Optimal matrix application methods for MS imaging

Importance of the Topic

Matrix application in MALDI imaging directly influences crystal morphology, analyte extraction and spatial fidelity, thus impacting sensitivity and resolution in diverse fields such as biomedical research and drug distribution studies.

Study Objectives and Overview

This study introduces the iMLayerTM Matrix Vapor Deposition System and evaluates three matrix application methods—spraying, vapor deposition and a patented 2-step approach combining both—to establish optimal protocols for high-resolution MS imaging.

Methodology and Used Instrumentation

• Matrices Tested: DHB, 9-AA and CHCA applied on ITO glass slides.
• Application Methods: manual spraying with iMLayer AERO, vapor sublimation via iMLayerTM, and the 2-step sequence.
• Instrumentation: iMLayerTM vapor deposition system, iMLayer AERO automatic sprayer, iMScopeTM QT imaging mass microscope, OLS4100 laser microscope, SPM-9700HT scanning probe microscope.
• Comparative Metrics: crystal size and uniformity, analyte delocalization, and extraction efficiency assessed on mouse liver and drug-treated hair samples.

Main Results and Discussion

• Spraying produced large (tens of micrometers), irregular crystals with significant analyte spreading (~200 μm) but high extraction efficiency.
• Vapor deposition yielded ultra-uniform crystals (<1 μm), no analyte delocalization, yet limited extraction.
• The 2-step method achieved small, homogenous crystals (~1 μm), minimal spreading (~5–20 μm) and enhanced extraction, boosting sensitivity by up to fourfold for certain analytes.
• In specific cases such as cholesterol imaging, vapor deposition alone provided superior signal compared to the 2-step approach, underscoring the need for analyte-specific method selection.

Benefits and Practical Applications

• High reproducibility and quantitative comparability via precise thickness control (0.1 μm increments).
• Enhanced spatial resolution suitable for 5–10 μm laser beam diameters.
• Reduced analyte delocalization and improved detection sensitivity in pharmacological and metabolomic studies.
• Streamlined workflows enabling both novice and expert users to apply matrices consistently.

Future Trends and Applications

• Integration with high-throughput and fully automated sample preparation platforms.
• Development of tailored matrix compounds to target emerging biomarkers.
• Coupling high-resolution MS imaging with machine learning for automated parameter optimization.
• Expansion to multimodal imaging combining MS data with complementary spectroscopic techniques.

Conclusion

The iMLayerTM system and the 2-step matrix application strategy offer versatile solutions for balancing crystal quality, analyte extraction and spatial fidelity in MS imaging. Method choice should be guided by target analyte properties and analytical objectives, with a preliminary vapor deposition test followed by optional spraying to achieve optimal results.