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High-spatial-resolution mass spectrometry imaging of biological tissues using a microfluidic probe

Nature Protocols (2025)Cite this article

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Abstract

Nanospray desorption electrospray ionization (nano-DESI) is a liquid extraction-based ambient ionization mass spectrometry imaging (MSI) technique that enables quantitative molecular mapping of biological samples in their native state with high spatial resolution. To facilitate the wider adoption of nano-DESI MSI by the scientific community, we have developed a robust and user-friendly microfluidic probe (MFP). The probe has been used to achieve high spatial resolution of 8–10 µm and up to 10-fold improvement in the experimental throughput, enabling imaging of large tissue sections with cellular resolution. Here, we provide detailed instructions for designing, fabricating and operating MFPs. In addition, we describe a complete workflow for nano-DESI MSI, covering every step from probe assembly to data acquisition and analysis. Although the fabrication of MFPs requires expertise in microfluidics and can take a few days, the process can be outsourced to qualified companies for manufacturing. Once the MFP is fabricated, the entire imaging workflow can be completed in several hours, depending on the sample size. For example, a sample with an area of 1 cm² can be analyzed in <10 h at a spatial resolution of 10 µm. The exceptional performance and ease of use of these probes will make high-resolution nano-DESI MSI more accessible to the scientific community.

Key points

  • This protocol covers the fabrication of the microfluidic probe via photolithography and selective laser-assisted etching, its assembly, and the nano-DESI MSI experiments for imaging different types of tissue.

  • An alternative method is matrix-assisted laser desorption/ionization (MALDI) that shows poor fragmentation of singly charged protein ions. DESI is an ambient ionization method that generates multiply charged protein ions, facilitating protein identification.

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Fig. 1: Setup of nano-DESI probes.
Fig. 2: Schematic illustrating the fabrication process of the MFP using photolithography.
Fig. 3: Schematic of the SLE-MFP.
Fig. 4: Effects of grinding and polishing on the shape of the MFP.
Fig. 5: Setup for polishing the SLE-MFP and assembling it with an external glass capillary.
Fig. 6: Examples of MFP holders.
Fig. 7: Optical image and representative positive- and negative-mode ion images of molecules in human pancreatic tissue acquired using the iMFP.
Fig. 8: Optical image and representative positive-mode ion images of molecules in mouse brain tissue acquired using the SLE-MFP.

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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 programs are available upon request. The MSIGen program can be accessed through GitHub at https://github.com/LabLaskin/MSIGen.

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Acknowledgements

L.-X.J., X.L. and J.L. acknowledge support from the National Institutes of Health (NIH) awards RF1MH128866 (BICCN), R01MH136394 and UH3CA255132 (HuBMAP) along with support from the National Science Foundation (NSF-2108729 and 2333734). M.P. and D.B. acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; FOR 2177-251124697). We are grateful to J. Chen, C. Matthews and M. Campbell-Thompson (Department of Pathology, Immunology and Laboratory Medicine, University of Florida) and Y. Zhu and P. Piehowski (Pacific Northwest National Laboratory) for providing human pancreatic tissue sections. We thank M. Yang from Purdue University for sectioning mouse brain tissue and J. H. Park from the Birck Nanotechnology Center at Purdue University for helpful discussions and technical assistance with the development of the photomask.

Author information

Authors and Affiliations

  1. James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, West Lafayette, IN, USA

    Li-Xue Jiang, Xiangtang Li & Julia Laskin

  2. Institute of Analytical Chemistry, Leipzig University, Leipzig, Germany

    Matthias Polack & Detlev Belder

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  1. Li-Xue Jiang
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  2. Xiangtang Li
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  3. Matthias Polack
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Contributions

J.L. and X.L. developed the iMFP procedure. J.L., D.B., L.-X.J. and M.P. developed the SLE-MFP procedure. X.L. and L.-X.J. performed imaging experiments and processed the data. J.L. and D.B. conceived the study and acquired funding. L.-X.J. and J.L. co-wrote the manuscript with assistance from X.L., D.B. and M.P.

Corresponding author

Correspondence to Julia Laskin.

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The authors declare no competing interests.

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Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Key references

Jiang, L. X. et al. Lab Chip 23, 4664–4673 (2023): https://doi.org/10.1039/D3LC00637A

Li, X. et al. Anal. Chim. Acta 1279, 341830 (2023): https://doi.org/10.1016/j.aca.2023.341830

Li, X. et al. Anal. Chem. 94, 9690–9696 (2022): https://doi.org/10.1021/acs.analchem.2c01093

Li, X. et al. Angew. Chem. Int. Ed. 59, 22388–22391 (2020): https://doi.org/10.1002/anie.202006531

This protocol is an extension to: Nat. Protoc. 14, 3445–3470 (2019): https://doi.org/10.1038/s41596-019-0237-4

Supplementary information

Reporting Summary

Supplementary Table 1

3D CAD design file for the SLE-MFP.

Supplementary Table 2

3D CAD design file for the SLE-MFP holder.

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Jiang, LX., Li, X., Polack, M. et al. High-spatial-resolution mass spectrometry imaging of biological tissues using a microfluidic probe. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01188-y

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