Mass spectrometry (MS) imaging (MSI) is an untargeted and label-free chemical imaging method that can map distributions of compounds, such as metabolites, lipids, proteins, and pharmaceutical compounds, in solid samples, such as tissues and cells. Infrared laser ablation ambient MS (IRLA-MS) methods can carry out MSI measurements at atmospheric pressure with minimal sample preparation, which allows straightforward analysis of biological samples in their native state, and which is not feasible with traditional vacuum-based MSI methods that typically require extensive sample preparation and treatment. IRLA-MS methods are best suited for the analysis of small <1500 Da molecules, and they can cover the analysis of small nonpolar, neutral polar, and charged polar molecules depending on the ionization method. IRLA-MS methods with electrospray ionization (e.g., laser ablation electrospray ionization, LAESI) can analyze a wide-range of medium polar, polar, and ionic compounds, whereas IRLA-MS with atmospheric pressure photoionization (i.e., laser ablation atmospheric pressure photoionization, LAAPPI) is suitable for the analysis of nonpolar and polar analytes. However, LAAPPI- and LAESI-MSI have thus far been limited by a relatively poor lateral resolution of 200–400 μm, which has limited the number of structures they can spatially resolve in samples, and which can make them an unappealing choice for biological research. This thesis and its four studies cover the early stage of LAAPPI-MSI research and the development of a novel LAAPPI/LAESI MSI platform for optimized imaging with sub-100 µm lateral resolution. The main objective of the study was to enhance the imaging quality of these IRLA-MS methods for biological research.