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

ABSTRACT

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.