Deep MALDI-MS spatial omics guided by quantum cascade laser mid-infrared imaging microscopy.

In spatial'omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry imaging (MSI)-based spatial 'omics must compromise between data acquisition speed and biochemical profiling depth. Here, we introduce fast, label-free quantum cascade laser mid-infrared imaging microscopy (QCL-MIR imaging) to guide MSI to high-interest tissue regions as small as kidney glomeruli, cultured multicellular spheroid cores or single motor neurons. Focusing on smaller tissue areas enables extensive spatial lipid identifications by on-tissue tandem-MS employing imaging parallel reaction monitoring-Parallel Accumulation-Serial Fragmentation (iprm-PASEF). QCL-MIR imaging-guided MSI allowed for unequivocal on-tissue elucidation of 157 sulfatides selectively accumulating in kidneys of arylsulfatase A-deficient mice used as ground truth concept and provided chemical rationales for improvements to ion mobility prediction algorithms. Using this workflow, we characterized sclerotic spinal cord lesions in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and identified upregulation of inflammation-related ceramide-1-phosphate and ceramide phosphatidylethanolamine as markers of white matter lipid remodeling. Taken together, widely applicable and fast QCL-MIR imaging-based guidance of MSI ensures that more time is available for exploration and validation of new biology by default on-tissue tandem-MS analysis.