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
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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.
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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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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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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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T.B. is an employee of Bruker Daltonics GmbH & Co. KG (Bremen). All other authors declare no competing interests.
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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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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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DOI: https://doi.org/10.1038/s41596-023-00864-1
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