High Speed, High Lateral Resolution Lipid Imaging using a timsTOF fleX

Importance of the Topic

Lipid molecules participate in key biological functions and their spatial distribution within tissues can reveal insights into disease mechanisms, drug effects and cellular processes. High-resolution, high-throughput imaging of lipids enables researchers to map these molecules in situ, bridging the gap between molecular analysis and histological detail and supporting applications in pharmaceutical development, neuroscience and pathology.

Objectives and Overview

This study evaluates the capabilities of a timsTOF fleX platform for MALDI-MSI of lipids. The goal is to demonstrate simultaneous achievement of high acquisition speed and true 20 µm lateral resolution, while maintaining robust mass accuracy and sensitivity, and to benchmark performance against established MALDI-TOF and magnetic resonance MS systems.

Methodology

Sample Preparation

• Fresh frozen rat and mouse brain sections cut at 10 µm and mounted on conductive slides
• Matrix application using robotic TM sprayer with either 15 mg/mL DHB in 90 ACN/H2O or 2.5 mg/mL ZSA in 70 ACN/H2O

Acquisition Parameters

• timsTOF fleX configured with Smartbeam 3D laser and fast-moving stage
• Acquisition range m/z 300–1000 (or up to 3000), 400 shots per pixel, 10 kHz laser frequency, 20 µm pitch
• Data processing and visualization performed in SCiLS Lab MVS, with anatomical referencing via the Allen Brain Atlas

Instrumentation

The timsTOF fleX platform combines a quadrupole time-of-flight mass analyzer with integrated high-speed MALDI source and Smartbeam 3D laser optics. The system’s precision stage supports rapid raster scanning at 20 µm pitch. SCiLS Lab MVS software handles large-scale image reconstruction, normalization and region annotation.

Results and Discussion

Performance Comparison

• Benchmark lipids such as PC(36:1) [M+K]+ were imaged on timsTOF fleX, a high-throughput axial MALDI-TOF and an MRMS instrument
• timsTOF fleX achieved resolving power (FWHM) above 40 000 and S/N ratios exceeding 500 for representative lipids, positioning its performance between the TOF and MRMS systems

High-Resolution Imaging

• True 20 µm lateral resolution confirmed by localizing PC(36:4) [M+K]+ to the cerebellar granule cell layer and co-localizing heme signals in adjacent vasculature
• Serial sections in positive and negative modes produced consistent spatial patterns across instruments, validating reproducibility

Stability and Robustness

• Approximately 1.5 million pixels acquired over 20 hours without lockmass calibration showed negligible signal decay or matrix sublimation
• Mass drift remained within ±5 ppm across four consecutive large-scale imaging runs, indicating long-term stability

Benefits and Practical Applications

High throughput at cellular resolution accelerates spatial lipidomics workflows in drug discovery and biomarker research. The robust performance supports routine QA/QC in pharmaceutical and clinical research environments. Integration with SpatialOMx approaches allows simultaneous mapping of lipids alongside other omics layers.

Future Trends and Applications

Emerging developments include combining ion mobility separation with MALDI-MSI for enhanced isomer discrimination, extending multiplexed imaging to metabolites and peptides, and integrating deep-learning-driven image analysis for automated region classification. Continued advances in laser technology and stage performance will push resolution below 10 µm while further reducing acquisition times.

Conclusion

The timsTOF fleX delivers a balanced combination of high speed, high spatial resolution and stable performance for lipid imaging. Its capabilities fill the gap between throughput and mass resolution, enabling detailed molecular mapping in biomedical research and industrial applications.

References

[1] Fülöp A et al Anal Chem 2013 Oct 1 85 19 9156-63