Comprehensive High-Spatial-Resolution Imaging Metabolomics Workflow for Heterogeneous Tissues.

Mass spectrometry imaging is a developing technique that maps the molecular composition of samples in a label-free manner. However, highly heterogeneous samples, including bones, usually cannot be easily analyzed due to challenging sample preparation, particularly in minimizing cracks and maintaining flatness. To comprehensively address these issues, we developed a sample preparation method for the fresh frozen heterogeneous samples such as knee joint and skull of murine, which includes complex structures and tissue types, such as neuronal tissue, peripheral nerve, muscle, tracks, connective tissue, cartilage, mineralized bone, and bone marrow. By controlling the sample thickness and employing an optimized drying method, we achieved minimal cracking. We found that a combination of lyophilization and 5 μm section thickness, when attached to a cryofilm, was readily achievable and significantly reduced cracking in bone tissue. Additionally, we implemented a contactless spin-flattening technique to ensure surface uniformity. Centrifuging the section at 7000 improved surface flatness, bringing the height variation within the range typically observed in soft tissues while also removing excess mounting medium and bubbles. This approach enhances the sample quality and reliability without requiring complex manual skills, making it more practical and reproducible for routine analysis. High molecular coverage was achieved, including small metabolites, metals, and lipids, by using the -(1-naphthyl) ethylenediamine dihydrochloride (NEDC) matrix. To further explore the potential of our workflow, high-resolution MSI was performed on rat tibial growth plates at different growth stages. Numbers of -acetyl disaccharide sulfate and PE (34:1) are found to be complementary expressed in growth plate cartilage and have different intensities at different growth stages. Our findings suggested the potential involvement of those metabolites in bone development. By addressing the challenges of sample preparation, including surface flatness, bubbles, and severe cracking, our approach significantly improves the quality of the MS imaging. Additionally, this method offers a broad detection range that encompasses both metal ions and metabolites. This advancement enables detailed and accurate molecular characterization of rigid biological samples, enhancing the potential for applications in biomedical research.