EP2660848A1 - Dreidimensionale molekulare Bildgebung mittels Infrarot-Laserablation-Elektrospray-Ionisation-Massenspektrometrie - Google Patents

Dreidimensionale molekulare Bildgebung mittels Infrarot-Laserablation-Elektrospray-Ionisation-Massenspektrometrie Download PDF

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EP2660848A1
EP2660848A1 EP13170238.3A EP13170238A EP2660848A1 EP 2660848 A1 EP2660848 A1 EP 2660848A1 EP 13170238 A EP13170238 A EP 13170238A EP 2660848 A1 EP2660848 A1 EP 2660848A1
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sample
laser
ions
imaging
laesi
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French (fr)
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Akos Vertes
Peter Nemes
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George Washington University
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George Washington University
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/14—Ion sources; Ion guns using particle bombardment, e.g. ionisation chambers
    • H01J49/145—Ion sources; Ion guns using particle bombardment, e.g. ionisation chambers using chemical ionisation
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0004—Imaging particle spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
    • H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step

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  • the field of the invention is atmospheric pressure mass spectrometry (MS), and more specifically a process and apparatus which combine infrared laser ablation with electrospray ionization (ESI) to provide three-dimensional molecular imaging of chemicals in specimens, for example, metabolites in live tissues or cells.
  • MS atmospheric pressure mass spectrometry
  • ESI electrospray ionization
  • Three-dimensional (3D) tissue or cell imaging of molecular distributions offers insight into the correlation between biochemical processes and the spatial organization of cells in a tissue.
  • Presently available methods generally rely on the interaction of electromagnetic radiation (e.g., magnetic resonance imaging and fluorescence or multiphoton microscopy) or particles (e.g., secondary ion mass spectrometry, SIMS) with the specimen.
  • electromagnetic radiation e.g., magnetic resonance imaging and fluorescence or multiphoton microscopy
  • particles e.g., secondary ion mass spectrometry, SIMS
  • coherent anti-Stokes Raman scattering provides vibrant lateral and depth resolution for in vivo imaging of lipid distributions on cellular or subcellular level. They, however, typically report on only a few species and often require the introduction of molecular labels. These obstacles are less pronounced in methods based on mass spectrometry (MS) that report the distributions for diverse molecular species.
  • MS mass spectrometry
  • Imaging by SIMS and matrix-assisted laser desorption ionization (MALDI) are appealing because they capture the two- and three-dimensional distributions of endogenous and drug molecules in tissue and whole-body sections. Characteristic to these methods is the requirement for delicate chemical and physical sample manipulation and the need to perform the imaging experiment in vacuum, preventing the study of live specimens.
  • Ambient MS circumvents these limitations by bringing the ionization step into the atmosphere while minimizing chemical and physical treatment to the sample.
  • this field has experienced rapid development providing us with an array of ambient ion sources.
  • Desorption electrospray ionization (DESI) in combination with MS has been successful in various applications, including the detection of drugs, metabolites and explosives on human fingers, and the profiling of untreated bacteria.
  • DESI and extractive electrospray ionization have been used in metabolomic fingerprinting of bacteria.
  • IR-MALDI and MALDESI a combination of MALDI and DESI, the energy necessary for the desorption and ionization of the analyte is deposited by a mid-IR and a UV laser, respectively.
  • electrospray laser desorption ionization ELDI
  • the efficiency of ion production by a UV laser is enhanced by postionization using an electrospray source.
  • LAESI Laser ablation electrospray ionization
  • a laser pulse at ⁇ 2.9 ⁇ m wavelength ablates a minute volume of the sample to eject fine neutral particles and/or molecules.
  • This laser plume is intercepted by an electrospray and the ablated material is efficiently ionized to produce mass spectra similar to direct electrospray ionization.
  • LAESI we have demonstrated metabolic analysis of less than 100 ng tissue material from volumes below 100 pL. As in LAESI the laser energy is absorbed by the native water in the sample, the photochemical damage of the biologically relevant molecules, such as DNA, peptides, proteins and metabolites is negligible.
  • Ambient imaging mass spectrometry captures the spatial distribution of chemicals with molecular specificity. Unlike optical imaging methods, IMS does not require color or fluorescent labels for successful operation.
  • 2D imaging A handful of MS-based techniques has demonstrated molecular two dimensional (2D) imaging in AP environment: AP IR-MALDI and DESI captured metabolite transport in plant vasculature and imaged drug metabolite distributions in thin tissue sections, respectively.
  • 2D imaging LAESI provided insight into metabolic differences between the differently colored sectors of variegated plants. The lateral resolution of these methods generally ranged from 100 to 300 ⁇ m.
  • volume distributions of molecules in organisms are of interest in molecular and cell biology. Recently LAESI MS showed initial success in depth profiling of metabolites in live plant tissues but 3D imaging is not yet available for the ambient environment.
  • a process and apparatus which combine infrared laser ablation with electrospray ionization (ESI) to provide three-dimensional molecular imaging of metabolites in live tissues or cells.
  • EESI electrospray ionization
  • the ions which can be analyzed using this process include but are not limited to metabolites, lipids and other biomolecules, pharmaceuticals, dyes, explosives, narcotics and polymers.
  • the invention starts with using a focused IR laser beam to irradiate a sample thus ablating a plume of ions and particulates. This plume is then intercepted with charged electrospray droplets. From the interaction of the laser ablation plume and the electrospray droplets, gas phase ions are produced that are detected by a mass spectrometer. This is performed at atmospheric pressure.
  • a method for the three-dimensional imaging of a live tissue or cell sample by mass spectrometry comprising: subjecting the live tissue or cell sample to infrared LAESI mass spectrometry, wherein the LAESI-MS is performed using a LAESI-MS device directly on the live tissue or cell sample wherein the sample does not require conventional MS pre-treatment and is performed at atmospheric pressure, wherein the LAESI-MS device is equipped with a scanning apparatus for lateral scanning of multiple points on a grid or following the cellular pattern or regions of interest that is defined on the live tissue or cell sample, and for depth profiling of each point on the grid or following the cellular pattern or regions of interest by performing multiple ablations at each point, each laser pulse of said ablations ablating a deeper layer of the live tissue or cell sample than a prior pulse, wherein the combination of lateral scanning and depth profiling provides three-dimensional molecular distribution imaging data.
  • an ambient ionization process for producing three-dimensional imaging of a sample which comprises: i) irradiating the sample with an infrared laser to ablate the sample; ii) intercepting this ablation plume with an electrospray to form gas-phase ions; and iii) analyzing the produced ions using mass spectrometry, wherein the LAESI-MS is performed using a LAESI-MS device directly on the live tissue or cell sample wherein the sample does not require conventional chemical/physical preparation and is performed at atmospheric pressure, wherein the LAESI-MS device is equipped with a scanning apparatus for lateral scanning of multiple points on a grid or following the cellular pattern or regions of interest that is defined on the live tissue or cell sample, and for depth profiling of each point on the grid or following the cellular pattern or regions of interest by performing multiple ablations at each point, each laser pulse of said ablations ablating a deeper layer of the live tissue or cell sample than a prior pulse, wherein the combination of
  • LAESI-MS detects ions from target molecules within the sample, said ions selected from the group consisting of pharmaceuticals, metabolites, dyes, explosives or explosive residues, narcotics, polymers, chemical warfare agents and their signatures, peptides, oligosaccharides, proteins, metabolites, lipids and other biomolecules, synthetic organics, drugs, and toxic chemicals.
  • a LAESI-MS device for three-dimensional imaging of a sample, comprising: i) a pulsed infrared laser for emitting energy at the sample; ii) an electrospray apparatus for producing a spray of charged droplets; iii) a mass spectrometer having an ion transfer inlet for capturing the produced ions; iv) and a scanning apparatus for lateral scanning of multiple points on a grid or following the cellular pattern or regions of interest that is defined on the sample, and for depth profiling of each point on the grid or following the cellular pattern or regions of interest by controlling the performing of multiple ablations at each point, each laser pulse of said ablations ablating a deeper layer of the sample than a prior pulse, wherein the combination of lateral scanning and depth profiling provides three-dimensional molecular distribution imaging data.
  • the device herein further comprising wherein the LAESI-MS is performed at atmospheric pressure.
  • the device herein further comprising an automated feedback mechanism to correct for variances in water content and tensile strength of the sample by continuously adjusting laser energy and/or laser wavelength while recording the depth of ablation for each pulse.
  • LAESI-MS detects ions from target molecules within the sample, said ions selected from the group consisting of pharmaceuticals, dyes, explosives or explosive residues, narcotics, polymers, chemical warfare agents and their signatures, peptides, oligosaccharides, proteins, metabolites, lipids, and other biomolecules, synthetic organics, drugs, and toxic chemicals.
  • a (parent) method for the direct chemical analysis of a sample by mass spectrometry comprising: subjecting a sample to infrared LAESI mass spectrometry, wherein the sample is selected from the group consisting of pharmaceuticals, dyes, explosives, narcotics, polymers, tissue or cell samples, and biomolecules, and wherein the LAESI-MS is performed using a LAESI-MS device directly on a sample wherein the sample does not require conventional MS pre-treatment and is performed at atmospheric pressure.
  • FIGS 1-4 Three-dimensional imaging with LAESI MS was demonstrated on leaf tissues of S. Lynise. The adaxial and the abaxial cuticles were marked with right angle lines and a spot colored in basic blue 7 and rhodamine 6G, respectively.
  • FIGURE 1 shows the top view of the interrogated area with an array of ablation marks. Some rhodamine 6G dye from the bottom surface is visible through the ablation holes. Brown discoloration surrounding the edges of the analysis area was linked to dehydration and/or oxidation. Combination of lateral scanning and depth profiling provided 3D molecular distributions.
  • FIGURE 2 shows the ion intensities from basic blue 7 ( m / z 478.3260 in blue), rhodamine 6G ( m / z 443.2295 in orange/wine) and leucine ( m / z 154.0819 in grey/ black) on false color scales.
  • the ion distributions for the two dyes paralleled the mock patterns shown in the optical image. Higher abundances of the endogenous metabolite leucine were observed in the top two layers.
  • FIGURE 3 shows the distribution of cyanidin/kaempferol rhamnoside glucoside ( m / z 595.1649 in grey).
  • FIGURE 4 The molecular distribution pattern for protonated chlorophyll a ( m / z 893.5425 in cyan/royal blue) showed accumulation in the spongy mesophyll region, in agreement with the known localization of chloroplasts within plant tissues.
  • FIGS 5-6 For the depth imaging of S . Lynise leaves, six successive single laser pulses were delivered to the adaxial surface. Mass analysis of the generated ions indicated varying tissue chemistry with depth. FIGURES 5 and 6 present representative mass spectra acquired for the first and second laser shots, respectively. They indicated that flavonoids ( m / z 383.1130) and cyanidin/kaempferol rhamnoside glucoside ( m / z 595.1649) were present at higher abundances in the top 30-40- ⁇ m section of the tissue.
  • FIGURE 7 Optical image of the variegation pattern on the leaf of A. Squarrosa. The metabolite makeup of the rastered area was probed by 3D LAESI IMS. The top view of the resulting array of circular 350 ⁇ m ablation marks can be seen in FIGURE 8 .
  • the 3D distribution of kaempferol-(diacetyl coumarylrhamnoside) with m/z 663.1731 included in FIGURE 9 was an example for accumulation in the mesophyll (third and fourth) layers with uniform distributions within these layers.
  • the protonated chlorophyll a ion with m/z 893.5457 also populated the mesophyll layers and is shown in cyan-royal color scale in FIGURE 10 .
  • Kaempferol/luteolin with m/z 287.0494 exhibited heterogeneity both laterally and in the cross section, and was most abundant in the second and third layers.
  • FIGURE 11 Acacetin with m/z 285.0759 belonged to a group of compounds with tissue-specificity not previously revealed in lateral imaging experiments due to the averaging of depth distributions.
  • Table 1 Tentative assignment of the observed ions was achieved on the basis of accurate mass measurement, collision-activated dissociation, isotope peak distribution analysis, and a wide plant metabolome data-base search.
  • the mass accuracy, ⁇ m is the difference between the measured and calculated monoisotopic masses.
  • Lynise epidermal cells were elliptical in shape with axes of ⁇ 20 and ⁇ 60 ⁇ m. The average height of the cells measured 15 ⁇ m. Thus, each ⁇ 4 nL imaging voxel sampled about 300 cells for analysis.
  • the top view of the leaf following LAESI 3D IMS can be seen in Figure 1 .
  • the interrogated area was marked by an array of ⁇ 350- ⁇ m-diameter ablation spots with a displacement of 500 ⁇ m in both directions.
  • This lateral step size yielded ⁇ 2-3 pixels to sample across the width of the lines drawn in basic blue 7.
  • a circular Rhodamine 6G dye pattern from the marking of the back side can be seen in the lower left corner of the image, indicating complete tissue removal in 6 laser pulses. Scanning electron microscopy images confirmed that the first laser pulse successfully removed the protective waxy cuticle layer.
  • Cyanidin rhamnoside and/or luteolinidin glucoside ( m / z 433.1125) and cyanidin/kaempferol rhamnoside glucoside ( m / z 595.1649) were generally observed at higher abundances in the top 40 ⁇ m section of the tissue.
  • the second pulse which sampled the layer between 40 ⁇ m and 80 ⁇ m from the top surface, new ions emerged in the m / z 600 to 1000 region of the spectrum. Singly charged ions characteristic to this section were observed at m / z 650.4, 813.5, 893.5, and 928.6.
  • Other ions, such as m / z 518.4, 609.4, 543.1, and 621.3 were observed at higher abundances during the third, fourth, fifth and six laser pulses, respectively.
  • the photosynthetic cycle is known to involve a variety of chlorophyll derivatives.
  • ions with m / z 813.4917, 852.5833, 860.5171, and 928.6321 exhibited similar 3D molecular patterns and isotopic distributions to that of [chlorophyll a + H] + .
  • Prolonged thermal treatment of vegetables has been described to yield m / z 813.5, a fragment of pyrochlorophyll a , supporting this scenario.
  • LAESI probes the neutrals and particulates that are ejected at a later phase when the sample is closer to thermal equilibrium with the environment.
  • the time frame of sampling and mass analysis is tens of milliseconds, which is at least four orders of magnitude shorter than those needed to cause extensive chlorophyll a decomposition.
  • Leaves of A. Squarrosa demonstrated a higher tensile strength and thickness than those of S. Lynise.
  • the incident laser energy was slightly increased to compensate for these effects and to obtain depth analysis with 6 laser pulses.
  • the thickness of the selected leaf area for analysis was generally ⁇ 300-350 ⁇ m, corresponding to a depth resolution of 50-60 ⁇ m/pulse.
  • the abaxial surface contained two parallel-running secondary veins that induced ⁇ 50-100 ⁇ m protrusions on the lower side of the lamina, producing a total thickness of 350-450 ⁇ m in these regions.
  • the 3D chemical makeup of an 11.5x7.5 mm 2 area was probed on a 24x16x6 grid resulting in 2,304 voxels.
  • Three-dimensional molecular imaging of mass-selected ions revealed a variety of distribution patterns for metabolites and indicated the coexistence of diverse metabolic pathways. These patterns could be grouped on the basis of lateral and cross-sectional molecular homogeneity.
  • the first group of metabolites demonstrated homogenous distributions in all three dimensions. For example, the protonated 7-oxocoumarin ( m / z 163.0373 measured), sodiated methoxy-hydroxyphenyl glucoside ( m / z 325.0919 measured), and acacetin diglucoronide ( m / z 637.0127 measured) fell in this category.
  • Another class of metabolites exhibited distributions with lateral heterogeneity. Such localization was observed in all the layers for the protonated kaempferol/luteolin and methoxy(kaempferol/luteolin) glucoronide ions with measured m / z values of 287.0494 and 493.0942, respectively. Shown in Figure 9 , both metabolites yielded higher intensities in the second and third layers. Kaempferol/luteolin ions were observed in ⁇ 90% of the variegation pattern area, indicating that this metabolite was characteristic to the cells of the achlorophyllous tissue sections.
  • a quantitative characterization of the relationship between tissue architecture and metabolite distributions is possible through the correlation between the intensity distribution of the tissue morphology acquired through, e.g., optical imaging, M ( r ), and the normalized distribution for the m / z ion obtained by, e.g., LAESI MS, I mi ( r ).
  • Pearson product-moment correlation coefficients, r m1m2 were calculated between the 3D spatial distributions of ion intensities, I m / z ( r ), for twelve selected m / z in an A . squarrosa leaf.
  • I m / z ( r ) ion intensities
  • r 301,317 0.88, i.e., the results confirmed the strong correlation between ion distributions placed in the same groups.
  • the degree of similarity was reflected for less clear cases.
  • the r 285.287 0.65, i.e., although both distributions reflect the variegation pattern, in layers two and three the m / z 285 distribution exhibits significant values in the green sectors, as well.
  • Another interesting example was the lack of spatial correlation between kaempferol/luteolin at m / z 287 and chlorophyll a at m / z 893.
  • the low value of the correlation coefficient, r 287.893 0.08, indicated that these two metabolites were not co-localized. They are also known to belong to different metabolic pathways. This and other examples showed that the correlation coefficients can be a valuable tool to identify the co-localization of metabolites in tissues and to uncover the connections between the metabolic pathways involved.
  • LAESI is an ambient ionization source for MS that enables the simultaneous investigation of a variety of biomolecules while eliminating the need for tailored reporter molecules that are generally required in classical biomedical imaging techniques.
  • a capability for quantitation, and lateral and depth profiling on the molecular scale are further virtues of this method with great potential in the life sciences.
  • the distribution of secondary metabolites presented in this work, for example, may be used to pinpoint the tissue specificity of enzymes in plants. Water-containing organs, tissue sections or cells from plants or animals, as well as medical samples can be subjected to 3D analysis for the first time.
  • the studies can be conducted under native conditions with a panoramic view of metabolite distributions captured by MS.
  • LAES1 is an ambient ionization source that enables the simultaneous investigation of a variety of biomolecules while eliminating the need for tailored reporter molecules that are generally required in classical biomedical imaging techniques.
  • In vivo analysis with low limits of detection, a capability for quantitation, and lateral and depth profiling on the molecular scale are further virtues of the method that forecast great potentials in the life sciences.
  • the distribution of secondary metabolites presented in this work, for example, may be used to pinpoint enzymes to tissue or cell specificity in plants. Water-containing organs or whole-body sections of plants, animals and human tissues or cells can be subjected to 3D analysis for the first time under native conditions with a panoramic view for ions offered by MS.
  • LAESI offers middle to low level of resolving power in comparison to optical imaging techniques.
  • Advances are promised by oversampling typically applied in MALDI experiments, aspherical lenses for light focusing, and fiber optics for direct light coupling into the sample. The latter two approaches have allowed us to analyze single cells with dimensions of ⁇ 50 ⁇ m diameter while maintaining good signal/noise ratios. Higher lateral and depth resolutions in three dimensions can dramatically enhance our understanding of the spatial organization of tissues and cells on the molecular level.
  • the electrospray source was identical to the one we have recently described.
  • a low-noise syringe pump (Physio 22, Harvard Apparatus, Holliston, MA) supplied 50 % methanol solution containing 0.1 % (v/v) acetic through a tapered tip metal emitter (100 ⁇ m i.d. and 320 ⁇ m o.d., New Objective, Woburn, MA).
  • Electrospray was initiated by directly applying stable high voltage through a regulated power supply (PS350, Stanford Research System, Inc., Sunnyvale, CA). The flow rate and the spray voltage were adjusted to establish the cone-jet mode. This axial spraying mode has been reported to be the most efficient for ion production.
  • Live leaf tissues of approximately 20 ⁇ 20 mm 2 area were mounted on microscope slides, positioned 18 mm below the electrospray axis.
  • the output of a Nd:YAG laser operated at a 0.2-Hz repetition rate (4-ns pulse duration) was converted to 2940 nm light via an optical parametric oscillator (Vibrant IR, Opotek Inc., Carlsbad, CA).
  • This mid-infrared laser beam was focused with a plano-convex focusing lens (50-mm focal length) and was used to ablate samples at right angle under 0° incidence angle, ⁇ 3-5 mm downstream from the tip of the spray emitter.
  • the average output energy of a laser pulse was measured to be 0.1 mJ ⁇ 15% and 1.2 mJ ⁇ 10%, respectively.
  • the ablated material was intercepted by the electrospray plume and the resulted ions were analyzed by an orthogonal acceleration time-of-flight mass spectrometer (Q-TOF Premier, Waters Co., Milford, MA) with a 1 s/spectrum integration time.
  • the original electrospray ion source of the mass spectrometer was removed.
  • the sampling cone of the mass spectrometer was located on axis with and 13 mm away from the tip of the spray emitter.
  • the ion optics settings of the instrument were optimized for best performance and were kept constant during the experiments. Metabolite identification was facilitated by tandem MS. Fragmentation was induced by CAD in argon collision gas at 4 ⁇ 10 -3 mbar pressure with the collision energy set between 15-30 eV.
  • a three-axis translation stage was positioned with precision motorized actuators (LTA-HS, Newport corp., Irvine, CA) to scan the sample surface while keeping all other components of the LAESI setup in place.
  • the actuators had a travel range of 50 mm and a minimum incremental motion of 0.1 ⁇ m.
  • the ultimate resolution was determined by the focusing of the incident laser beam and the dimensions of the ablation craters ( ⁇ 350 ⁇ m in diameter).
  • the sample surface was scanned at a step size of 500 ⁇ m in the X and Y directions. At each coordinate, the cross-section of the live tissues were analyzed with 6 laser pulses while the generated ions were recorded for 30 seconds with the mass spectrometer.
  • Glacial acetic acid (TraceSelect grade) and gradient grade water and methanol were obtained from Sigma Aldrich and were used as received.
  • the Easter lily Spathiphyllum Lynise
  • Zebra plant Aphelandra Squarrosa
  • the plants were watered every 2 days with ⁇ 300 mL tap water to keep their soil moderately moist to touch. No fertilizer was used during the experiments. Temperature and light conditions were 20-25 °C in light shade, protected from direct sun.

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EP13170238.3A 2008-11-25 2009-11-25 Dreidimensionale molekulare Bildgebung mittels Infrarot-Laserablation-Elektrospray-Ionisation-Massenspektrometrie Withdrawn EP2660848A1 (de)

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US12/323,276 US7964843B2 (en) 2008-07-18 2008-11-25 Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US26411909P 2009-11-24 2009-11-24
EP09832362.9A EP2356668B1 (de) 2008-11-25 2009-11-25 Dreidimensionale molekulare bildgebung mittels infrarot-laserablation-elektrospray-ionisation-massenspektrometrie

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CN102590089B (zh) * 2012-01-13 2014-03-26 浙江大学 室内光谱观测三维载物台及其应用
WO2021104855A1 (en) * 2019-11-27 2021-06-03 Thermo Fisher Scientific (Bremen) Gmbh Systems and methods for imaging and ablating a sample
WO2022080412A1 (ja) * 2020-10-13 2022-04-21 花王株式会社 コムギの収量予測方法
WO2023037536A1 (ja) * 2021-09-13 2023-03-16 株式会社島津製作所 質量分析装置

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