<p dir="ltr">Mass spectrometry imaging (MSI) is a label-free approach for mapping the spatial distribution of individual molecules on sample surfaces. Multiple MSI techniques that utilize various ionization approaches have been developed for mapping of multiple classes of biomolecules, including lipids, metabolites, peptides, and glycans, on biological tissue sections. However, spatial mapping of individual intact proteoforms still remains challenging due to the relatively low extraction, ionization, ion transmission, and fragmentation efficiency. Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that utilizes localized liquid extraction, enabling molecular mapping in tissue sections with high sensitivity and spatial resolution down to 10 µm. Relative quantification of individual molecules in tissue functional units can be performed by normalization of the signal intensity of the analytes to that of the internal standards integrated into the extraction solvent. Nano-DESI generates multiply charged ions of intact proteins, providing special opportunities for untargeted characterization and mapping of intact proteoforms directly from tissue samples. In this thesis, we developed a novel platform combining nano-DESI MSI with on-tissue tandem mass spectrometry (MS/MS) for spatial mapping of individual proteoforms on tissue sections. We demonstrated for the first time the differential expression of individual proteoforms with different post-translation modifications (PTMs) across tissue functional units, which provides unique insights into differences in biochemical pathways associated with different PTMs. Furthermore, we optimized sample preparation and the design of nano-DESI capillary probes and employed an oversampling strategy to improve the spatial resolution down to ~7 µm with improved sensitivity for proteoform mapping at the cellular-level spatial resolution. The developed methodology expands the molecular coverage and establishes nano-DESI MSI as a powerful tool for studying the state of diverse cell types. Moreover, we integrated the two different nano-DESI imaging workflows for metabolites/lipids and peptides/proteins into one platform to enable a correlative MSI approach for spatial multi-omics analysis. We applied this multimodal imaging approach to study the molecular signatures in specific cell types of healthy animal tissue samples and investigate the molecular pathology of diseased animal samples. The improvements in the nano-DESI MSI technique and its application provide new insights into the molecular phenotypes and the related cellular processes, opening up unique opportunities in clinical research.</p>