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

Nature Protocols volume 14, pages 3445–3470 (2019)Cite this article

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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.

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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.

References

  1. Norris, J. L. & Caprioli, R. M. Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research. Chem. Rev. 113, 2309–2342 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Vaysse, P.-M., Heeren, R., Porta, T. & Balluff, B. Mass spectrometry imaging for clinical research–latest developments, applications, and current limitations. Analyst 142, 2690–2712 (2017).

    CAS  PubMed  Google Scholar 

  3. Chaurand, P. Imaging mass spectrometry of thin tissue sections: a decade of collective efforts. J. Proteom. 75, 4883–4892 (2012).

    CAS  Google Scholar 

  4. Wu, C., Dill, A. L., Eberlin, L. S., Cooks, R. G. & Ifa, D. R. Mass spectrometry imaging under ambient conditions. Mass Spectrom. Rev. 32, 218–243 (2013).

    CAS  PubMed  Google Scholar 

  5. Laskin, J. & Lanekoff, I. Ambient mass spectrometry imaging using direct liquid extraction techniques. Anal. Chem. 88, 52–73 (2015).

    PubMed  PubMed Central  Google Scholar 

  6. Feider, C. L., Krieger, A., DeHoog, R. J. & Eberlin, L. S. Ambient ionization mass spectrometry: recent developments and applications. Anal. Chem. 91, 4266–4290 (2019).

    CAS  PubMed  Google Scholar 

  7. Rubakhin, S. S., Jurchen, J. C., Monroe, E. B. & Sweedler, J. V. Imaging mass spectrometry: fundamentals and applications to drug discovery. Drug Discov. Today 10, 823–837 (2005).

    CAS  PubMed  Google Scholar 

  8. Greer, T., Sturm, R. & Li, L. Mass spectrometry imaging for drugs and metabolites. J. Proteom. 74, 2617–2631 (2011).

    CAS  Google Scholar 

  9. 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).

    CAS  PubMed  Google Scholar 

  10. Zavalin, A. et al. Direct imaging of single cells and tissue at sub-cellular spatial resolution using transmission geometry MALDI MS. J. Mass Spectrom. 47, 1473–1481 (2012).

    PubMed  Google Scholar 

  11. Laskin, J., Heath, B. S., Roach, P. J., Cazares, L. & Semmes, O. J. Tissue imaging using nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 84, 141–148 (2011).

    PubMed  PubMed Central  Google Scholar 

  12. Lanekoff, I. et al. Automated platform for high-resolution tissue imaging using nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 84, 8351–8356 (2012).

    CAS  PubMed  Google Scholar 

  13. Lanekoff, I. et al. Imaging nicotine in rat brain tissue by use of nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 85, 882–889 (2013).

    CAS  PubMed  Google Scholar 

  14. Lanekoff, I., Thomas, M. & Laskin, J. Shotgun approach for quantitative imaging of phospholipids using nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 86, 1872–1880 (2014).

    CAS  PubMed  Google Scholar 

  15. Nguyen, S. N. et al. Towards high-resolution tissue imaging using nanospray desorption electrospray ionization mass spectrometry coupled to shear force microscopy. J. Am. Soc. Mass. Spectrom. 29, 316–322 (2017).

    PubMed  PubMed Central  Google Scholar 

  16. Yin, R. et al. High spatial resolution imaging of mouse pancreatic islets using nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 90, 6548–6555 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Bergman, H.-M., Lundin, E., Andersson, M. & Lanekoff, I. Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization. Analyst 141, 3686–3695 (2016).

    CAS  PubMed  Google Scholar 

  18. Duncan, K. D. & Lanekoff, I. Oversampling to improve spatial resolution for liquid extraction mass spectrometry imaging. Anal. Chem. 90, 2451–2455 (2018).

    CAS  PubMed  Google Scholar 

  19. Lanekoff, I. et al. Trp53 deficient mice predisposed to preterm birth display region-specific lipid alterations at the embryo implantation site. Sci. Rep. 6, 33023 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Nguyen, S. N., Liyu, A. V., Chu, R. K., Anderton, C. R. & Laskin, J. Constant-distance mode nanospray desorption electrospray ionization mass spectrometry imaging of biological samples with complex topography. Anal. Chem. 89, 1131–1137 (2016).

    PubMed  Google Scholar 

  21. Lanekoff, I. & Laskin, J. Imaging of lipids and metabolites using nanospray desorption electrospray ionization mass spectrometry. Methods Mol. Biol. 1203, 99–106 (2015).

    CAS  PubMed  Google Scholar 

  22. Ballesteros Katemann, B., Schulte, A. & Schuhmann, W. Constant-distance mode scanning electrochemical microscopy (SECM)—part I: adaptation of a non-optical shear-force-based positioning mode for SECM tips. Chem. Eur. J. 9, 2025–2033 (2003).

    PubMed  Google Scholar 

  23. Ndobo-Epoy, J. P., Lesniewska, E. & Guicquero, J. P. Shear force microscopy with a nanoscale resolution. Ultramicroscopy 103, 229–236 (2005).

    CAS  PubMed  Google Scholar 

  24. Wang, H. & Dey, S. K. Roadmap to embryo implantation: clues from mouse models. Nat. Rev. Genet. 7, 185–199 (2006).

    PubMed  Google Scholar 

  25. Heeren, R. M., Smith, D. F., Stauber, J., Kukrer-Kaletas, B. & MacAleese, L. Imaging mass spectrometry: hype or hope? J. Am. Soc. Mass Spectrom. 20, 1006–1014 (2009).

    CAS  PubMed  Google Scholar 

  26. Hankin, J. A. & Murphy, R. C. Relationship between MALDI IMS intensity and measured quantity of selected phospholipids in rat brain sections. Anal. Chem. 82, 8476–8484 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lanekoff, I., Stevens, S. L., Stenzel-Poore, M. P. & Laskin, J. Matrix effects in biological mass spectrometry imaging: identification and compensation. Analyst 139, 3528–3532 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Duncan, K. D., Bergman, H.-M. & Lanekoff, I. A pneumatically assisted nanospray desorption electrospray ionization source for increased solvent versatility and enhanced metabolite detection from tissue. Analyst 142, 3424–3431 (2017).

    CAS  PubMed  Google Scholar 

  29. Bodzon‐Kulakowska, A. & Suder, P. Imaging mass spectrometry: instrumentation, applications, and combination with other visualization techniques. Mass Spectrom. Rev. 35, 147–169 (2016).

    PubMed  Google Scholar 

  30. Gamble, L. J. & Anderton, C. R. Secondary ion mass spectrometry imaging of tissues, cells, and microbial systems. Micros Today 24, 24–31 (2016).

    PubMed  PubMed Central  Google Scholar 

  31. Tian, H. et al. Gas cluster ion beam time-of-flight secondary ion mass spectrometry high-resolution imaging of cardiolipin speciation in the brain: identification of molecular losses after traumatic injury. Anal. Chem. 89, 4611–4619 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Tian, H. et al. Secondary-ion mass spectrometry images cardiolipins and phosphatidylethanolamines at the subcellular level. Angew. Chem. 131, 3188–3193 (2019).

    Google Scholar 

  33. Zavalin, A., Yang, J., Hayden, K., Vestal, M. & Caprioli, R. M. Tissue protein imaging at 1 μm laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS. Anal. Bioanal. Chem. 407, 2337–2342 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Rao, W., Pan, N. & Yang, Z. High resolution tissue imaging using the single-probe mass spectrometry under ambient conditions. J. Am. Soc. Mass Spectrom. 26, 986–993 (2015).

    CAS  PubMed  Google Scholar 

  35. Campbell, D. I., Ferreira, C. R., Eberlin, L. S. & Cooks, R. G. Improved spatial resolution in the imaging of biological tissue using desorption electrospray ionization. Anal. Bioanal. Chem. 404, 389–398 (2012).

    CAS  PubMed  Google Scholar 

  36. Kim, J. Y. et al. Atmospheric pressure mass spectrometric imaging of live hippocampal tissue slices with subcellular spatial resolution. Nat. Commun. 8, 2113 (2017).

    PubMed  PubMed Central  Google Scholar 

  37. Luxembourg, S. L., Mize, T. H., McDonnell, L. A. & Heeren, R. M. High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface. Anal. Chem. 76, 5339–5344 (2004).

    CAS  PubMed  Google Scholar 

  38. Schwartz, S. A., Reyzer, M. L. & Caprioli, R. M. Direct tissue analysis using matrix‐assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. J. Mass Spectrom. 38, 699–708 (2003).

    CAS  PubMed  Google Scholar 

  39. Stoeckli, M., Staab, D. & Schweitzer, A. Compound and metabolite distribution measured by MALDI mass spectrometric imaging in whole-body tissue sections. Int. J. Mass Spectrom. 260, 195–202 (2007).

    CAS  Google Scholar 

  40. Nguyen, S. N. et al. Lipid coverage in nanospray desorption electrospray ionization mass spectrometry imaging of mouse lung tissues. Anal. Chem. 91, 11629–11635 (2019).

    CAS  PubMed  Google Scholar 

  41. Duncan, K. D. et al. Quantitative mass spectrometry imaging of prostaglandins as silver ion adducts with nanospray desorption electrospray ionization. Anal. Chem. 90, 7246–7252 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wu, C., Ifa, D. R., Manicke, N. E. & Cooks, R. G. Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization. Anal. Chem. 81, 7618–7624 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Garza, K. Y. et al. Desorption electrospray ionization mass spectrometry imaging of proteins directly from biological tissue sections. Anal. Chem. 90, 7785–7789 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Hsu, C. C., Chou, P. T. & Zare, R. N. Imaging of proteins in tissue samples using nanospray desorption electrospray ionization mass spectrometry. Anal. Chem. 87, 11171–11175 (2015).

    CAS  PubMed  Google Scholar 

  45. Burnum, K. E. et al. Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation. J. Lipid Res. 50, 2290–2298 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Song, H. et al. Cytosolic phospholipase A2alpha is crucial for ‘on-time’ embryo implantation that directs subsequent development. Development 129, 2879–2889 (2002).

    CAS  PubMed  Google Scholar 

  47. Brash, A. R., Boeglin, W. E. & Chang, M. S. Discovery of a second 15S-lipoxygenase in humans. Proc. Natl. Acad. Sci. USA 94, 6148–6152 (1997).

    CAS  PubMed  Google Scholar 

  48. Lim, H. et al. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208 (1997).

    CAS  PubMed  Google Scholar 

  49. Walsh, S. W. Evidence for 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4(LTC4) in the onset of labor. Ann. NY Acad. Sci. 622, 341–354 (1991).

    CAS  PubMed  Google Scholar 

  50. Lanekoff, I. et al. Three-dimensional imaging of lipids and metabolites in tissues by nanospray desorption electrospray ionization mass spectrometry. Anal. Bioanal. Chem. 407, 2063–2071 (2015).

    CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. Department of Chemistry, Purdue University, West Lafayette, IN, USA

    Ruichuan Yin & Julia Laskin

  2. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA

    Kristin E. Burnum-Johnson

  3. Division of Reproductive Sciences, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Xiaofei Sun & Sudhansu K. Dey

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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.

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Correspondence to Julia Laskin.

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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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