liquid level height information to the liquid path sample injection module, and the liquid path sample injection module controls the flow rate of the single-cell suspension into the sample cell according to the liquid level height information.
Preferably, the sample holder further comprises at least one tissue sample fixation device comprising a slide and a support spring;
the sample cell also comprises a three-dimensional moving table, and the sample support is arranged on the three-dimensional moving table.
Preferably, the three-dimensional mobile station further comprises an illumination light source, and the bottom of the sample support is made of high light transmission material.
Preferably, the sample cell further comprises a sample cup, a carrier gas outlet and a carrier gas inlet, and the sample cup is arranged above the sample support.
The invention also provides a use method of the femtosecond laser ablation mass spectrometry flow type all-in-one machine, wherein the sample is a cell suspension, and the use method comprises the following steps:
step S1: preparing a cell suspension: processing the sample into a single cell suspension;
step S2: marking: labeling the single cell suspension with an antibody having a metal isotope label for generating a mass spectrometry detection signal;
step S3: adjusting the focusing position and the light spot size of the ablation laser to enable the ablation area to cover the single-cell ablation opening;
step S4: the liquid path sample injection module slowly conveys the single-cell suspension obtained in the step S2 to the single-cell ablation port at a stable flow rate;
step S5: the laser ablation module performs whole-cell laser ablation on single cells reaching a single-cell ablation port;
step S6: the aerosol generated after laser ablation is sent to a mass spectrum detection module through carrier gas, and detection signals are obtained;
step S7: and (5) recording and analyzing data.
In another aspect, the present invention provides a method for using the femtosecond laser ablation mass spectrometry flow machine, where the sample is a tissue slice, and the method includes:
step S1: preparing a tissue section;
step S2: marking: labeling the tissue section with an antibody having a metal isotope label for generating a mass spectrometry detection signal;
step S3: fixing the labeled cell tissue slice on a tissue sample fixing device;
step S4: introducing carrier gas and shielding gas into the sample cell, and adjusting related condition parameters;
step S5: the laser ablation module performs laser ablation scanning on the cell tissue slice;
step S6: the aerosol generated after laser ablation is sent to a mass spectrum detection module through carrier gas, and detection signals are obtained;
step S7: and (5) recording and analyzing data.
The invention has the following beneficial effects:
1) According to the cell suspension flow analysis method of the femtosecond laser ablation mass spectrometry flow type integrated machine, the existing mass spectrometry flow type argon high-speed atomization technology (part of the method also adopts a high-temperature heating atomization technology) is replaced by the femtosecond laser ablation, the atomization efficiency is close to 100%, and the sensitivity and the reliability of mass spectrometry flow cytometry are improved;
2) The invention further integrates the three-dimensional imaging function, and the whole coverage of a single-cell sampling area is realized by applying the femtosecond laser and the triaxial high-speed galvanometer, so that omission is avoided basically, and whole-cell mass spectrum information of single cells can be obtained completely; the second laser ablation efficiency is extremely high, so that the sampling amount and the analysis speed of cells are greatly improved, and the transmission efficiency can reach more than 90%;
3) The device is two-in-one equipment, integrates the flow type of the femtosecond laser ablation mass spectrum and the tissue imaging mass spectrum, improves the analysis efficiency, reduces the cost of a user, and can be used for imaging two-dimensional tissue elements and three-dimensional tissue elements by utilizing the device.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIGS. 1-4 are schematic diagrams of flow cytometry of the prior art;
FIG. 5 is a schematic structural diagram of a femtosecond laser ablation mass spectrometry flow type all-in-one machine of the invention;
FIG. 6 is a schematic diagram of a three-dimensional galvanometer module according to the invention;
FIG. 7 is a schematic structural view of a tissue sample fixation device of the present invention;
FIG. 8 is a flow chart of a method of using the femtosecond laser ablation mass spectrometry-flow-type all-in-one machine of the invention;
wherein:
100. a liquid path sample injection module; 101. a laser emitter; 102. a syringe pump; 200. a laser ablation module; 201. degrading the laser emitter; 202. a three-dimensional galvanometer module; 2021. moving the lens; 2022. a focusing lens; 2023 An X-axis vibrating mirror; 2024 A Y-axis vibrating mirror; 203. a field lens; 204. a laser beam; 205. a carrier gas; 206. a shielding gas; 207. a sample cup; 208. a sample holder; 209. an illumination light source; 300. a mass spectrum detection module; 301. a sample; 302. a detector; 303. a beam-splitting prism; 304. a three-dimensional mobile station; 305. a bandpass filter; 306. a computer; 401. a cell; 402. single cell suspensions; 403. a sample is added after the marking; 404. an atomizer; 405. a plasma torch; 406. an aerosol; 407. heating the atomizer; 408. laser ablation; 501. a glass slide; 502. a support spring; 503. degrading the tube; 504. single cell ablation ports; 505. a liquid level sensor; 506. a high light transmission material.
Detailed Description
One of the cores of the invention is to provide a femtosecond laser ablation mass spectrometry flow type all-in-one machine and a use method thereof, which can greatly improve the atomization efficiency through laser ablation, improve the sensitivity and the reliability of mass spectrometry flow cytometry, realize full coverage of a single cell sampling area and greatly improve the sampling amount and the analysis speed of cells.
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring first to fig. 5, the femtosecond laser ablation mass spectrometry flow-type all-in-one machine disclosed in the present embodiment includes: the device comprises a liquid path sample injection module 100, a laser ablation module 200, a mass spectrum detection module 300 and a sample cell; the liquid path sample injection module 100 is used for acquiring single-cell suspension 402 with a mass spectrum detection tag and sending the single-cell suspension into a sample cell; the laser ablation module 200 is used for generating ablation laser to perform laser ablation on a sample 301 in the sample cell, and sending the obtained aerosol 406 into the mass spectrum detection module 300, and the laser ablation module 200 comprises a coaxial observation module which is used for observing a focusing position; the mass detection module 300 is used for generating a mass detection signal.
In this embodiment, the liquid path sampling module 100: consists of a sample tube, a working solution tube, a high-precision injection pump 102, a quantitative ring, a flushing station and the like. The sample tube stores the labeled single cell suspension 402 and the working solution tube stores the carrier fluid and the working fluid. The high precision syringe pump 102 can accurately regulate the sample flow rate and operate at very low steady flow rates, such as 10 mul/min.
The sample cell comprises a three-dimensional moving table and a sample support 208, the sample support 208 is arranged on the three-dimensional moving table 304, as shown in fig. 6, the sample support 208 of the embodiment is of a concave structure, the sample support 208 comprises an ablation tube 503 and a liquid level sensor 505, the ablation tube 503 is a right-angle elbow, one end of the ablation tube 503 is connected with the liquid path sampling module 100 (i.e. in the sampling direction of single cell suspension 402 in the figure), and the center of the other end of the ablation tube 503 is coaxial with ablation laser and is provided with a single cell ablation port 504; the liquid level sensor 505 is disposed adjacent to the single cell ablation port 504, and the liquid level sensor 505 is configured to feed back liquid level information to the liquid path sampling module 100, and the liquid path sampling module 100 controls a flow rate of the single cell suspension into the sample cell according to the liquid level information. In this embodiment, the bottom of the sample support 208 is made of a high light-transmitting material 506 (such as high light-transmitting organic glass), and the three-dimensional moving table 304 is provided with an illumination light source 209, preferably, the bottom of the sample support 208 is also provided with uniformly distributed small holes, so as to be matched with mushroom nails, and be used for fixing samples or sample containers with various shapes; the sample cell further comprises a sample cup 207, a carrier gas outlet and a carrier gas inlet, the sample cup 207 being arranged above the sample holder 208. To facilitate detection of the tissue sample, the sample holder 208 further comprises at least one tissue sample holding device comprising a slide 501 and a support spring 502
In the present embodiment, the laser ablation module 200 includes a three-dimensional galvanometer module for adjusting the focus position of ablation laser at high speed, and as shown in fig. 7, the three-dimensional galvanometer module 202 includes a moving lens 2021, a focusing lens 2022, an X-axis galvanometer 2023, and a Y-axis galvanometer 2024; the movable lens 2021 can axially move, and the movable lens 2021 adjusts the distance between the movable lens 2021 and the focusing lens 2022 to change the focusing position of the ablation laser on the surface of the sample 301 along the Z axis; the X-axis galvanometer 2023 and the Y-axis galvanometer 2024 are reciprocally rotatable around axes at high frequencies, respectively, and the X-axis galvanometer 2023 and the Y-axis galvanometer 2024 are used to adjust a focus position in a horizontal direction on the surface of the sample 301. In this embodiment, the laser ablation module 200 uses argon as the carrier gas 205 and helium as the shielding gas 206 (which gas may be selected as the case may be); the fundamental frequency of the femtosecond laser module laser is 1030nm, 515nm and 343nm are output through a frequency multiplier, the pulse width is smaller than 600 fs, and the repetition frequency is 1KHz-1MHz.
Because the three-dimensional galvanometer module 202 capable of switching the focusing position at high speed is adopted in the embodiment, the focusing position can be scanned at high speed in the range of the ablation area during flow detection so as to improve the flux of laser ablation, thereby improving the speed of ablation detection.
The embodiment also discloses a use method of the femtosecond laser ablation mass spectrometry flow type integrated machine, and for a conventional sample such as a cell suspension (namely, a femtosecond laser ablation mass spectrometry flow cytometry method), as shown in fig. 8, the method is as follows:
1. preparation of cells: samples (including non-adherent cells from cultures, bacteria, yeast, blood and tissue, etc.) are cultured and processed into single cell suspensions 402.
2. Labeling the antibody: cells are labeled with an antibody labeled with a metal isotope to form a single cell suspension 402 with a label.
3. The sample cell is filled with carrier gas 205 and shielding gas 206;
4. adjusting the height of the three-dimensional moving table 304 through a coaxial observation system to enable laser to be focused at the center of an ablation area of the ablation tube;
5. setting laser ablation parameters, such as laser frequency, energy density, spot size, carrier gas flow rate and the like, so as to ensure that an ablated region can completely cover single cells;
6. the femtosecond laser ablation mass spectrometry flow cytometry device starts working according to preset parameters under the control of a computer;
7. the fluid line injection module 100 loads the single cell suspension 402 by the syringe pump 102 and precisely delivers the single cell suspension 402 to the ablation detection zone at an extremely low steady flow rate;
8. the laser ablation module 200 generates ablation laser which is focused on cells at high speed through a light path system by the three-dimensional galvanometer module 202 to perform single-cell whole-cell ablation;
9. after the cells are ablated by the laser, tiny nanoparticles are directly formed, and an aerosol 406 is formed with the carrier gas 205 and transmitted along a pipeline to the mass spectrum detection module 300, for example: ICP-TOFMS;
10. the aerosol 406 is subjected to Inductively Coupled Plasma (ICP) source, and after being plasmatized, enters a time-of-flight mass spectrum for elemental detection.
For the case where the sample is a tissue section (i.e., femtosecond laser ablation tissue imaging mass spectrometry), the method is as follows:
1. firstly, marking a biological tissue slice by adopting a metal tag antibody in a sample preparation chamber;
2. moving the labeled tissue section onto a slide 501; (in the case of tissue targets, no movement onto the slide is required)
3. Fixing the slide (or tissue target) to the sample holder using the support spring 502 and the mushroom pins;
4. the sample cell is filled with carrier gas 205 and shielding gas 206;
5. replacing the field lens 203 with an objective lens;
6. adjusting the height of the three-dimensional moving table 304 through a coaxial observation system to focus laser on the center of a tissue slice and select an imaging area;
7. setting laser ablation parameters such as laser frequency, energy density, spot size (less than 1 um) and carrier gas flow rate,
8. the computer sends out a laser ablation instruction and simultaneously analyzes mass spectrum;
9. the laser ablation module 200 generates ablation laser, and the ablation laser passes through the optical path system and is focused on cells at a high speed through the three-dimensional galvanometer module 202 to perform high-speed 2D or 3D ablation on the cells in a selected area;
10. after the cells are ablated by the laser, tiny particles are directly formed, and an aerosol 406 is formed with the carrier gas 205, and the aerosol 406 is transferred to the mass spectrum detection module 300 along a pipeline, for example: ICP-TOFMS;
11. the aerosol 406 is subjected to Inductively Coupled Plasma (ICP) source, plasma treatment, and then enters a mass spectrum for element detection;
12. the detection data can form a tissue two-dimensional or three-dimensional element imaging chart after being processed by software.
Compared with the prior art, the embodiment has the following advantages:
1. solves the problems of signal interference of traditional immunofluorescence cross color and tissue autofluorescence in principle
2. The characteristics of short pulse, very high instantaneous power and the like of the femtosecond laser reduce the fractional effect of elements;
3. the femto-second laser ablation is adopted to replace the current mass flow type argon high-speed atomization technology (part of the high-temperature heating atomization technology is also adopted), the atomization efficiency is close to 100%, and the sensitivity and the reliability of mass flow cytometry are improved;
4. the whole coverage of a single-cell sampling area is realized by applying the femtosecond laser and the triaxial high-speed galvanometer, so that omission is avoided basically, and whole-cell mass spectrum information of single cells can be obtained completely; the second laser ablation efficiency is extremely high, so that the sampling amount and the analysis speed of cells are greatly improved, and the transmission efficiency can reach more than 90%;
5. the device is two-in-one equipment, integrates the femtosecond laser ablation mass spectrum flow type and the tissue imaging mass spectrum, improves the analysis efficiency, reduces the cost of a user, can image two-dimensional tissue elements and three-dimensional tissue elements by utilizing the device, and can carry out cross-validation on the same tissue by adopting two methods;
6. the method can detect the metal mark of the cell and detect almost all elements (including heavy metal elements) in the cell, and provides a powerful tool for diagnosis of diseases and deep research of the cell.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A femtosecond laser ablation mass spectrometry flow machine, comprising: the device comprises a liquid path sample injection module, a laser ablation module, a mass spectrum detection module and a sample cell;
the liquid path sample injection module is used for acquiring single-cell suspension with a mass spectrum detection tag and sending the single-cell suspension into the sample cell;
the laser ablation module is used for generating ablation laser to perform laser ablation on a sample in the sample cell and sending the obtained aerosol into the mass spectrum detection module, and comprises a coaxial observation module and a three-dimensional galvanometer module, wherein the coaxial observation module is used for observing a focusing position; the three-dimensional galvanometer module is used for adjusting the focusing position of the ablation laser at a high speed within a preset ablation range; the size of the ablation range is at least one single cell volume; the laser ablation module is a femtosecond laser ablation module;
the mass spectrum detection module is used for generating a mass spectrum detection signal;
the sample cell comprises a sample support, and the sample support is of a concave structure; the sample support comprises an ablation tube and a liquid level sensor, wherein the ablation tube is a right-angle bent tube, one end of the ablation tube is connected with the liquid path sampling module, and the center of the other end of the ablation tube is coaxial with the ablation laser and is provided with a single-cell ablation port; the liquid level sensor is arranged at a position adjacent to the single-cell ablation opening and is used for feeding back liquid level height information to the liquid path sample injection module, and the liquid path sample injection module controls the flow rate of the single-cell suspension into the sample cell according to the liquid level height information.
2. The femtosecond laser ablation mass spectrometry integration machine of claim 1, wherein the sample holder further comprises at least one tissue sample fixture comprising a slide and a support spring;
the sample cell also comprises a three-dimensional moving table, and the sample support is arranged on the three-dimensional moving table.
3. The femtosecond laser ablation mass spectrometry integration machine according to claim 2, wherein the three-dimensional mobile station further comprises an illumination light source, and the bottom of the sample support is made of a high light transmission material.
4. The femtosecond laser ablation mass spectrometry integration machine of claim 2, wherein the sample cell further comprises a sample cup, a carrier gas outlet, and a carrier gas inlet, the sample cup being disposed above the sample support.
5. A method of using the femtosecond laser ablation mass spectrometry integration machine of any one of claims 1 to 4, wherein the sample is a cell suspension comprising:
step S1: preparing a cell suspension: processing the sample into a single cell suspension;
step S2: marking: labeling the single cell suspension with an antibody having a metal isotope label for generating a mass spectrometry detection signal;
step S3: adjusting the focusing position and the light spot size of the ablation laser to enable the ablation area to cover the single-cell ablation opening;
step S4: the liquid path sample injection module slowly conveys the single-cell suspension obtained in the step S2 to the single-cell ablation port at a stable flow rate;
step S5: the laser ablation module performs whole-cell laser ablation on single cells reaching a single-cell ablation port;
step S6: the aerosol generated after laser ablation is sent to a mass spectrum detection module through carrier gas, and detection signals are obtained;
step S7: and (5) recording and analyzing data.
6. A method of using the femtosecond laser ablation mass spectrometry integration machine of any one of claims 2 to 4, wherein the sample is a tissue slice comprising:
step S1: preparing a tissue section;
step S2: marking: labeling the tissue section with an antibody having a metal isotope label for generating a mass spectrometry detection signal;
step S3: fixing the labeled cell tissue slice on a tissue sample fixing device;
step S4: introducing carrier gas and shielding gas into the sample cell, and adjusting related condition parameters;
step S5: the laser ablation module performs laser ablation scanning on the cell tissue slice;
step S6: in the scanning process, aerosol generated after laser ablation is sent to a mass spectrum detection module through carrier gas, and detection signals are obtained;
step S7: and (5) recording and analyzing data.
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