Tenfold Expansion Submicrometer MALDI Mass Spectrometry Imaging of Tissues and Cultured Cells

Achieving  submicrometer lateral resolution in matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is essential for molecular characterization at the single-cell and subcellular levels but is fundamentally limited by optical focusing and matrix crystallization. Here, we present tenfold expansion mass spectrometry imaging (10X ExMSI), an expansion MALDI-MSI workflow that reaches an effective lateral resolution of ∼500 nm on standard commercial instruments using a 5 µm acquisition step size without pixel oversampling. The SDS-free digestion protocol minimizes lipid leaching and supports broad detection of major lipid classes, including glycerophospholipids and sphingolipids, although a reduction in primary amine-containing lipids is observed due to cross-linking. Using 10X ExMSI, we resolve subcellular structures in mouse brain tissues, such as dendritic arborizations, that are challenging to access with existing MALDI-MSI implementations. We further demonstrate expansion-based MSI on cultured A549 cells, achieving subcellular-level lipid mapping. The method is fully compatible with standard MALDI-MSI systems, providing an accessible and scalable route to high-resolution, label-free molecular imaging. These capabilities open new opportunities for studying cellular architecture and molecular heterogeneity in biological and biomedical research.