An Improved Workflow for DESI Imaging Mass Spectrometry Incorporating Waters High Definition Imaging Software

Importance of the Topic

DESI imaging mass spectrometry enables spatially resolved chemical analysis of biological and material samples without extensive preparation, offering ambient‐pressure operation, high sensitivity, and micrometer‐scale resolution. This capability is critical for lipidomics, metabolomics, pharmacokinetic studies, and clinical tissue analysis, where spatial distribution of molecules reveals functional and pathological insights.

Goals and Overview of the Study

This technology brief presents enhancements in Waters High Definition Imaging (HDI) software and its integration with MassLynx instrument control, aimed at streamlining DESI imaging workflows. The article details a new user interface, automated experiment queuing, and batch processing to improve throughput and data consistency for multiple sequential imaging experiments.

Methodology and Instrumentation

• Optical imaging: Photographic capture or flatbed scanning of sample slides
• Sample handling: Mounting on Prosolia 2D DESI source slide holders or microtiter plate holder
• Image co-registration: Alignment of optical image corners with DESI stage via HDI
• Region definition: Setting of imaging area, stage speed, and automatic MS scan rate calculation
• Mass spectrometry settings: Configuration of experiment type (MS-Tof, MS/MS-Tof, IMS, HDMS(E)), mass range, collision energy, polarity, and analyzer mode
• Data processing: Automated parameter selection for HDI processing, including ion mobility integration when available
• Software integration: Export of experiment as a MassLynx sample list to control the DESI stage coordinates and data acquisition

Main Results and Discussion

• Batch acquisition: Demonstrated negative and positive ion mode imaging on the same mouse brain section through sequential experiments
• High throughput: Six imaging runs (treated and untreated mouse skin sections) were queued and executed overnight with no user intervention
• Automated processing: Raw data processed in HDI for visual overlay of DESI ion images with optical images
• Multivariate analysis: Integration of unsupervised (PCA) and supervised (OPLS-DA) methods to distinguish molecular profiles between sample conditions

Benefits and Practical Applications of the Method

• Intuitive workflow: Simplified setup reduces training time and manual error
• Enhanced throughput: Batch mode execution and automated processing maximize data acquisition during unattended runs
• Reproducibility: Standardized experiment templates and integrated control improve consistency across samples
• Versatility: Applicable to diverse sample types for metabolomic, lipidomic, and pharmaceutical imaging studies

Future Trends and Potential Applications

• Further automation: AI-assisted experiment design and real-time data quality assessment
• Expanded ion mobility: Deeper integration of HDMS(E) separation for complex sample matrices
• Higher spatial resolution: Advances in DESI source technology to reach sub-30 µm pixel sizes
• Cross-platform compatibility: Integration with additional mass spectrometer models and third-party imaging modalities
• Clinical translation: Routine use in histopathology and diagnostic laboratories for rapid tissue phenotyping

Conclusion

The integration of improved HDI software with MassLynx instrument control for DESI imaging offers a streamlined, high‐throughput workflow that reduces manual intervention, enhances reproducibility, and supports advanced data analysis. These developments facilitate broader adoption of DESI imaging in research and clinical settings.

Used Instrumentation

• Waters High Definition Imaging (HDI) Software
• Waters MassLynx Software
• Prosolia 2D DESI source
• SYNAPT G2-Si and Xevo G2-XS mass spectrometers

References

No literature references were provided in the source document.