Delineation of Subcellular Molecular Heterogeneity in Single Cells via Ultralow-Flow-Rate Desorption Electrospray Ionization Mass Spectrometry Imaging.

Single-cell analysis uncovers cellular heterogeneity and dynamic metabolism at the individual cell level, providing fundamental insights into the physiological and pathological mechanisms of life. Spatial molecular mapping of single cells requires advanced techniques that achieve high spatial resolution and comprehensive molecular coverage while preserving the cells' native state with minimal preparation. Here, we report an ultralow-flow-rate desorption electrospray ionization mass spectrometry imaging (u-DESI-MSI) platform for biomolecule mapping of single cells with subcellular spatial resolution. The u-DESI-MSI single-cell platform utilizes a stable and ultralow-solvent-flow-rate (150 nL/min) system with optimized geometrical settings, avoiding complicated hardware modifications on the ESI emitter. The capability of u-DESI-MSI was demonstrated by spatial molecular mapping of human pancreatic cells from different lineages. An unprecedented spatial resolution was achieved for molecular mapping of the single cells under a rastering step size of 5 μm, even revealing lipid distribution in subcellular regions. Using this high-spatial-resolution u-DESI-MSI platform, we effectively visualized both intercellular and intracellular molecular heterogeneity in single cells. Notably, u-DESI-MSI provides a versatile tool for direct molecular imaging of single cells in their native states under ambient conditions, eliminating the need for extensive sample preparation. We anticipate that this platform will facilitate the exploration of single-cell heterogeneity, offering valuable insights into cellular variability and metabolism.