Optimized Workflow for High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to pathological alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto ITO coated glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracks, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was leveraged to characterize lipid distributions in fresh-frozen bone, and complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Furthermore, we discovered that [sphingomyelin(42:1) + H]+ was abundant in megakaryocytes, whereas [sphingomyelin(42:2) + H]+ was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Therefore, this application of multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage in a preserved native bone microenvironment.