Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful tool for imaging lipids, proteins, N-glycans, metabolites, and drugs in situ. A key step in MALDI MSI is matrix deposition, which must balance the extraction of molecules from the surface of the tissue without inducing excessive analyte delocalization. Further, MALDI MSI spatial resolution is limited by the size of the matrix crystals formed during matrix deposition. Minimizing crystal size is paramount to obtaining detailed images and refined data. Sublimation has been shown to be a reliable method of depositing matrix while minimizing crystal size. Since sublimation does not use any solvents to deposit the matrix, it is also theorized to minimize analyte delocalization. However, these same solvents are thought to aid in analyte extraction, which may limit detection by sublimation. In this study, we compared crystal size, analyte delocalization, and signal intensity differences when using a commercial automatic pneumatic sprayer and a sublimator for lipid imaging analyses. We tested three matrices: α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxyacetophenone (DHAP), and 2,5-dihydroxybenzoic acid (DHB) on mouse brain, heart, and kidney. Our results revealed that crystal size depended upon the matrix and whether the crystals formed on or off tissue. Our data also showed that only a subset of the lipid spectrum was susceptible to increased delocalization from spraying and decreased signal intensity from sublimation.