Single-Cell Resolution DESI Mass Spectrometry Imaging through 10-Fold Sample Expansion

Desorption electrospray ionization mass spectrometry imaging (DESI MSI) is a valuable tool for label-free, spatially resolved molecular analysis of biological tissues. However, its spatial resolution has been limited to tens of micrometers due to constraints in spray geometry and solvent flow, hindering single-cell imaging. Inspired by expansion microscopy, we developed a tenfold DESI expansion MSI (10x-DESI ExMSI) technique to achieve cellular-level resolution. In this study, tissue sections were embedded in a swellable polyelectrolyte gel, and physically enlarged by approximately tenfold without compromising molecular integrity. After drying of the sample, mass spectrometry imaging was performed using a custom-built DESI ion source coupled with an Orbitrap mass spectrometer. Using mouse brain tissues as representative samples, 10x-DESI ExMSI achieved a spatial resolution of approximately 5 {micro}m, comparable to the highest levels attained by advanced commercial MALDI-MSI systems. Enhanced resolution allowed clear visualization of fine structural details, such as the granule cell layer of the dentate gyrus and the pyramidal layer of the Cornu Ammonis, which were unresolved in unexpanded samples under a 100 m step size. Lipid profiling demonstrated high retention and detection efficiency, confirming the techniques suitability for comprehensive molecular imaging. The 10x-DESI ExMSI significantly advances DESI MSI by enabling single-cell level resolution without modifying existing mass spectrometers. The method is simple, cost-effective, and easily implementable in most laboratories, making high-resolution DESI MSI accessible for a wide range of biological applications. This approach overcomes longstanding limitations of DESI imaging and holds great potential for detailed molecular studies at the cellular level.