CN114354737A - A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization - Google Patents

Abstract

The invention relates to a mass spectrum imaging device with normal-pressure laser desorption ionization and secondary photoionization, and belongs to the technical field of mass spectrum imaging. Comprises a laser desorption ionization mechanism, a secondary light ionization mechanism and a gas-phase dopant introduction mechanism. The improvement lies in that: a secondary photoionization mass spectrometry mechanism and a gas phase dopant introduction mechanism are additionally arranged. The secondary photoionization mechanism comprises a tubular photoionization chamber and a vacuum ultraviolet discharge lamp; the vacuum ultraviolet discharge lamp comprises a discharge tube filled with rare gas; the gas-phase dopant introducing mechanism comprises a bubbling tank, a first conduit and a second conduit; the bubbling tank is internally provided with a dopant. During detection and analysis, the object to be detected in the biological tissue enters the sampling capillary after being analyzed by normal pressure laser, wherein unionized neutral components can be further ionized by photoinduced direct ionization and photoinduced ion molecule reaction, and high-resolution and high-sensitivity mass spectrum imaging under normal pressure is realized. The imaging technology of the invention can be used for analyzing the action mechanism of the medicine, the tumor tissue, the cell metabolism and the like.

Description

Translated fromChinese

一种具有常压激光解析电离和二次光电离的质谱成像装置A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization

技术领域technical field

本发明属于质谱成像技术领域，具体涉及一种结合常压激光解析电离和二次光电离质谱成像装置。The invention belongs to the technical field of mass spectrometry imaging, in particular to a mass spectrometry imaging device combining atmospheric pressure laser desorption ionization and secondary photoionization.

背景技术Background technique

质谱成像技术是样品平台在软件程序的控制下按照一定的规律运动，通过质谱直接扫描生物样品成像，根据所测质荷比（m/z）来分析生物分子的空间分布的成像方法。与传统的光学生物成像技术相比，质谱成像技术属于分子信息成像，是研究生物组织及活体动物中分子成像的新型分析技术。与传统的荧光分子成像、免疫标记分子成像技术相比，质谱成像可以在不用标记，无需复杂预处理的条件下实现分子成像，而且可以在同一张组织切片上同时分析数百种生物分子的空间分布特征，还可以与生物组织病理学分析结果对照用于生物病理学研究。目前，质谱成像技术已经广泛应用于蛋白质组，脂质组学以及药物代谢组学等领域，同时也已经在病理学，临床医学以及疾病诊断中展现了巨大的应用潜力。Mass spectrometry imaging technology is an imaging method in which the sample platform moves according to a certain rule under the control of a software program, directly scans the biological sample imaging by mass spectrometry, and analyzes the spatial distribution of biomolecules according to the measured mass-to-charge ratio (m/z). Compared with traditional optical bioimaging technology, mass spectrometry imaging technology belongs to molecular information imaging, and is a new analysis technology for studying molecular imaging in biological tissues and living animals. Compared with traditional fluorescent molecular imaging and immunolabeling molecular imaging techniques, mass spectrometry imaging can realize molecular imaging without labeling and complex preprocessing, and can simultaneously analyze the space of hundreds of biomolecules on the same tissue section. The distribution characteristics can also be compared with the results of biological histopathological analysis for biopathological research. At present, mass spectrometry imaging technology has been widely used in the fields of proteomics, lipidomics and pharmacometabolomics, and has also shown great application potential in pathology, clinical medicine and disease diagnosis.

目前，主要的质谱成像技术主要包括基质辅助激光解吸电离（MALDI）质谱、解析电喷雾电离（DESI）质谱和二次离子电离质谱（SIMS），这三种技术分别是通过激光、带电的小液滴和离子束将待测物从组织的表面解析电离，都属于直接解析电离的分析方法。一种结合解吸电喷雾电离的光电离质谱成像装置，通过电喷雾针喷出的溶剂解析固体被测物，通过取样毛细管进入到光电离室内，经光源的二次离子化后进入到质谱仪进行分析。但是基于带电液滴的解析电喷雾电离质谱成像技术空间分辨率只能达到200μm，高空间分辨的质谱成像技术对于研究组织微区甚至细胞级别的空间结构具有非常重要的意义。基于激光的质谱成像技术可以实现10-50μm左右的空间分辨，可以满足大部分需求，同时激光解析电离技术是一种比二次离子电离更“软”的离子化技术，因此应用最为广泛。但是，在质谱成像领域，灵敏度与分辨率是相互制约的，在更高的空间分辨条件下，实现同样的灵敏度是极具挑战的。此外，由于生物组织自身复杂的基质环境，即使在基质辅助的情况下，生物组织中内源性化学成分的激光解析电离效率依然小于1/1000。而且，高丰度且离子化效率强的化合物会抑制其他种类化合物的电离。例如，在正离子模式下的，生物组织的MALDI质谱图中脂质类化合物主要以磷脂酰胆碱类化合物（PC）为主，而同样含量丰富的糖酯类化合物却很少被测到。At present, the main mass spectrometry imaging techniques mainly include matrix-assisted laser desorption ionization (MALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry and secondary ion ionization mass spectrometry (SIMS). Droplet and ion beam analytical ionization of the analyte from the surface of the tissue are both analytical methods of direct analytical ionization. A photoionization mass spectrometry imaging device combined with desorption electrospray ionization. The solid object to be tested is resolved by the solvent sprayed by the electrospray needle, and then enters the photoionization chamber through the sampling capillary, and then enters the mass spectrometer after the secondary ionization of the light source. analyze. However, the spatial resolution of analytical electrospray ionization mass spectrometry imaging technology based on charged droplets can only reach 200 μm. High spatial resolution mass spectrometry imaging technology is of great significance for studying the spatial structure of tissue micro-regions and even cells. Laser-based mass spectrometry imaging technology can achieve spatial resolution of about 10-50 μm, which can meet most needs. At the same time, laser desorption ionization technology is a “softer” ionization technology than secondary ion ionization, so it is the most widely used. However, in the field of mass spectrometry imaging, sensitivity and resolution are mutually restricted, and it is extremely challenging to achieve the same sensitivity under higher spatial resolution conditions. In addition, due to the complex matrix environment of biological tissues, the laser desorption ionization efficiency of endogenous chemical components in biological tissues is still less than 1/1000 even with matrix assistance. Furthermore, compounds with high abundance and high ionization efficiency inhibit the ionization of other compounds. For example, in the MALDI mass spectrum of biological tissues in positive ion mode, the lipid compounds are mainly phosphatidylcholine compounds (PC), while the sugar esters, which are also abundant, are rarely detected.

2015年，Soltwisch等人提出为了提高MALDI中难电离化合物的离子化效率，可在氮气冷却的中等压力下（2.0-2.5 mbar），激光解析出的扩散粒子与二次光电离激光交叠，经过二次离子化之后经离子传输系统被飞行时间质谱检测，该方法被成为MALDI-2（Science）2015, 348 (6231), 211-215。In 2015, Soltwisch et al. proposed that in order to improve the ionization efficiency of refractory compounds in MALDI, the diffused particles desorbed by the laser can overlap with the secondary photoionization laser under moderate pressure (2.0-2.5 mbar) cooled by nitrogen gas. After secondary ionization, it was detected by time-of-flight mass spectrometry through an ion transmission system, and the method was called MALDI-2 (Science) 2015, 348 (6231), 211-215.

发明内容SUMMARY OF THE INVENTION

为了实现在常压条件下，基于激光解析电离技术结合，光诱导的离子分子反应二次电离，实现生物组织中不同种类化合物高分辨高灵敏成像，本发明提供一种具有常压激光解析电离和二次光电离质谱的成像装置。In order to realize the secondary ionization of light-induced ion molecule reaction under normal pressure conditions, based on the combination of laser desorption ionization technology, and realize high-resolution and high-sensitivity imaging of different types of compounds in biological tissues, the present invention provides an atmospheric pressure laser desorption ionization and Imaging device for secondary photoionization mass spectrometry.

一种具有常压激光解析电离和二次光电离的质谱成像装置包括激光解析电离机构，所述激光解析电离机构包括质谱仪、激光器1、扩束镜3、反光镜4、聚焦镜5、载物台6和取样毛细管8；A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization includes a laser desorption ionization mechanism, the laser desorption ionization mechanism includes a mass spectrometer, alaser 1, a beam expander 3, areflector 4, a focusingmirror 5, acarrier Stage 6 andsampling capillary 8;

还包括二次光电离机构9和气相掺杂剂引入机构10；Also includes a secondary photoionization mechanism 9 and a gas-phase dopant introduction mechanism 10;

所述二次光电离机构包括管状的光电离室95和真空紫外放电灯；The secondary photoionization mechanism includes atubular photoionization chamber 95 and a vacuum ultraviolet discharge lamp;

所述光电离室95的上端内设有同轴的导流管97，所述导流管97的上端口与质谱仪的传输毛细管98的出口对应，与导流管97外周对应的光电离室95一侧连通着抽真空管96的一端；光电离室95下部一侧连通着取样毛细管8的一端；光电离室95的下端连接着同轴的下盖板91，所述下盖板91的一侧连通着掺杂剂引入管94的出口端；The upper end of thephotoionization chamber 95 is provided with acoaxial guide tube 97 , the upper port of theguide tube 97 corresponds to the outlet of the transmission capillary 98 of the mass spectrometer, and the photoionization chamber corresponding to the outer periphery of theguide tube 97 One end of the evacuatedtube 96 is connected to one side of 95; the lower side of thephotoionization chamber 95 is connected to one end of thesampling capillary 8; the lower end of thephotoionization chamber 95 is connected to the coaxiallower cover plate 91. The side is connected to the outlet end of thedopant introduction tube 94;

所述真空紫外放电灯包括放电管99、环形负极910和窗片911；所述环形负极910设于放电管99的上端，所述窗片911设于环形负极910中心的通孔处，所述放电管99的下部外圆周上均布设有环形正极凸块93；环形正极凸块93与环形负极910之间的电压差使放电管99内稀有气体放电产生真空紫外光，真空紫外光子通过窗片911进入光电离室95；所述环形负极910外圆围上设有同轴的环状的上盖板92，通过下盖板91和上盖板92固定连接实现光电离室95和放电管99密封连接；放电管99中填充稀有气体，得到不同能量的真空紫外光；The vacuum ultraviolet discharge lamp includes adischarge tube 99, an annularnegative electrode 910 and awindow 911; the annularnegative electrode 910 is arranged on the upper end of thedischarge tube 99, and thewindow 911 is arranged at the through hole in the center of the annularnegative electrode 910. The lower outer circumference of thedischarge tube 99 is evenly provided with annularpositive bumps 93; the voltage difference between the annularpositive bump 93 and the annularnegative electrode 910 makes the rare gas discharge in thedischarge tube 99 to generate vacuum ultraviolet light, and the vacuum ultraviolet photons pass through thewindow 911 Enter thephotoionization chamber 95; the outer circumference of the annularnegative electrode 910 is provided with a coaxial annularupper cover plate 92, and thephotoionization chamber 95 and thedischarge tube 99 are sealed by the fixed connection of thelower cover plate 91 and theupper cover plate 92. connection; thedischarge tube 99 is filled with rare gas to obtain vacuum ultraviolet light of different energies;

所述气相掺杂剂引入机构包括鼓泡罐104、第一导管103和第二导管106；所述鼓泡罐104内设有掺杂剂105；所述第一导管103的一端和第二导管106的一端均插设入鼓泡罐104，且第一导管103的端口插设在掺杂剂105内，第二导管106的端口位于掺杂剂105上方的鼓泡罐104内；第一导管103的另一端连接着进气管101；The gas-phase dopant introduction mechanism includes abubbling tank 104, afirst conduit 103 and asecond conduit 106; thebubbling tank 104 is provided with adopant 105; one end of thefirst conduit 103 and the second conduit One end of 106 is inserted into thebubbling tank 104, and the port of thefirst conduit 103 is inserted in thedopant 105, and the port of thesecond conduit 106 is located in thebubbling tank 104 above thedopant 105; the first conduit The other end of 103 is connected to theintake pipe 101;

所述第一导管103上串联着第一流量控制计102，所述第二导管106上串联着第二流量控制计107；A firstflow control meter 102 is connected in series on thefirst conduit 103, and a secondflow control meter 107 is connected in series on thesecond conduit 106;

工作时，将载有待测样品11的玻片7放置于载物台6上；所述光电离室95温度为250～380 ℃，真空度为2×10³～5×10⁴ Pa。During operation, theglass slide 7 carrying the sample to be tested 11 is placed on thestage 6 ; the temperature of thephotoionization chamber 95 is 250-380° C., and the vacuum degree is 2×10³ ˜5×10⁴ Pa.

进一步地技术方案如下：Further technical solutions are as follows:

所述光电离室95的内径和导流管97的内径连接处为喇叭状管光滑过渡连接；所述光电离室95的内径和导流管97的外径之间的间距为1-2 mm；光电离室95的内径为4-8 mm；导流管97的内径为1.2-1.6 mm，取样毛细管8内径为0.5-1.5 mm。The connection between the inner diameter of thephotoionization chamber 95 and the inner diameter of the guidingtube 97 is a smooth transition connection of a trumpet tube; the distance between the inner diameter of thephotoionization chamber 95 and the outer diameter of the guidingtube 97 is 1-2 mm ; The inner diameter of thephotoionization chamber 95 is 4-8 mm; the inner diameter of theguide tube 97 is 1.2-1.6 mm, and the inner diameter of thesampling capillary 8 is 0.5-1.5 mm.

所述玻片7与取样毛细管8出口之间的距离不大于5mm；取样毛细管8进口与所述窗片911表面的距离小于8mm，掺杂剂引入管94出口与所述窗片911表面的距离也小于8mm；所述激光波长为紫外光、可见光或红外光波段。The distance between theglass slide 7 and the outlet of the sampling capillary 8 is not more than 5 mm; the distance between the inlet of the sampling capillary 8 and the surface of thewindow 911 is less than 8 mm, and the distance between the outlet of thedopant introduction tube 94 and the surface of thewindow 911 It is also less than 8mm; the laser wavelength is ultraviolet light, visible light or infrared light band.

所述载物台6为导体，玻片7的表面喷涂导电材料氧化铟锡时，正离子模式下选择性施加0V至10KV电压，负离子模式下选择性施加-10KV至0V。Thestage 6 is a conductor. When the conductive material indium tin oxide is sprayed on the surface of theglass slide 7, a voltage of 0V to 10KV is selectively applied in positive ion mode, and -10KV to 0V is selectively applied in negative ion mode.

所述窗片911材料为氟化镁（Mg2F）或氟化锂（LiF）。The material of thewindow 911 is magnesium fluoride (Mg2F) or lithium fluoride (LiF).

检测时，在所述待测样品11表面喷涂基质以增加激光解析电离效率，所述基质为α-氰基-4-羟基肉桂酸或2,5-二羟基苯甲酸或二氧化钛。During detection, a substrate is sprayed on the surface of the sample to be tested 11 to increase the laser desorption ionization efficiency, and the substrate is α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid or titanium dioxide.

所述第一流量控制计102的流量小于第二流量控制计107的流量，通过控制两者的流量差值可调节气相掺杂剂的引入量。The flow rate of the firstflow control meter 102 is smaller than the flow rate of the secondflow control meter 107 , and the introduction amount of the gas-phase dopant can be adjusted by controlling the flow difference between the two.

所述掺杂剂为甲苯、丙酮、苯甲醚、氯苯、溴苯、二硫化碳中的一种。The dopant is one of toluene, acetone, anisole, chlorobenzene, bromobenzene and carbon disulfide.

所述稀有气体为氦气、氖气、氪气、氙气、氮气中一种。The rare gas is one of helium, neon, krypton, xenon and nitrogen.

本发明的有益技术效果体现在以下方面：The beneficial technical effect of the present invention is embodied in the following aspects:

1. 本发明与结合解吸电喷雾电离的光电离质谱成像装置相比：（1）掺杂剂通过掺杂剂鼓泡系统以气相形式直接引入光电离室，更加高效、稳定；（2）在解吸电喷雾电离的光电离质谱成像装置中，电喷雾针出口端与取样毛细管之间的水平距离为10～40mm，然而本发明取样口距离激光解析位点小于2mm，取样效率高；（3）解吸电喷雾电离的光电离质谱成像装置需要用高速雾化气从雾化器中喷出以雾化溶剂，在高速雾化气吹扫下取样毛细管入口处温度会显著降低，影响取样效率，而本发明不存在上述问题；（4）解吸电喷雾电离的光电离质谱成像技术的成像空间分辨率仅可达到200μm，如图6所示，本发明可实现≤20μm空间分辨的质谱成像。1. Compared with the photoionization mass spectrometry imaging device combined with desorption electrospray ionization, the present invention: (1) the dopant is directly introduced into the photoionization chamber in the form of gas phase through the dopant bubbling system, which is more efficient and stable; In the photoionization mass spectrometry imaging device for desorption electrospray ionization, the horizontal distance between the outlet end of the electrospray needle and the sampling capillary is 10-40 mm, but the distance between the sampling port of the present invention is less than 2 mm from the laser desorption site, and the sampling efficiency is high; (3) The photoionization mass spectrometry imaging device of desorption electrospray ionization needs to spray the high-speed atomizing gas from the atomizer to atomize the solvent. Under the high-speed atomizing gas purging, the temperature at the inlet of the sampling capillary will be significantly reduced, which will affect the sampling efficiency. The present invention does not have the above problems; (4) the imaging spatial resolution of the photoionization mass spectrometry imaging technology of desorption electrospray ionization can only reach 200 μm, as shown in FIG.

2．本发明与MALDI-2相比：（1） MALDI-2是一种真空里的解析电离技术，更换样品时需将样品从真空中取出，而本发明是常压激光解析，操作更加方便；（2）虽然MALDI-2与本发明都采用紫外光作为二次电离方式，但是MALDI-2是利用光致直接电离，本发明是利用光致诱导的离子分子反应和光致直接电离的总和实现二次电离，离子分子反应可显著提高光电离效率，这在之前的文献中已有报道（Anal. Chim. Acta, 2015, 891, 203-210）；（3）二次电离室在本发明（2×10²～5×10⁴Pa）可实现比MALDI-2（2.0-2.5 mbar）更高的真空度，根据理想气体状态方程PV=nRT,相同温度，相同体积空间内，真空度P越高，待测分子物质的量n越大，即待测分子体积密度越大，可电离得到更高密度的待测离子。2. Compared with MALDI-2, the present invention: (1) MALDI-2 is an analytical ionization technology in a vacuum, and the sample needs to be taken out from the vacuum when replacing the sample, while the present invention is a normal-pressure laser analysis, and the operation is more convenient; ( 2) Although both MALDI-2 and the present invention use ultraviolet light as the secondary ionization method, MALDI-2 uses photo-induced direct ionization, and the present invention uses the sum of photo-induced ion-molecule reaction and photo-induced direct ionization to achieve secondary ionization. ionization, the ion-molecule reaction can significantly improve the photoionization efficiency, which has been reported in the previous literature (Anal. Chim. Acta, 2015, 891, 203-210); (3) The secondary ionization chamber is used in the present invention (2× 10² ~ 5×10⁴ Pa) can achieve a higher vacuum degree than MALDI-2 (2.0-2.5 mbar). According to the ideal gas state equation PV=nRT, the same temperature, in the same volume space, the higher the vacuum degree P, The larger the amount n of the molecular substance to be tested is, that is, the larger the bulk density of the molecular substance to be tested is, and the higher density of the tested ions can be obtained by ionization.

附图说明Description of drawings

图1为本发明结构示意图。Figure 1 is a schematic structural diagram of the present invention.

图2为图1的局部放大图。FIG. 2 is a partial enlarged view of FIG. 1 .

图3为图1的二次光电离机构剖视放大图。FIG. 3 is an enlarged cross-sectional view of the secondary photoionization mechanism of FIG. 1 .

图4为图1的气相掺杂剂引入机构剖视放大图。FIG. 4 is an enlarged cross-sectional view of the gas-phase dopant introduction mechanism of FIG. 1 .

图5为红色水彩笔涂在玻片上，经常压激光解析电离和常压激光解析电离-二次光电离质谱分析得到的正离子模式质谱图，其中图5中A为常压激光解析电离图，图5中B为常压激光解析电离-二次光电离质谱分析红色水彩笔中罗丹明6G的质谱图。Figure 5 is a positive ion mode mass spectrum obtained by applying a red watercolor pen on a glass slide, atmospheric pressure laser desorption ionization and atmospheric pressure laser desorption ionization-secondary photoionization mass spectrometry, in which A in Figure 5 is the atmospheric pressure laser desorption ionization spectrum, B in FIG. 5 is the mass spectrum of rhodamine 6G in red watercolor pen analyzed by atmospheric pressure laser desorption ionization-secondary photoionization mass spectrometry.

图6为利用本发明中常压激光解析电离和常压激光解析电离-二次光电离质谱分析得到的小鼠小脑组织胆固醇(m/z 369)的分布图。6 is a distribution diagram of cholesterol (m/z 369) in mouse cerebellum tissue obtained by atmospheric pressure laser desorption ionization and atmospheric pressure laser desorption ionization-secondary photoionization mass spectrometry in the present invention.

图1-4中序号：激光器1、激光2、扩束镜3、反光镜4、聚焦镜5、载物台6、玻片7、取样毛细管8、二次光电离机构9、气相掺杂剂引入机构10、待测样品11、下盖板91、上盖板92、环形正极凸块93、放电管99、环形负极910、窗片911、掺杂剂引入管94、光电离室95、抽真空管96、导流管97、质谱传输毛细管98、进气管101、第一流量控制计102、第一导管103、鼓泡罐104、掺杂剂105、第二导管106、第二流量控制计107。Serial numbers in Figure 1-4:Laser 1,Laser 2,Beam Expander 3,Reflector 4, FocusingMirror 5,Stage 6,Glass Slide 7,Sampling Capillary 8, Secondary Photoionization Mechanism 9, Gas Phase Dopant Introduction mechanism 10, sample to be tested 11,lower cover 91,upper cover 92, annularpositive bump 93,discharge tube 99, annular negative 910,window 911,dopant introduction tube 94,photoionization chamber 95,pump Vacuum tube 96 ,draft tube 97 , massspectrometer transmission capillary 98 ,air inlet tube 101 , firstflow control meter 102 ,first conduit 103 , bubblingtank 104 ,dopant 105 ,second conduit 106 , secondflow control meter 107 .

具体实施方式Detailed ways

下面结合附图，通过实施例对本发明作进一步地描述。Below in conjunction with the accompanying drawings, the present invention will be further described through embodiments.

实施例Example

参见图1，一种具有常压激光解析电离和二次光电离的质谱成像装置包括激光解析电离机构；激光解析电离机构包括质谱仪、激光器1、扩束镜3、反光镜4、聚焦镜5、载物台6和取样毛细管8；还包括二次光电离机构和气相掺杂剂引入机构。Referring to FIG. 1, a mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization includes a laser desorption ionization mechanism; the laser desorption ionization mechanism includes a mass spectrometer, alaser 1, abeam expander 3, areflector 4, and a focusingmirror 5 , astage 6 and asampling capillary 8; it also includes a secondary photoionization mechanism and a gas-phase dopant introduction mechanism.

参见图2，激光器1输出的349nm紫外光先经扩束镜3扩束的激光2，经反光镜4反射，然后，通过聚焦镜5聚焦至生物组织11的背侧。玻片7选用喷涂导电氧化铟锡材料的石英玻片，90%以上349nm紫外光可穿过该玻片7。载物台6为导电材料，加上5KV电压，待测样品11表面距离取样毛细管8的取样口之间的间距1mm，待测样品11为生物组织切片时，其厚度为10μm。Referring to FIG. 2 , the 349 nm ultraviolet light output by thelaser 1 is firstly beamed by thelaser beam 2 expanded by thebeam expander 3 , reflected by thereflector 4 , and then focused to the back side of thebiological tissue 11 by the focusingmirror 5 . Theglass slide 7 is a quartz glass slide sprayed with conductive indium tin oxide material, and more than 90% of the 349 nm ultraviolet light can pass through theglass slide 7 . Thestage 6 is a conductive material, and a voltage of 5KV is applied. The distance between the surface of thesample 11 and the sampling port of thesampling capillary 8 is 1 mm. When thesample 11 is a biological tissue slice, its thickness is 10 μm.

参见图3，二次光电离机构包括管状的光电离室95和真空紫外放电灯。Referring to FIG. 3 , the secondary photoionization mechanism includes atubular photoionization chamber 95 and a vacuum ultraviolet discharge lamp.

光电离室95的上端内设有同轴的导流管97，光电离室95的内径和导流管97的内径连接处为喇叭管状光滑过渡连接。导流管97的上端口与质谱仪的传输毛细管98的出口对应，与导流管97外周对应的光电离室95一侧连通着抽真空管96的一端；光电离室95下部一侧连通着取样毛细管8的一端；光电离室95的下端连接着同轴的下盖板91，下盖板91的一侧连通着掺杂剂引入管94的出口端。The upper end of thephotoionization chamber 95 is provided with acoaxial guide tube 97, and the connection between the inner diameter of thephotoionization chamber 95 and the inner diameter of theguide tube 97 is a horn-shaped smooth transition connection. The upper port of theguide tube 97 corresponds to the outlet of thetransmission capillary 98 of the mass spectrometer, and the side of thephotoionization chamber 95 corresponding to the outer periphery of theguide tube 97 is connected to one end of the evacuatedtube 96; the lower side of thephotoionization chamber 95 is connected to the sampling One end of thecapillary 8; the lower end of thephotoionization chamber 95 is connected to the coaxiallower cover plate 91, and one side of thelower cover plate 91 is connected to the outlet end of thedopant introduction tube 94.

真空紫外放电灯包括放电管99、环形负极910和窗片911；所述环形负极910设于放电管99的上端，所述窗片911设于环形负极910中心的通孔处，所述放电管99的下部外圆周上均布设有环形正极凸块93；环形正极凸块93与环形负极910之间的电压差使放电管99内稀有气体放电产生真空紫外光，真空紫外光子通过窗片911进入光电离室95；所述环形负极910外圆围上设有同轴的环状的上盖板92，通过下盖板91和上盖板92固定连接实现光电离室95和放电管99密封连接。放电管99中填充稀有气体氦气。The vacuum ultraviolet discharge lamp includes adischarge tube 99, an annularnegative electrode 910 and awindow 911; the annularnegative electrode 910 is arranged at the upper end of thedischarge tube 99, and thewindow 911 is arranged at the through hole in the center of the annularnegative electrode 910. The lower outer circumference of 99 is evenly provided with annularpositive bumps 93; the voltage difference between the annularpositive bumps 93 and the annular negative 910 makes the rare gas discharge in thedischarge tube 99 to generate vacuum ultraviolet light, and the vacuum ultraviolet photons enter the photoelectric through thewindow 911 There is a coaxial annularupper cover plate 92 on the outer circumference of the annularnegative electrode 910, and thephotoionization chamber 95 and thedischarge tube 99 are sealedly connected by the fixed connection of thelower cover plate 91 and theupper cover plate 92. Thedischarge tube 99 is filled with the rare gas helium.

光电离室95的内径和导流管97的外径之间的间距为1.2 mm；光电离室95的内径为5 mm；导流管97的内径为1.6 mm，取样毛细管8内径为1.5 mm。The distance between the inner diameter of thephotoionization chamber 95 and the outer diameter of the guidingtube 97 is 1.2 mm; the inner diameter of thephotoionization chamber 95 is 5 mm; the inner diameter of the guidingtube 97 is 1.6 mm, and the inner diameter of thesampling capillary 8 is 1.5 mm.

取样毛细管8出口与窗片911之间的距离1.5 mm，掺杂剂引入管94的出口与窗片911之间的距离为1 mm。The distance between the outlet of thesampling capillary 8 and thewindow 911 is 1.5 mm, and the distance between the outlet of thedopant introduction tube 94 and thewindow 911 is 1 mm.

参见图4，气相掺杂剂引入机构包括鼓泡罐104、第一导管103和第二导管106；所述鼓泡罐104内设有掺杂剂105。Referring to FIG. 4 , the gas-phase dopant introduction mechanism includes a bubblingtank 104 , afirst conduit 103 and asecond conduit 106 ; the bubblingtank 104 is provided with adopant 105 .

第一导管103的一端和第二导管106的一端均插设入鼓泡罐104，且第一导管103的端口插设在掺杂剂105内，第二导管106的端口位于掺杂剂105上方的鼓泡罐104内；第一导管103的另一端连接着进气管101；第一导管103上串联着第一流量控制计102，所述第二导管106上串联着第二流量控制计107。掺杂剂105为甲苯。One end of thefirst conduit 103 and one end of thesecond conduit 106 are inserted into the bubblingtank 104 , the port of thefirst conduit 103 is inserted into thedopant 105 , and the port of thesecond conduit 106 is located above thedopant 105 The other end of thefirst conduit 103 is connected to theair inlet pipe 101; thefirst conduit 103 is connected in series with a firstflow control meter 102, and thesecond conduit 106 is connected in series with a secondflow control meter 107. Thedopant 105 is toluene.

参见图4，通过第一导管103引入气体在鼓泡罐104内鼓泡使液态的掺杂剂甲苯挥发成气体进入到第二导管106内，进而使掺杂剂能够经掺杂剂引入管94进入到光电离室95内与待测物反应促进离子化，掺杂剂引入管94处于取样毛细管8与光源之间，从而确保样品能够在掺杂剂甲苯的辅助下进行离子化。第二流量控制计107的流速比第一流量控制计102的流速大8ml/min。Referring to FIG. 4 , the gas introduced through thefirst conduit 103 is bubbling in the bubblingtank 104 to volatilize the liquid dopant toluene into gas into thesecond conduit 106 , so that the dopant can be introduced into thedopant 94 through the dopant. Entering into thephotoionization chamber 95, it reacts with the analyte to promote ionization, and thedopant introduction tube 94 is located between the samplingcapillary 8 and the light source, so as to ensure that the sample can be ionized with the aid of the dopant toluene. The flow rate of the secondflow control meter 107 is greater than the flow rate of the firstflow control meter 102 by 8 ml/min.

本发明的工作原理详细说明如下：The working principle of the present invention is described in detail as follows:

待测样品11在聚焦的激光光斑作用下，从玻片7表面解析，解析出的化学物质离子化效率小于0.1%，带电离子和中性分子在真空抽吸作用下，同时从取样毛细管8进入光电离室95。在低于大气压的压力氛围下，未离子化的中性分子可在掺杂剂甲苯的辅助下，继续被从真空紫外灯窗片911发射出的真空紫外光二次电离，二次电离产生的离子与激光解析产生的带电离子一起通过导流管97和质谱传输毛细管98进入质谱仪被检测。Thesample 11 to be tested is analyzed from the surface of theglass slide 7 under the action of the focused laser spot, and the ionization efficiency of the analyzed chemical substances is less than 0.1%. The charged ions and neutral molecules enter from thesampling capillary 8 simultaneously under the action of vacuum suction.Photoionization chamber 95. Under the pressure atmosphere below atmospheric pressure, the unionized neutral molecules can continue to be secondary ionized by the vacuum ultraviolet light emitted from the vacuumultraviolet lamp window 911 with the assistance of the dopant toluene, and the ions generated by the secondary ionization Together with the charged ions generated by laser desorption, they enter the mass spectrometer through thedraft tube 97 and the mass transfer capillary 98 to be detected.

参见图5，将红色水彩笔涂覆在玻片7上，在常压激光解析电离和常压激光解析-二次光电离两种模式下分析得到的质谱分析结果。与常压激光解析电离模式相比，激光解析电离-二次光电离模式下，环形正极凸块93与环形负极910之间施加1500V起伏电压以点亮真空紫外放电灯，同时打开气相掺杂剂引入机构的第一流量控制计102和第二流量控制计107，引入掺杂剂甲苯。如图5中A所示，在传统的常压激光解析电离质谱图中，红色水彩笔中罗丹明6G（[M-Cl]⁺，m/z 443）的信号只有180；但是，在二次光电离辅助下的常压激光解析-二次光电离质谱图中（图5中B），罗丹明6G（[M-Cl]⁺，m/z 443）的信号达到了180000，以该物质为例说明，二次光电离可将待测样品11信号提高三个量级。Referring to FIG. 5 , a red watercolor pen is coated on theglass slide 7, and the obtained mass spectrometry analysis results are analyzed in two modes of atmospheric pressure laser desorption ionization and atmospheric pressure laser desorption-secondary photoionization. Compared with the normal pressure laser analytical ionization mode, in the laser analytical ionization-secondary photoionization mode, a 1500V undulating voltage is applied between the annularpositive electrode bump 93 and the annularnegative electrode 910 to light the vacuum ultraviolet discharge lamp, while turning on the gas phase dopant The firstflow control meter 102 and the secondflow control meter 107 of the introduction mechanism introduce the dopant toluene. As shown in A in Figure 5, in the traditional atmospheric pressure laser desorption ionization mass spectrometer, the signal of Rhodamine 6G ([M-Cl]⁺ , m/z 443) in the red watercolor pen is only 180; however, in the secondary In the atmospheric pressure laser desorption-secondary photoionization mass spectrometer assisted by photoionization (B in Figure 5), the signal of rhodamine 6G ([M-Cl]⁺ , m/z 443) reached 180000, and the substance was For example, secondary photoionization can increase the signal of the sample to be tested 11 by three orders of magnitude.

参见图6，二次光电离对待测物在待测样品11表面的空间可视化分析也有显著的影响，以胆固醇（m/z 369）在小鼠小脑组织成像结果为例，该成像图单像素点尺寸为20μm×20μm，该空间分辨率远远优于解吸电喷雾电离的光电离质谱成像分辨率（约200μm）。此外，常压激光解析电离-二次光电离质谱成像（y轴1.7-3.5mm），与传统的常压激光解析电离质谱成像（y轴0-1.7mm）相比，位于m/z 369处的胆固醇（[M+H-H₂O]⁺）信号显著提高，该实验证明，二次光电离增强待测样品11表面的待测物信号后，待测物在待测样品11表面空间分布的精细结构也更加清晰，成像质量显著提高。Referring to Figure 6, secondary photoionization also has a significant impact on the spatial visualization analysis of the test object on the surface of thetest sample 11. Taking the imaging results of cholesterol (m/z 369) in the mouse cerebellum as an example, the imaging image has a single pixel With a size of 20 μm × 20 μm, this spatial resolution is far superior to the photoionization mass spectrometry imaging resolution of desorption electrospray ionization (about 200 μm). In addition, atmospheric pressure laser desorption ionization-secondary photoionization mass spectrometry imaging (y-axis 1.7-3.5mm) is located at m/z 369 compared to conventional atmospheric-pressure laser desorption ionization-mass imaging (y-axis 0-1.7mm) The signal of cholesterol ([M+HH₂ O]⁺ ) is significantly improved. This experiment proves that after the signal of the analyte on the surface of thesample 11 to be tested is enhanced by the secondary photoionization, the spatial distribution of the analyte on the surface of thesample 11 to be tested is fine. The structure is also sharper, and the imaging quality is significantly improved.

本领域的技术人员容易理解，以上实施例仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。Those skilled in the art can easily understand that the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection scope of the present invention.

Claims (9)

Translated fromChinese

1.一种具有常压激光解析电离和二次光电离的质谱成像装置，包括激光解析电离机构，所述激光解析电离机构包括质谱仪、激光器（1）、扩束镜（3）、反光镜（4）、聚焦镜（5）、载物台（6）和取样毛细管（8），其特征在于：1. A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization, comprising a laser desorption ionization mechanism, the laser desorption ionization mechanism comprising a mass spectrometer, a laser (1), a beam expander (3), a reflector (4), a focusing mirror (5), a stage (6) and a sampling capillary (8), characterized in that:还包括二次光电离机构（9）和气相掺杂剂引入机构（10）；Also includes a secondary photoionization mechanism (9) and a gas-phase dopant introduction mechanism (10);所述二次光电离机构包括管状的光电离室（95）和真空紫外放电灯；The secondary photoionization mechanism includes a tubular photoionization chamber (95) and a vacuum ultraviolet discharge lamp;所述光电离室（95）为管状，光电离室（95）的上端内设有同轴的导流管（97），所述导流管（97）的上端口与质谱仪的传输毛细管（98）的出口对应，与导流管（97）外周对应的光电离室（95）一侧连通着抽真空管（96）的一端；光电离室（95）下部一侧连通着取样毛细管（8）的一端；光电离室（95）的下端连接着同轴的下盖板（91），所述下盖板（91）的一侧连通着掺杂剂引入管（94）的出口端；The photoionization chamber (95) is tubular, the upper end of the photoionization chamber (95) is provided with a coaxial guide tube (97), and the upper port of the guide tube (97) is connected to the transmission capillary of the mass spectrometer ( 98), the side of the photoionization chamber (95) corresponding to the outer periphery of the guide tube (97) is connected to one end of the vacuum tube (96); the lower side of the photoionization chamber (95) is connected to the sampling capillary (8) one end; the lower end of the photoionization chamber (95) is connected to the coaxial lower cover plate (91), and one side of the lower cover plate (91) is communicated with the outlet end of the dopant introduction tube (94);所述真空紫外放电灯包括放电管（99）、环形负极（910）和窗片（911）；所述环形负极（910）设于放电管（99）的上端，所述窗片（911）设于环形负极（910）中心的通孔处，所述放电管（99）的下部外圆周上均布设有环形正极凸块（93）；环形正极凸块（93）与环形负极（910）之间的电压差使放电管（99）内稀有气体放电产生真空紫外光，真空紫外光子通过窗片（911）进入光电离室（95）；所述环形负极（910）外圆围上设有同轴的环状的上盖板（92），通过下盖板（91）和上盖板（92）固定连接实现光电离室（95）和放电管（99）密封连接；所述放电管（99）中填充稀有气体，得到不同能量的真空紫外光；The vacuum ultraviolet discharge lamp includes a discharge tube (99), an annular negative electrode (910) and a window (911); the annular negative electrode (910) is arranged on the upper end of the discharge tube (99), and the window (911) is arranged on the upper end of the discharge tube (99). At the through hole in the center of the annular anode (910), annular anode bumps (93) are evenly distributed on the lower outer circumference of the discharge tube (99); between the annular anode bump (93) and the annular anode (910) The voltage difference caused by the discharge tube (99) causes the rare gas discharge in the discharge tube (99) to generate vacuum ultraviolet light, and the vacuum ultraviolet photons enter the photoionization chamber (95) through the window (911); A ring-shaped upper cover plate (92), through which the lower cover plate (91) and the upper cover plate (92) are fixedly connected to achieve a sealed connection between the photoionization chamber (95) and the discharge tube (99); in the discharge tube (99) Filling with rare gas to obtain VUV light of different energies;所述气相掺杂剂引入机构包括鼓泡罐（104）、第一导管（103）和第二导管（106）；所述鼓泡罐（104）内设有掺杂剂（105）；所述第一导管（103）的一端和第二导管（106）的一端均插设入鼓泡罐（104），且第一导管（103）的端口插设在掺杂剂（105）内，第二导管（106）的端口位于掺杂剂（105）上方的鼓泡罐（104）内；第一导管（103）的另一端连接着进气管（101）；The gas-phase dopant introduction mechanism includes a bubbling tank (104), a first conduit (103) and a second conduit (106); the bubbling tank (104) is provided with a dopant (105); the One end of the first conduit (103) and one end of the second conduit (106) are inserted into the bubbling tank (104), and the port of the first conduit (103) is inserted into the dopant (105), and the second conduit (103) is inserted into the dopant (105). The port of the conduit (106) is located in the bubbling tank (104) above the dopant (105); the other end of the first conduit (103) is connected to the air inlet pipe (101);所述第一导管（103）上串联着第一流量控制计（102），所述第二导管（106）上串联着第二流量控制计（107）；A first flow controller (102) is connected in series on the first conduit (103), and a second flow controller (107) is connected in series on the second conduit (106);工作时，将载有待测样品（11）的玻片（7）放置于载物台（6）上；所述光电离室（95）温度为250～380 ℃，真空度为2×10³～5×10⁴ Pa。During operation, the glass slide (7) carrying the sample to be tested (11) is placed on the stage (6); the temperature of the photoionization chamber (95) is 250-380°C, and the vacuum degree is 2×10³ ~5×10⁴ Pa.2.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述光电离室（95）的内径和导流管（97）的内径连接处为喇叭状管光滑过渡连接；所述光电离室（95）的内径和导流管（97）的外径之间的间距为1-2 mm；光电离室（95）的内径为4-8 mm；导流管（97）的内径为1.2-1.6 mm，取样毛细管（8）内径为0.5-1.5 mm。2. A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: the inner diameter of the photoionization chamber (95) and the inner diameter of the guide tube (97) The connection is a smooth transition connection of a trumpet tube; the distance between the inner diameter of the photoionization chamber (95) and the outer diameter of the guide tube (97) is 1-2 mm; the inner diameter of the photoionization chamber (95) is 4 mm -8 mm; the inner diameter of the draft tube (97) is 1.2-1.6 mm, and the inner diameter of the sampling capillary (8) is 0.5-1.5 mm.3.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述玻片（7）与取样毛细管（8）出口之间的距离不大于5mm；取样毛细管（8）进口与所述窗片（911）表面的距离小于8mm，掺杂剂引入管（94）出口与所述窗片（911）表面的距离也小于8mm；所述激光波长为紫外光、可见光或红外光波段。3. A mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: the distance between the glass slide (7) and the outlet of the sampling capillary (8) is different. greater than 5mm; the distance between the inlet of the sampling capillary (8) and the surface of the window (911) is less than 8mm, and the distance between the outlet of the dopant introduction tube (94) and the surface of the window (911) is also less than 8mm; the laser The wavelengths are ultraviolet, visible or infrared.4.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述载物台（6）为导体，玻片（7）的表面喷涂导电材料氧化铟锡时，正离子模式下选择性施加0V至10KV电压，负离子模式下选择性施加-10KV至0V。4. The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: the stage (6) is a conductor, and the surface of the glass slide (7) is sprayed When the conductive material is indium tin oxide, 0V to 10KV is selectively applied in positive ion mode, and -10KV to 0V is selectively applied in negative ion mode.5.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述窗片（911）材料为氟化镁（Mg2F）或氟化锂（LiF）。5. The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: the material of the window (911) is magnesium fluoride (Mg2F) or lithium fluoride (LiF).6.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：检测时，在所述待测样品（11）表面喷涂基质以增加激光解析电离效率，所述基质为α-氰基-4-羟基肉桂酸或2,5-二羟基苯甲酸或二氧化钛。6. The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: during detection, a matrix is sprayed on the surface of the sample to be tested (11) to increase laser desorption Ionization efficiency, the matrix is α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid or titanium dioxide.7.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述第一流量控制计（102）的流量小于第二流量控制计（107）的流量，通过控制两者的流量差值可调节气相掺杂剂的引入量。7. The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1, characterized in that: the flow rate of the first flow control meter (102) is smaller than that of the second flow control meter ( 107), the introduction amount of the gas-phase dopant can be adjusted by controlling the flow difference between the two.8.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述掺杂剂为甲苯、丙酮、苯甲醚、氯苯、溴苯、二硫化碳中的一种。8 . The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1 , wherein the dopant is toluene, acetone, anisole, chlorobenzene, and bromobenzene. 9 . , one of carbon disulfide.9.根据权利要求1所述的一种具有常压激光解析电离和二次光电离的质谱成像装置，其特征在于：所述稀有气体为氦气、氖气、氪气、氙气、氮气中一种。9 . The mass spectrometry imaging device with atmospheric pressure laser desorption ionization and secondary photoionization according to claim 1 , wherein the rare gas is one of helium, neon, krypton, xenon, and nitrogen. 10 . kind.

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