Mass Spectrometry Imaging

Abstract

Mass spectrometry imaging (MSI) maps the spatial distributions of chemicals on chemically complex surfaces. MSI offers unrivaled sensitivity and information density with each pixel comprising a mass spectrum. Over the past three decades, numerous technological developments have enabled MSI to evolve into a mainstream technique for untargeted molecular and elemental imaging with wide-spread applications ranging from material analysis to life sciences and clinical diagnostics. Here, we review the field of MSI with a focus on key technological advancements. We examine different image acquisition modes and the most popular ionization methods in MSI, including matrix-assisted laser desorption/ionization (MALDI), laser ablation inductively coupled plasma (LA-ICP), laser ablation electrospray ionization (LAESI), secondary ion mass spectrometry (SIMS), and desorption electrospray ionization (DESI). For each method, we discuss figures of merit, such as spatial resolving power and sensitivity, the ionization mechanism, sample preparation, advantages, and disadvantages, including ways to overcome them wherever applicable. We subsequently discuss more aspects of MSI instrumentation, such as commonly used mass analyzers, tandem mass spectrometry, ion mobility, and advancements in imaging throughput. Based on these technological developments, targeted MSI strategies are explained, including imaging mass cytometry (IMC), multiplexed ion beam imaging (MIBI), and stable isotope labeling (SIL), as well as approaches for multimodal imaging. Last, we present selected application examples of MSI in cancer research, single cell analysis, and drug distribution studies. We target this review to provide researchers with an interest in recent developments in MSI with a concise technological understanding of the different main approaches to MSI.

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Operating principles of TOF-based microprobe-mode MSI and mass microscopy. In microprobe-mode MSI (a) a focused ionizing beam scans the sample and sequentially acquires mass spectra for every pixel. In mass microscopy (b) a defocused ionizing beam irradiates a large part of the sample causing the formation of an ion image on the surface. This image is then extracted into the gas phase, preserved during mass analysis, and magnified onto a fast, spatially sensitive detector. The ionizing beams are depicted in blue, and ions of different mass-to-charge are for illustrative purposes annotated m₁, m₂, and m₃, respectively.

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Ionization pathways in MALDI. A laser irradiates a sample surface coated with matrix crystals causing the collective desorption of matrix and analytes into the gas phase. In the ‘lucky survivors’ model ionization occurs on the surface, and the laser merely causes desorption of charged clusters, from which analyte ions can be released and detected. In the MALDI plume proton transfer model , analyte molecules undergo ionization via gas phase collisions with other ionized particles, for instance matrix molecules.

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An example sample preparation workflow for MALDI MSI of fresh-frozen tissue. First, a biological sample is retrieved from an organism and flash-frozen via plunging into cryogenic isopentane. Then, if necessary, the tissue is embedded in gelatin to facilitate its sectioning into thin slices, which are then mounted onto microscopy slides. The obtained slides are defrosted under vacuum in a desiccator, which removes volatile compounds, e.g. water, from the tissue and, moreover, prevents water condensation from the atmosphere. Washing tissue slices to remove salts and/or lipids may be required depending on the analytes of interest. Last, matrix is applied to the sample.

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Transmission mode MALDI combined with MALDI-2 enhances spatial resolving power in MALDI MSI. In reflection geometry (a, top) the MALDI laser is focused onto the sample from a larger distance and angle. As a result, laser spot size is usually limited to 4–5 μm using common MALDI wavelengths. In transmission geometry (a, bottom) the laser is focused directly below the microscope slide allowing the use of lens objectives with higher numerical aperture and thus for smaller spot sizes than in reflection geometry. In combination with additional ionization enhancement, for instance via MALDI-2, transmission mode improves spatial resolving power in MALDI MSI (b, adapted with permission from Macmillan Publishers Ltd: NATURE METHODS, Niehaus, M.; Soltwisch, J.; Belov, M. E.; Dreisewerd, K. Nat. Methods 2019, 16, 925–931 (ref ). Copyright 2019.). MALDI-2 features a laser ionization step of a MALDI plume at EP (depicted in gray, c) to enhance ion yield by 2–3 orders of magnitude, including ions that were not detectable with MALDI alone (d, adapted with permission from Soltwisch, J.; Kettling, H.; Vens-Cappell, S.; Wiegelmann, M.; Müthing, J.; Dreisewerd, K. Mass Spectrometry Imaging with Laser-Induced Postionization. Science (80-. ). 2015, 348 (6231), 211–215 (ref ). Copyright 2015 AAAS.).

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Example of a LA-ICP MSI setup. A high energy 193 nm laser beam is homogenized and focused onto the sample under helium atmosphere, causing ablation. The emitted aerosols disperse in the chamber and are transported by a laminar argon flow to the ICP torch where they are ionized and transferred into a mass spectrometer.

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A secondary ion mass spectrometry (SIMS, a) setup drawn with a liquid metal ion source (LMIS) and a reflectron time-of-flight (TOF) mass spectrometer. Primary ion pulses from the LMIS are accelerated and focused onto the surface where they cause the emission of secondary ions. Next to the LMIS, gas cluster ion beams (GCIBs) are commonly used for removing sample layers. As GCIBs cannot be pulsed effectively, using them for SIMS analysis would require a different mass spectrometer, for instance an orthogonal TOF or an Orbitrap. In (b) a molecular dynamics simulation (adapted with permission from Computational View of Surface Based Organic Mass Spectrometry, Garrison, B. J.; Postawa, Z. Mass Spectrom. Rev. Vol. 27, Issue 4 (ref ). Copyright 2008 Wiley.) shows the impact of a primary Au⁺ ion at different time points. The ion perforates the surface and causes the displacement of atoms and molecules in a collision cascade. Material from the upper layers can also be ejected and, when charged, be detected with MS. The colors refer to the degree of displacement: red >2 nm, yellow 1.6 nm, green 1.2 nm, cyan 0.8 nm, blue 0.4 nm, and gray <0.4 nm. In (c) molecular dynamics simulations show Ga⁺, Au₃ ⁺, C₆₀ ⁺, and Ar₈₇₂ ⁺ primary ions hitting a surface at different times after primary ion impact (adapted with permission from Vickerman, J. C.; Briggs, D. TOF-SIMS: MATERIALS ANALYSIS BY MASS SPECTROMETRY, 2^nd ed.; IM Publications LLP and SurfaceSpectra Limited, 2013 (ref ). Copyright 2013 Surface Spectra.). The numbers in the lower row indicate the amount of sputtered material for each primary ion. Primary cluster ions cause less subsurface damage and fragmentation while achieving higher sputtering yields than monatomic primary ions. Furthermore, Sputtering yield and direction with gas cluster ion beams depend on the primary ion angle of incidence (AOI, d, adapted with permission from Kański, M.; Postawa, Z. Effect of the Impact Angle on the Kinetic Energy and Angular Distributions of β-Carotene Sputtered by 15 KeV Ar2000 Projectiles. Anal. Chem. 2019, 91 (14), 9161–9167 (ref ). Copyright 2019 American Chemical Society.). Unlike other ion beams, highest sputter yield for Ar₂₀₀₀ ⁺ is achieved at 45° AOI and ∼60° angle of ion collection. In (e) a nanoscale SIMS is depicted, in which primary ions pass through the same ion optic as secondary ions to achieve nanoscale ion beam focusing by having a short working distance to the sample and an angle of 0° to the surface normal.

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Setup of desorption electrospray ionization (DESI) MSI. The sample is scanned with an ESI tip, which shoots charged droplets onto the surface. There, a wet film forms, which extracts analytes from the sample. Subsequent droplet impacts cause the emission of charged secondary droplets and, following their decomposition, ions. These droplets are collected and transferred to a MS via a heated capillary. Further technical details on the setup of DESI may be found in Table .

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Advanced time-of-flight (TOF) and ion mobility spectrometers. In (a), an open-path multireflectron TOF with planar gridless ion mirrors and refocusing lenses is shown. In (b), a stigmatic triple ion focusing TOF (TRIFT) is depicted. Analogue to an optical telescope, two lenses magnify an ion image and project it after TOF separation onto a fast pixelated detector. Similar to a reflectron, three electrostatic analyzers equipped with Herzog shunts and Matsuda plates (not shown) elongate the flight path to 2 m and provide 1^st order energy correction. Mass and spatial resolving power are limited by aberrations and can be increased at the cost of ion transmission with an energy slit and a contrast diaphragm, respectively. , In (c and d), a travelling wave and a trapped ion mobility spectrometer are displayed (TWIMS and TIMS, respectively). In TWIMS, ions are radially confined and pushed forward by periodic DC waves against a stationary gas. In TIMS, ions are accumulated in an ion trap while a counter directed gas flow prevents ions from propelling ions forward and distributes ions spatially separated in the ion trap. After an accumulation period, the ion trap is blocked against further ions, and the trapping potential is gradually lowered, allowing ions with ever smaller collision-cross section to elude the ion trap. Another TIMS design consists of two ion traps in series, allowing for parallel accumulation in the first and IMS separation in the second ion trap. This design allows for a 100% duty cycle without the need for blocking the TIMS cell, and acquiring multiple MS/MS spectra in one go via PASEF. ,

1. Double Bond Specific MS/MS Reactions

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An example of targeted IHC MSI – LA-ICP MSI of mouse duodenum of an in-vitro injected ¹⁵⁹Tb-marked polysarcosine-modified dendrimer drug-delivery system (S-Dend) followed by immunostaining and imaging mass cytometry (IMC) of 24 proteins (adapted with permission from Strittmatter, N.; England, R. M.; Race, A. M.; Sutton, D.; Moss, J. I.; Maglennon, G.; Ling, S.; Wong, E.; Rose, J.; Purvis, I.; Macdonald, R.; Barry, S. T.; Ashford, M. B.; Goodwin, R. J. A. Method to Investigate the Distribution of Water-Soluble Drug-Delivery Systems in Fresh Frozen Tissues Using Imaging Mass Cytometry. Anal. Chem. 2021, 93 (8), 3742–3749. (ref ). Copyright 2021 American Chemical Society.). Since S-Dend is water soluble and washed away by immunostaining, Strittmatter et al. first performed LA-ICP MSI of S-Dend (a; (b) is an overlay of (a) with the mass image of an adjacent tissue section), and then IMC on the same tissue section (c–h). In (c) the region previously ablated to measure S-Dend is highlighted by a white box. In (d), (a) is overlaid onto (c) with 50% opacity. The images (e) and (f) are of connective tissue and mucosa substructures, while (g) and (h) are zoom-ins into the areas marked with dashed boxes in (c) and (d), respectively. A white vertical line in (g) separates previously ablated (right) from nonablated tissue (left). The white arrow in (h) highlights a large blood vessel. In (c), (d), (g), and (h) the colors cyan, yellow, red, green, blue, and white correspond to CD45, E-cadherin, αSMA, pan-CK, collagen I, and S-Dend, respectively. In (e), yellow, blue, and magenta represent collagen I, vimentin, and desmin, while in (f) green stands for β-catenin, yellow for E-cadherin, red for EpCam, and magenta for tenascin C, respectively. The scale bars are 200 (a–f) and 50 μm (g and h), respectively.

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AP MALDI MSI and fluorescence in situ hybridation (FISH) imaging of gill filament tissue sections of a Bathymodiolus deep-sea mussel (adapted with permission from Macmillan Publishers Ltd: NATURE MICROBIOLOGY, Geier, B.; Sogin, E. M.; Michellod, D.; Janda, M.; Kompauer, M.; Spengler, B.; Dubilier, N.; Liebeke, M. Nat. Microbiol. 2020, 5 (3), 498–510 (ref ). Copyright 2020.). A micro-computed tomography model (a) depicts the location of the gills. Parts of the gill tissue, the bacteriocytes (bc), are co-populated by sulfur and methane oxidizing symbionts (SOX and MOX, respectively), while the ciliated edge (ce) consists only of host cells. The symbionts and their host were imaged with FISH (b). The colors cyan, magenta, and yellow correspond to the host DNA, MOX, and SOX, respectively. In (c), the spatial distributions of phosphatidylcholine PC(32:1), 35-aminobacteriophane-32,33,34-triol (ABHTr), and phosphonethanolamine ceramide PnE-Cer(34:2), were measured with AP MALDI MSI at 3 μm pixel size. Cyan, magenta, and yellow correspond to PC(32:1), ABHTr, and PnE-Cer(34:2), respectively. A white box in (b) highlights the region chosen for normalized zoom-ins (d–g). ABHTr can only be observed in the bacteriocytes whereas PnE-Cer(34:2) is solely found in symbiont-free host tissue. This multimodal approach allowed the authors to map chemical interactions between host and symbiont. The scale bars correspond to 150 (b and c) and 50 μm (d–g), respectively.