Single Cell Image Resolution with DESI XS Coupled to a Xevo G3 Mass Spectrometer

Significance of the topic

The ability to image and chemically characterize individual cells is transforming our understanding of biological heterogeneity. Conventional mass spectrometry imaging often lacks the spatial resolution to resolve single cells, obscuring cell‐to‐cell variations in lipid metabolism and other molecular signatures. Achieving true single‐cell resolution opens the door to detailed studies of cellular interactions, disease mechanisms and personalized medicine approaches.

Objectives and study overview

This study demonstrates a commercially available desorption electrospray ionization (DESI) configuration coupled to a Xevo G3 QTof mass spectrometer for high‐resolution imaging at 5 µm pixel size. The main goals were:

• To visualize individual cells in tissue sections and non‐confluent cell cultures using DESI imaging.
• To identify and profile lipid distributions at the single‐cell level.
• To apply statistical tools for assessing cellular heterogeneity in two human cell lines (THP‐1 and PC‐3).

Methodology and instrumentation

Cell cultures of THP‐1 macrophages and PC‐3 prostate cancer cells were grown to 70 % confluency, treated with oleic acid, then fixed and stored at –80 °C. Murine brain tissue was cryosectioned at 18 µm. Slides were analyzed without matrix application using a DESI XS source equipped with a low‐flow DESI kit on a Xevo G3 QTof MS. Key instrumental parameters included:

• Solvent flow tuned for nano‐flow DESI (kit p/n 186011378) with ACQUITY M-Class delivery.
• High-performance sprayer, heated transfer line and source geometry optimized for 5 µm pixels.
• Survey scanning and targeted ‘microscope mode’ reacquisition in HDI v1.8.

Data processing involved MassLynx for initial review, HDI v1.8 for image generation and ROI selection, Lipostar for lipid annotation (<6 ppm), and MetaboAnalyst 6.0 for multivariate statistics.

Main results and discussion

Imaging at 5 µm pixel size revealed clear differentiation of single cells in murine cerebellum white matter and cultured THP‐1/PC‐3 cells. Comparison of 10 µm and 5 µm pixels showed substantial gains in image fidelity at higher resolution. ‘Microscope mode’ accelerated acquisition by combining a rapid 25 µm survey with focused 5 µm scans of selected regions. On average, ~50 lipids per cell were putatively identified, totaling 192 unique lipid annotations across both cell types. A principal component analysis separated THP‐1 and PC‐3 cells and highlighted intra‐cell line heterogeneity, demonstrating the platform’s ability to resolve biological variance at single‐cell level.

Benefits and practical applications

• Commercially available DESI XS kit supports routine 5 µm imaging, enabling single‐cell MS profiling.
• Non‐destructive survey followed by targeted high‐resolution scans reduce overall analysis time.
• Robust lipid signal intensity permits deep molecular coverage and statistical interrogation of cell populations.

Future trends and opportunities

Advances may further shrink pixel dimensions below 5 µm, integrate real‐time single‐cell MS analyses and combine DESI imaging with transcriptomic or proteomic assays. Automated ROI selection and machine‐learning‐driven data processing will enhance throughput and enable comprehensive cell atlas projects.

Conclusion

This work establishes a practical DESI‐MS imaging workflow for single‐cell analysis, delivering 5 µm spatial resolution with in‐depth lipid profiling. The approach offers a powerful tool for exploring cellular heterogeneity, disease pathology and drug responses at the individual cell level.

Reference

1. Seydel C. Nat Methods. 2021;18:1452–1456.
2. Carter B, Zhao K. Nat Rev Genet. 2021;22:235–250.
3. Towers M, Reid L, Jones E, Ballantyne J. Pushing the Boundaries of DESI Imaging with High Spatial Resolution. Waters Application Note 720008551. 2024.
4. Pang Z, Lu Y, Zhou G, et al. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing. Nucleic Acids Res. 2024; doi:10.1093/nar/gkae253.
5. Fruttiger M, et al. Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice. Development. 1999;126(3):457–467.
6. Jäkel S, Agirre E, Mendanha Falcão A, et al. Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature. 2019;566:543–547.
7. Tsuchiya S, et al. Int J Cancer. 1980;26:171–176.
8. Chanput W, Mes JJ, Wichers HJ. Int Immunopharmacol. 2014;23:37–45.
9. Kaighn ME, et al. Invest Urol. 1979;17:16–23.