This thesis presents efforts to improve the methodology of matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) as a method for analysis of metabolites from plant tissue samples. The first chapter consists of a general introduction to the technique of MALDI-MSI, and the sixth and final chapter provides a brief summary and an outlook on future work.

The second chapter (following a general introduction) illustrates how MALDI-MSI of metabolites can be combined with genomics knowledge to infer functional genomic information. Wild-type Arabidopsis flower petals were investigated for their content of three flavonoids (kaempferol, quercetin, and isorhamnetin) and their glucosides, and compared to a well-understood flavonoid mutant (tt7). Spatially non-uniform accumulation/depletion of flavonoids is observed between the wild-type and mutant, and metabolite abundances are compared to infer information about localized gene expression.

The third chapter demonstrates a novel small molecule application for a matrix previously used for MALDI-MSI of lipids (1,5-diaminonaphthalene, DAN). DAN was compared to other common MALDI matrices for its efficiency in ionization of several plant metabolite standards, and was found to be comparable or superior for all tested metabolites. DAN was then applied to image a range of classes of metabolites from a cross-section of corn leaf.

The fourth chapter presents a novel acquisition method, in which high-mass resolution and tandem MS scans are acquired in alternating polarities during a single MALDI-MSI experiment. This methodology yields highly information-rich datasets, allowing for the generation of images from either high-resolution, accurate mass measurements or specific MS/MS or MSn transitions in both polarities, greatly expanding the number of detectable metabolites.

The fifth chapter describes modifications made to the instrument laser optics to significantly enhance the achievable spatial resolution for MALDI-MSI on the system. A combination beam expander/spatial filter and aspheric focusing optics are used to reduce the laser spot size at the sample surface. This high-resolution configuration is then used to image metabolites from a corn leaf cross-section at 5 µm resolution, revealing single-cell metabolite localizations within the tissue.