Achieving Single-Cell Resolution via Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) on...

Desorption electrospray ionization (DESI) is a widely used ambient mass spectrometry imaging (MSI) technique valued for its minimal sample preparation and ability to preserve native chemical states. However, achieving single-cell resolution with DESI has been challenging due to relatively low efficiencies of molecular ionization and ion transmission at small spatial scales. Here, we present four distinct implementations that enable single-cell DESI imaging of cultured cells through a combination of optimized experimental parameters and modular hardware integration. In the first platform, a Waters system consisting of a DESI XS source and a Synapt G2-Si Q-TOF mass spectrometer was used with a customized heated ion-transfer capillary and carefully optimized key parameters, including heating temperature, sprayer-to-surface distance, and solvent flow rate, for improved desolvation and ion transmission. In the second platform, a home-built sampling and ionization setup, including a DESI XS sprayer, motorized XYZ-stage, microscope, and ion-source interface, was coupled to a Thermo LTQ Orbitrap XL mass spectrometer. In the third platform, a similar setup containing a DESI XS sprayer was integrated with a Thermo Exploris 240 Orbitrap mass spectrometer. In the fourth platform, a similar setup was coupled to a Thermo Orbitrap Fusion Lumos mass spectrometer. All four platforms allowed MSI studies of metabolites in single cells with heterogeneous populations. Integration with Orbitrap systems provided higher mass resolution and improved spatial resolution, facilitating a demonstration of DESI-based single-cell MSI. Among all four platforms, combining DESI XS source with Exploris 240 resulted in the smallest pixel size (2.7 μm × 10 μm) and largest number of detected molecular features. Together, these results establish a flexible and reproducible framework for adapting DESI across platforms for high-resolution ambient MSI and reveal distinct chemical differences between neighboring cells under native conditions.