Immunohistochemistry-compatible gel-assisted mass spectrometry imaging

While lipids are fundamental to cellular signaling and structure, probing native lipid compositions and distributions across complex tissue architectures at single-cell resolution remains challenging due to inherent trade-offs in spatial resolution, sensitivity, and molecular coverage in current spatial lipidomics methods. Here, we report a multimodal imaging approach that seamlessly integrates gold-standard immunohistochemical labeling with gel-assisted mass spectrometry imaging to enable cell-type-specific, single-cell spatial lipidomics with modern instrumentation. Using intact brain tissue as a testbed, we demonstrate in situ measurement of cerebellar Purkinje cells and spatially mapped lipids across timepoints and cerebellar subregions within the pathological landscape of a neurodegenerative, lysosomal storage disorder. With single-cell lipidomic profiling, we delineated spatiotemporally distinct accumulation of specific glycosphingolipids and phospholipids within Purkinje cells and non-Purkinje cells in the diseased brain. Furthermore, unsupervised single-cell lipidomic clustering elucidated disease-progression- and subregion-dependent molecular divergence between healthy and neurodegenerative states within the cerebellum.