Pushing the Boundaries of DESI Imaging With High Spatial Resolution

Significance of the Topic

Desorption electrospray ionization (DESI) imaging is a powerful ambient mass spectrometry technique that enables molecular mapping of biological tissues with minimal preparation. Achieving high spatial resolution is critical for resolving cellular and subcellular features, informing studies in biomarker discovery, pathology, and pharmaceutical research.

Objectives and Overview

This study aims to extend the spatial resolution limits of DESI imaging by implementing a low-flow configuration on a commercially available DESI XS source. The work demonstrates imaging at pixel sizes down to 5 µm and introduces a targeted reacquisition workflow using the Microscope Mode in HDI Software v1.8 to balance resolution and acquisition time.

Methodology and Instrumentation

Sections of porcine liver and murine brain (18 µm thickness) were analyzed without additional treatment. A Xevo G3 QTof mass spectrometer was coupled to a DESI XS source equipped with a high-precision sprayer and heated transfer line. Solvent (95% methanol/5% water) was delivered at flow rates of 250–500 nL/min via an ACQUITY M-Class µBSM pump, with backpressure maintained by a C18 column and reduced-id capillary tubing. Key operational parameters included:

• Capillary voltage: 0.6 kV
• Nitrogen nebulizing gas: 19.5 psi
• Transfer line temperature: 450 °C (negative mode), 200 °C (positive mode)
• Pixel sizes: 5–100 µm
• Scan rates: 10–20 scans per second

Data processing was performed in MassLynx and HDI Software for image visualization and ROI selection.

Results and Discussion

Optimization reduced extraction area to below 10 µm by adjusting solvent flow, nebulizing gas, and sprayer proximity. Comparison of conventional (500 nL/min) and low-flow (250 nL/min) configurations across pixel sizes (50, 20, 10, 5 µm) showed:

• Conventional flow yielded limited fidelity improvement below 20 µm and poor quality at 5 µm.
• Low-flow configuration improved image resolution at 5 µm, confirming a reduction in effective spot size.

To mitigate lengthy acquisitions at high resolution, the Microscope Mode workflow employs an initial survey scan (e.g., 100 µm pixels) followed by targeted reacquisition of ROIs at finer resolutions. This non-destructive approach preserved tissue integrity and yielded consistent spectral intensities across sequential scans. High-resolution images of murine cerebellum highlighted oligodendrocytes (~10–15 µm) using a lipid ion (m/z 760.5848). At 5 µm pixels, individual cells were clearly differentiated, demonstrating subcellular resolution.

Benefits and Practical Applications

• Enables commercial DESI systems to achieve pixel sizes as small as 5 µm without extensive hardware modifications.
• Improves image fidelity and sensitivity on a QTof platform at accelerated scan rates.
• Supports a non-destructive, data-driven survey and targeted imaging workflow to optimize analytical throughput.
• Facilitates cellular-level molecular mapping for research in neuroscience, pathology, and drug distribution studies.

Future Trends and Potential Applications

Continued development of low-flow DESI and advanced software workflows is expected to push spatial resolution further into the subcellular domain. Future directions include integration with quantitative imaging, correlative microscopy, and automated ROI selection. These advances will expand applications in clinical diagnostics, single-cell metabolomics, and high-throughput tissue profiling.

Conclusion

The combination of a low-flow DESI XS configuration and the Microscope Mode in HDI Software v1.8 enables high-fidelity imaging at pixel sizes down to 5 µm. This workflow offers a practical solution for balancing resolution and acquisition speed, unlocking near-single-cell mass spectrometry imaging on commercially available instrumentation.

References

• Fruttiger M. et al. Defective Oligodendrocyte Development and Severe Hypomyelination in PDGF-A Knockout Mice. Development. 1999;126(3):457–467.
• Jäkel S., Agirre E., Mendanha Falcão A. et al. Altered Human Oligodendrocyte Heterogeneity in Multiple Sclerosis. Nature. 2019;566:543–547.