<p> Mass spectrometry imaging (MSI) is an advanced analytical technique that renders spatially defined images of complex label-free samples. Nanospray desorption electrospray ionization (nano-DESI) MSI is an ambient ionization direct liquid extraction technique in which analytes are extracted by means of a continuous liquid flow between two fused-silica capillaries. The droplet generated between the two capillaries is controlled by a delicate balance of solvent flow, solvent aspiration, capillary angles, and distance from the surface. This technique produces reproducible ion images with up to 10 µm resolution and can be used to identify and quantify multiple analytes on a given surface.  This thesis discusses some of the applications of this technique to biological systems, as well as the work done to develop methodology to further improve this technique’s specificity and sensitivity. Herein, applications that push the limits of the current capabilities of nano-DESI are presented, such as the high-resolution imaging of lipid species in skeletal muscle at the single-fiber level, and the quantification of low-abundance drug metabolites.  The second theme of this thesis, developing new capabilities, introduces ion mobility mass spectrometry imaging. This integrated technique increases the selectivity previously possible with MSI. To support these efforts, the work in this thesis has generated data analysis workflows that not only make these experiments possible but also further endeavor to increase sensitivity and combat instrument limitations on mobility resolution. Finally, this thesis present streamlined workflows for tandem MS experiments and modifications to a recently introduced microfluidic variant of the nano-DESI technique. In all, this thesis showcases the current capabilities of the nano-DESI technique and lays the groundwork for future improvements and capabilities.      </p>