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Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling

Abstract

Label-free molecular imaging techniques such as matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enable the direct and simultaneous mapping of hundreds of different metabolites in thin sections of biological tissues. However, in host–microbe interactions it remains challenging to localize microbes and to assign metabolites to the host versus members of the microbiome. We therefore developed a correlative imaging approach combining MALDI-MSI with fluorescence in situ hybridization (FISH) on the same section to identify and localize microbial cells. Here, we detail metaFISH as a robust and easy method for assigning the spatial distribution of metabolites to microbiome members based on imaging of nucleic acid probes, down to single-cell resolution. We describe the steps required for tissue preparation, on-tissue hybridization, fluorescence microscopy, data integration into a correlative image dataset, matrix application and MSI data acquisition. Using metaFISH, we map hundreds of metabolites and several microbial species to the micrometer scale on a single tissue section. For example, intra- and extracellular bacteria, host cells and their associated metabolites can be localized in animal tissues, revealing their complex metabolic interactions. We explain how we identify low-abundance bacterial infection sites as regions of interest for high-resolution MSI analysis, guiding the user to a trade-off between metabolite signal intensities and fluorescence signals. MetaFISH is suitable for a broad range of users from environmental microbiologists to clinical scientists. The protocol requires ~2 work days.

Key points

  • A procedure for spatial metabolomics of host–microbe interactions, including tissue preparation, matrix application, MSI data acquisition, on-tissue hybridization using nucleic acid probes, fluorescence microscopy and data integration into a correlative image dataset.

  • MALDI-MSI enables single-cell-level mapping of metabolites by revealing their spatial distribution. Alternatively, laser-capture microdissection can be combined with LC–MS, or metaFISH combines spatial metabolomics with FISH.

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Fig. 1: Workflow for visualization of metabolites and microbes at high spatial resolution using MALDI–MSI and in situ fluorescence labeling (metaFISH).
Fig. 2: Combined analysis of spatial metabolome and microscopy data to associate metabolites to host or microbes.
Fig. 3: Trimming of CMC block before cryosectioning.
Fig. 4: Tissue section and microscopy slide documentation to follow the protocol.
Fig. 5: Laser intensity and shot number influence MSI and FISH signal intensity.
Fig. 6: Identifying bacterial infection sites for high-resolution MALDI–MSI.
Fig. 7: Metabolites co-localize with a patch of bacterial cells in earthworm tissue.
Fig. 8: metaFISH reveals metabolite distributions correlated with the presence of symbionts and parasites at 10 µm and 3 µm resolution.

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Data availability

All data presented in this paper have been deposited in the METASPACE project protocol (https://metaspace2020.eu/project/metaFISH). Individual datasets are deposited as follows: Fig. 1, MPIMM_193_QE_P_BC_CF (https://metaspace2020.eu/dataset/2019-11-28_11h08m15s); Fig. 5, 20210518_b_child_nix_s1_dhap_maldi2_tof_5um_laser90%_50shots (https://metaspace2020.eu/dataset/2021-07-08_13h54m26s), 20210518_b_child_nix_s1_dhap_laser70%_shots50 (https://metaspace2020.eu/dataset/2021-05-30_18h51m15s), 20210518_b_child_nix_s1_dhap_5um_maldi2_tof_laser50%_100 (https://metaspace2020.eu/dataset/2021-05-30_18h08m20s), 20210518_b_child_nix_s1_dhap_5um_maldi2_tof_laser30%_150 (https://metaspace2020.eu/dataset/2021-05-30_18h08m14s), and MPIMM_299_TTF_M2_Grid (https://metaspace2020.eu/dataset/2023-01-26_10h37m44s); Fig. 6, 20210706_bchild_nix_n25_tm_sl108 (https://metaspace2020.eu/dataset/2021-07-08_15h03m30s); Fig. 7, MTBLS2639; and Fig. 8, MPIMM_054_QE_P_BP_CF (https://metaspace2020.eu/dataset/2017-03-28_16h40m57s) and MPIMM_193_QE_P_BC_CF (https://metaspace2020.eu/dataset/2019-11-28_11h08m15s).

Code availability

Open-source scripts for the implementation of the MSI and microscopy co-registration have been published previously6 and are available on GitHub (R scripts, https://github.com/esogin/miniature-octo-fiesta; MATLAB, https://github.com/BenediktSenorDingDong/MALDI-FISHregistration).

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Acknowledgements

We acknowledge M. Sadowski (MPI Bremen) and J. Beckmann (MPI Bremen) for technical assistance with FISH and MSI, and Bruker Daltonics for access to timsTOF fleX instrumentation. P.B., B.G. and M.L. thank the Max Planck Society for funding. J.S. and K.D. are grateful for funding from Deutsche Forschungsgemeinschaft (DFG): DR 416/12-1 and SO976/41, SO976/5-1 and DR416/13-1, and CRC TRR332 (Z1). B.G. thanks the Human Frontier in Science Program for postdoctoral funding via a long-term fellowship (LT0015/2022-L).

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Contributions

P.B., B.G., T.B. and V.S. recorded MSI data. V.S., J.S., T.B. and K.D. assisted in the interpretation of results and writing the manuscript. T.B., V.S. and P.B. conducted the protocol validation experiments. P.B., B.G. and M.L. conceived and designed the study. P.B., B.G. and M.L. wrote the manuscript.

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Correspondence to Manuel Liebeke.

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T.B. is an employee of Bruker Daltonics GmbH & Co. KG (Bremen). All other authors declare no competing interests.

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Nature Protocols thanks Laura Sanchez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Geier, B. et al. Nat. Microbiol. 5, 498–510 (2020): https://doi.org/10.1038/s41564-019-0664-6

Geier, B. et al. Proc. Natl Acad. Sci. USA 118, e2023773118 (2021): https://doi.org/10.1073/pnas.2023773118

Bien, T. et al. Proc. Natl Acad. Sci. USA 119, e2114365119 (2022): https://doi.org/10.1073/pnas.2114365119

Niehaus, M. et al. Nat. Methods 16, 925–931 (2019): https://doi.org/10.1038/s41592-019-0536-2

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Supplementary Information

Supplementary Methods 1–4, Tables 1–13.

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Bourceau, P., Geier, B., Suerdieck, V. et al. Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling. Nat Protoc 18, 3050–3079 (2023). https://doi.org/10.1038/s41596-023-00864-1

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