| dc.description.abstract | MALDI IMS is a powerful technology that combines multiplexed omics data with in situ spatial information. Additional pairing with compatible imaging modalities increases the biological context of the biomolecular distributions throughout a tissue sample. This thesis aimed to establish a multimodal MALDI IMS workflow for fresh-frozen bone and to leverage this advancement for understanding the spatially oriented lipidome of a S. aureus-infected femur. Notably, fresh-frozen murine femurs are difficult to prepare for MALDI IMS due to its mineralized and heterogeneous nature. Methods were investigated to cryosection femurs, thaw frozen sections, and deposit MALDI matrices to achieve reproducible lipid signal and dual polarity coverage for 10 µm spatial resolution imaging. The established imaging workflow could then be used to spatially map biomolecules involved in inflammatory bone diseases like S. aureus osteomyelitis. This debilitating infection was of interest due to its spatially defined abscess pathology and its current medical relevancy regarding the high rate of treatment failure. First, molecular profiles or atlases were created for tissue types throughout an infected femur, including surrounding soft tissue, bone-specific structures, tissue altered by inflammatory cell influx, and SACs. In all, nearly 250 lipids were mapped to morphological features throughout the femur using IMS and supplementary LC-MS/MS. Next, significant lipid alterations were identified between two global regions of the abscess: the inner neutrophilic tissue and outer fibrous tissue. Ether lipids were discovered to closely associate with leukocytes throughout the intramedullary cavity, and lipids with PUFAs, like arachidonic acid, were most abundant in the fibrous tissue. These data allude to the unique membrane composition of innate leukocytes following a myelopoietic response and provide information on potential lipid substrates for lipid-mediated inflammatory signaling between cells. Finally, the lipidome was characterized for a unique population of cells in the fibrotic border of abscesses. In situ lipid distributions suggest a lipid-laden phenotype for macrophages in chronic bone marrow abscesses, highlighting cellular functions that cannot be discerned with traditional microscopy. | |
