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The 3D OrbiSIMS—label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power

Abstract

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters—gamma-aminobutyric acid, dopamine and serotonin—with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

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Figure 1: The 3D OrbiSIMS spectrometer and modes of operation.
Figure 2: 3D MS imaging using dual beam and dual spectrometer (mode 10) of single rat alveolar macrophage cell incubated in media with the drug amiodarone.
Figure 3: 3D OrbiSIMS MS images of the hippocampal region of a coronal section of mouse brain at tissue scale, cellular scale and subcellular scale.
Figure 4: Identification of lipids and mapping of their spatial distribution requires simultaneous high spatial resolution and high mass resolution as well as MS/MS.
Figure 5: Mapping of neurotransmitters in the hippocampus.
Figure 6: Single-cell metabolomics for macrophages incubated with different concentrations of amiodarone.

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Acknowledgements

The authors thank N. Harrison, R. Reid, A. Harling and M. Skingle for their support during the project and T. Heller, M. Krehl, A. Dütting, P. Hörster, K. Strupat, S. Möhring, F. Czemper, A. Venckus, S. Kanngiesser, O. Lange and A. Kühn for excellent technical support. The authors also thank M. Tiddia for AFM measurement of frozen hydrated cell heights. This work forms part of the “3D nanoSIMS” project (ISG) in the Life-science and Health programme of the National Measurement System of the UK Department of Business, Energy and Industrial strategy. This work has received funding from the 3DMetChemIT project (ISG) of the EMPIR programme cofinanced by the Participating States and from the European Union's Horizon 2020 research and innovation programme.

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Affiliations

Authors

Contributions

M.K.P., A.P. and R.H. performed experiments. M.K.P. analyzed data. P.S.M., C.F.N. and A.W. prepared tissue and cell experiments and H & E pathology. F.K. designed continuous mode Bi LMIG. R.M., A.M., D.G., E.N. designed interface to hybridize ToF and Orbitrap spectrometers. A.P., M.K.P., R.M., A.M., E.N., R.H. optimized performance of 3D OrbiSIMS. H.A. developed computer interfacing and computational methods. R.M. and E.N. designed cryo sample handling. A.W., P.S.M. and C.T.D. direction of pharmaceutical studies. M.R.A., S.H. and E.N. gave technical leadership at Thermo Fisher Scientific and ION-TOF, respectively. I.S.G. original design concept and supervised the project. M.K.P. and I.S.G. wrote the paper. All authors read and commented on the paper.

Corresponding author

Correspondence to Ian S Gilmore.

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Competing interests

EN is a director and shareholder of ION-TOF GmbH Muenster, Germany. AP, RM, FK, and HA are employees of ION-TOF GmbH. DG, SH and AM are employees of Thermo Fisher Scientific, the corporation that produces Orbitrap mass spectrometers. CN, PM, AW and CD (at the time of this study) are employees of GlaxoSmithKline.

Integrated supplementary information

Supplementary Figure 1 Resolving crystal violet isotope fine structure with high mass resolving power of the Orbitrap.

A) Positive ion mass spectra of crystal violet ([C25H30N3]+ at m/z 372.24371) at nominal resolution settings 240,000 (blue) and 480,000 (red) mass resolving power (mode 2) normalized to the molecular ion peak. B) Mass resolving power for secondary ions in the mass spectrum with fits of the expected m−0.5 relationship. At m/z 200 the mass resolving power is approximately 253,000 (0.5 s transient) and 417,000 (1 s transients). C) The isotopic distribution of the crystal violet molecular ion. D-F) Annotated spectra for the M+1, M+2 and M+3 isotope peaks for crystal violet spanning a dynamic range of five orders of magnitude (100% abundance to 0.001 % abundance). Results presented are from a single measurement.

Supplementary Figure 2 In situ tandem MS of cholesterol.

Orbitrap tandem MS of cholesterol fragment [M-H2O+H]+ from the corpus callosum of a mouse brain section. Peaks are annotated with chemical formulae and mass deviation. The fragment ion peaks were identified by their accurate mass. Result presented is from a single measurement.

Supplementary Figure 3 Lateral resolution measurement for the focused GCIB primary ion beam (with the secondary ion extraction potential off).

A) Ion induced secondary electron image of an electroformed mesh grid over a hole obtained with the Ar3000+ primary ion beam. Red lines along the x and y axes denote the location of linescans used to measure the resolution. B and C) Representative line scans of the secondary electron intensity across the edge of the grid for the x-axis and y-axis, respectively. D) The distribution of the FWHM lateral resolution measurements with a fit to a normal distribution function (dotted line, bin size 0.1 μm). The average FWHM lateral resolution was 1.72 μm ± 0.24 μm (μ ± 1σ) (n=246, grey bars) across the x-axis and 1.04 μm ± 0.16 μm (μ ± 1σ) (n=246, red bars) across the y-axis. Results presented are from a single image.

Supplementary Figure 4 Lateral resolution measurement for the focused GCIB primary ion beam (with the secondary ion extraction potential on, as in all SIMS analyses).

A) Ion induced secondary electron image of an electroformed mesh grid over a hole obtained with the Ar3000+ primary ion beam. Red lines along the x and y axes denote the location of linescans used to measure the resolution. B and C) Representative line scans of the secondary electron intensity across the edge of the grid for the x-axis and y-axis, respectively. D) The distribution of the FWHM lateral resolution measurements with a fit to a normal distribution function (dotted line, bin size 0.05 μm). The average FWHM lateral resolution was 2.49 μm ± 0.36 μm (μ ± 1σ) (n=243, grey bars) across the x-axis and 1.84 μm ± 0.36 μm (μ ± 1σ) (n=254, red bars) across the y-axis. Results presented are from a single image.

Supplementary Figure 5 Lateral resolution measurement for the focused 20 keV Ar3000+ GCIB primary ion beam for biomolecules.

A) Overlay ion image of selected sulfatide peaks (green), phosphatidylinositol peaks (blue) and nuclear markers (red). [Sulfatide peaks: C22(OH) ([C46H88NO12SO] at m/z 878.6035, (0.3 ppm)), C24:1 ([C48H90NO11S] at m/z 888.6242 (0.3 ppm)) and C24:1(OH) ([C48H90NO12S] at m/z 904.6192 (0.3 ppm)); PI peaks: PI(38:4) ([C47H82O13P] at m/z 885.5502 (0.3 ppm)) and PI head-group ([C6H10PO8] at m/z 241.0120 (0.6ppm)); Nuclear markers [C4N3] at m/z 90.0095 (2.8 ppm), [CN2O2P] at m/z 102.9702 (0.6 ppm), [C4H2N4] at m/z 106.0285 (0.3 ppm), [C4H3N4] at m/z 107.0207 (0.2 ppm), [C5HN4] at m/z 117.0207 (0.2 ppm), [C5H3N4] at m/z 119.0363 (0.3 ppm), [C5HN4O] at m/z 133.0156 (0.1 ppm) and [C5H4N5] at m/z 134.0472 (6 ppb). B) Polychromatic ion image of the nuclear markers with regions of interest outlined in white. C) Detail of one nucleus from B) with line scans across the x-axis and y-axis. D) as C) for the second nucleus. Average resolution determined to be 1.34 μm ± 0.24 μm (n=5).

Supplementary Figure 6 Measurement of the Bi LMIG simultaneous lateral resolution and mass resolving power.

(A) Total ion images of ZrO2 crystals (mode 7) obtained with the Bi LMIG source with insert showing detail of thin nanostructures. B) Ion image of the [ZrO]+ peak at m/z 105.8990 (0.5 ppm). C) Ion induced SE image of the ZrO2 crystal before analysis. D) Total ion images of ZrO2 crystals (mode 7) obtained with the Bi LMIG source and insert region of interest for resolution measurement outlined in white. Linescans were obtained along the y-axis of the ion image across the ZrO2 crystal edge. E) A representative line scan shows the total ion intensity across the ZrO2 interface.. F) The distribution of lateral resolution measurements with a fit a normal distribution function (red dotted line, bin size 0.01 μm). The average FWHM lateral resolution was 172 nm ± 61 nm (μ ± 1σ) (n=95). G) The [ZrO]+ peak at m/z 105.8991 (0.2 ppm) with a FWHM width of 0.3 mDa and mass resolving power of 355,000. Results presented are from a single image.

Supplementary Figure 7 3D imaging of an Irganox delta layer reference material.

A) Orbitrap MS (mode 3), 5 keV Ar2000+ sputtering and analysis, intensity depth profile of the Irganox 1010 molecular ion, [C73H107O12] at m/z 1175.776 (solid line), and fragment ion from Irganox 3114, [C33H46N3O5] at m/z 564.344 (dashed line). B) ToF MS (mode 9), 5 keV Ar2000+ sputtering and 30 keV Bi3+ analysis intensity depth profile, as A) with 3D reconstruction from ToF MS data and C) Dual analyser, dual beam mode (10) with 5 keV Ar2000+ sputter beam. Intensity depth profiles as A) for Orbitrap MS Ar2000+ analysis beam and ToF MS 30 keV Bi3+ beam, with 3D reconstruction from ToF MS data. Results presented are from a single measurement.

Supplementary Figure 8 20 keV Arn GCIB Orbitrap negative ion MS of reference lipid C24:1 Mono-Sulfo Galactosyl(ß) Ceramide (d18:1/24:1) (C48H90NO11S).

(A) n = 1000, (B) n = 5500 and (C) n = 10000. Inset shows detail of the molecular ion, C48H90NO11S, revealing little fragmentation for this particular species. Results presented are from single measurements.

Supplementary Figure 9 20 keV Arn GCIB Orbitrap positive ion MS of reference lipid 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (C29H58NO8P), normalised to the C12H22O2 peak intensity.

(A) n = 1000, (B) n = 5500 and (C) n = 10000. Results presented are from single measurements.

Supplementary Figure 10 Lipid region from the negative ion image from Figure 3D in the main text (mode 7).

The spectra was summed over the entire ion image. See Supplementary Tables 2 and 3 for annotations. Result presented is from a single measurement.

Supplementary Figure 11 Positive ion Orbitrap MS obtained from the CA3 vicinity of the hippocampus on a coronal brain section.

Supplementary Figure 12 Comparison of resolving power of ToF MS (mode 1) and Orbitrap MS (mode 2) for intact lipids from mouse hippocampus.

(A) Negative ion mass spectra between m/z 902 – m/z 910 (red = Orbitrap MS (mode 2), black = ToF MS (mode 1)). B) Detail of spectra between m/z 904.2 – m/z 905.0. Result presented is from a single measurement.

Supplementary Figure 13 20 keV Ar3000+ GCIB Orbitrap negative ion MS/MS of reference neurotransmitters using 20 eV collision energy.

(A) GABA [M-H], (B) dopamine [M-H] and (C) serotonin [M-H]. Inset images show the co-localised spatial distribution of these secondary ions from the data in Figure 5. Results presented are from single measurements.

Supplementary Figure 14 Orbitrap MS of lipids in control and amiodarone treated macrophage cells.

The average positive ion mass spectra for control cells (n=8) and treated cells (6.25 μg/ml (n=3) and 9.38 μg/ml (n=7)).

Supplementary Figure 15 Full MS and tandem MS spectra of reference sample of amiodarone.

20 keV Ar3000+ Orbitrap MS (mode 2) spectrum (blue, positive intensity scale) and tandem MS of the [M+H]+ peak (red, negative intensity scale). Results presented are from single measurements.

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Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1–9 (PDF 3454 kb)

Life Sciences Reporting Summary

Life Sciences Reporting Summary (PDF 160 kb)

Supplementary Protocol

Label-free Imaging of Biomolecules in Murine Brain Sections Using the 3D OrbiSIMS (PDF 682 kb)

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Passarelli, M., Pirkl, A., Moellers, R. et al. The 3D OrbiSIMS—label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power. Nat Methods 14, 1175–1183 (2017). https://doi.org/10.1038/nmeth.4504

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