We report an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) setup with a lateral resolution of 1.4 μm, a mass resolution greater than 100,000, and accuracy below ±2 p.p.m. We achieved this by coupling a focusing objective with a numerical aperture (NA) of 0.9 at 337 nm and a free working distance of 18 mm in coaxial geometry to an orbitrap mass spectrometer and optimizing the matrix application. We demonstrate improvement in image contrast, lateral resolution, and ion yield per unit area compared with a state-of-the-art commercial MSI source. We show that our setup can be used to detect metabolites, lipids, and small peptides, as well as to perform tandem MS experiments with 1.5-μm2 sampling areas. To showcase these capabilities, we identified subcellular lipid, metabolite, and peptide distributions that differentiate, for example, cilia and oral groove in Paramecium caudatum.
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Financial support from the Deutsche Forschungsgemeinschaft (DFG) under grant Sp314/13-1 is gratefully acknowledged. S.H. is grateful to the German National Academy of Sciences Leopoldina (LPDR 2014-01) for a postdoctoral scholarship. The authors are grateful to W. Kummer (Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany) and his group members for providing mouse brain and kidney samples.
B.S. is a consultant of TransMIT GmbH Giessen. The PhD work of M.K. is funded by TransMIT GmbH.
Integrated supplementary information
a) Experimental setup to visualize the laser beam profile at the focal plane of the centrally bored new objective. (b) Single laser pulse focus at 337 nm visualized with a calibrated beam profile system. (c) Laser ablation craters on red dye after moving the stage with 10 μm increments in x and y direction. 15 laser pulses with ~20 nJ per pulse were applied to each spot. (d) Focal depth determination on red pencil dye with 10 laser pulses of 20 nJ per pulse. The focal plane was reached between 4-6 μm z-distance increase.
(a) Optical images of mouse brain tissue sections after matrix application. Sublimation of CHCA (left); sublimation+recrystallization of CHCA (right). (b) Optical images of mouse brain tissue sections after matrix application. Pneumatic spraying of DHB (left) and CHCA (right).
MALDI mass spectra averaged over 100 pixels, obtained from mouse brain tissue using 20 laser pulses per pixel with 10 nJ per pulse, thereby probing ~1.5 μm2 of the tissue per ablation spot. Lipid intensities obtained by (MA)LDI MS from mouse brain tissue (a) without matrix, (b) after sublimation coating with DHB matrix, (c) after sublimation coating followed by recrystallization of DHB matrix and (d) after pneumatic spraying of DHB matrix. Selected lipid ion signals were labeled with the corresponding m/z values. Absolute signal intensity gain is reflected by scale values.
MS gray scale images of lipid ions shown in Fig. 3 of the manuscript. Mass spectra obtained from a single-pixel during MSI experiments, shown for the AP-SMALDI10 and the experimental AP MSI setup, respectively. Red lines indicate the data points used for contrast and edge width analysis.
Supplementary Figure 5 Single-cell MSI measurement and corresponding optical images of Paramecium caudatum.
(a) Optical Image of Paramecium caudatum revealing irregularities on the surface of the organism. (b-d) Corresponding gray scale MS images (100x100 pixels) obtained with 3 μm step size. A total of 30 laser pulses per spot with 15 nJ per pulse were applied. [DG(31:0)+NH4]+ (m/z 572.5240; red), [PC(34:1)+Na]+ (m/z 782.5668; green) and [Cer(d35:2)+H]+ (m/z 550.5195; blue) were selected for the three images. (e) Paramecium caudatum after the MSI measurement shown in Fig. 5a. Scale bars in all images are 100 μm.
Supplementary Figure 6 MS images of metabolites and peptides from Paramecium caudatum at 3-μm step size (100 × 100 pixels).
(1-8) Gray scale MS images: the ion signal assignment is given in Supplementary Table 2 according to the number code. Notably, metabolites, peptides and phospholipids were detected that are solely localized in the Paramecium caudatum organism. Signals assigned to the same analyte with differing ionic attachment show similar lateral distributions (see (1) and (2)). Scale bars in all images are 100 μm.
Supplementary Figure 7 Grayscale MS, optical, and RGB MS images of Paramecium caudatum and Rotifera.
(a) Optical image before MSI measurement of Paramecium caudatum in the process of being devoured by a Rotifera predator. (b) RGB image (175x150 pixels) obtained with 2 μm step size from Paramecium caudatum being devoured by Rotifera. A total of 30 laser pulses per spot with 10 nJ per pulse were used. [PE(35:2)+H]+ (m/z 730.5377; red) and [PC(34:1)+Na]+ (m/z 782.5668; green) were superimposed. (c-d) Corresponding gray scale images highlighting that the imaged lipids are characteristic for Paramecium caudatum and Rotifera, respectively. Scale bars in all images are 100 μm.
The setup features a (1) N2-laser system; (2) a beam shaping setup; (3) 45° mirror with central bore; (4) newly developed focusing objective with central bore; (5) an aperture; (6) a high precision sample movement stage with sample holder and (7, 8) a camera-mirror assembly to visualize the sample surface. Upon laser irradiation ions are ejected in reflection-mode and transferred into the inlet capillary (9). A DC voltage (HV) is applied between sample holder and inlet capillary of the mass spectrometer. The entire setup is attached to a Q Exactive mass spectrometer. Ion source and mass spectrometer are not to scale.
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Kompauer, M., Heiles, S. & Spengler, B. Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution. Nat Methods 14, 90–96 (2017). https://doi.org/10.1038/nmeth.4071
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