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High spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometry

Abstract

Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology, MSI of tissue samples at single-cell resolution remains challenging. Herein, we describe a protocol for robust imaging of tissue sections with a high (better than 10-μm) spatial resolution using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry, an ambient ionization technique that does not require sample pretreatment before analysis. In this protocol, mouse uterine tissue is used as a model system to illustrate both the workflow and data obtained in these experiments. We provide a detailed description of the nano-DESI MSI platform, fabrication of the nano-DESI and shear force probes, shear force microscopy experiments, spectral acquisition, and data processing. A properly trained researcher (e.g., technician, graduate student, or postdoc) can complete all the steps from probe fabrication to data acquisition and processing within a single day. We also describe a new strategy for acquiring both positive- and negative-mode imaging data in the same experiment. This is achieved by alternating between positive and negative data acquisition modes during consecutive line scans. Using our imaging approach, hundreds of high-quality ion images were obtained from a single uterine section. This protocol enables sensitive and quantitative imaging of lipids and metabolites in heterogeneous tissue sections with high spatial resolution, which is critical to understanding biochemical processes occurring in biological tissues.

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Fig. 1: Overall workflow for high-resolution imaging of biological tissues using nano-DESI MSI.
Fig. 2: Platform for high-resolution imaging of biological tissues using nano-DESI MSI.
Fig. 3: Shear force microscopy.
Fig. 4: Representative ion images of endogenous molecules acquired in uterine tissues.
Fig. 5: Compensation for matrix effects in nano-DESI MSI by adding internal standards to the solvent.
Fig. 6: Representative line profiles of PC 32:0, SM 16:0, and PI 36:2 acquired on uterine tissue.
Fig. 7: Quantitative analysis of endogenous PC species in different types of uterine cells.

Data availability

The data presented in this study are available from the corresponding author upon reasonable request.

Code availability

The LabVIEW and MSI QuickView programs are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under award UG3HL145593 (HuBMAP Program, J.L. and K.E.B.-J.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD, grant R21 HD084788 to K.E.B.-J.), and NIH grants (DA006668 and HD068524 to S.K.D.).

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Contributions

J.L. and R.Y. developed the procedure. R.Y. performed the imaging experiments and processed the data. K.E.B.-J. and J.L. conceived the study and assisted with data analysis. X.S. and S.K.D. provided mouse uterine sections and were involved in the interpretation of the results. R.Y. and J.L. wrote the manuscript with assistance from K.E.B.-J., X.S., and S.K.D.

Corresponding author

Correspondence to Julia Laskin.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Pierre Chaurand and Livia Schiavinato Eberlin for their contribution to the peer review of this work.

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Key references using this protocol

Nguyen, S. N. et al. J. Am. Soc. Mass Spectrom. 29, 316–322 (2018): https://doi.org/10.1007/s13361-017-1750-8

Yin, R. et al. Anal. Chem. 90, 6548–6555 (2018): https://doi.org/10.1021/acs.analchem.8b00161

Nguyen, S. N., Liyu, A. V., Chu, R. K., Anderton, C. R. & Laskin, J. Anal. Chem. 89, 1131–1137 (2016): https://doi.org/10.1021/acs.analchem.6b03293

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Yin, R., Burnum-Johnson, K.E., Sun, X. et al. High spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometry. Nat Protoc 14, 3445–3470 (2019). https://doi.org/10.1038/s41596-019-0237-4

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