High-spatial resolution mass spectrometry imaging (MSI) is crucial for the mapping of chemical distributions at the cellular and subcellular level. In this work, we improved our previous laser optical system for matrix-assisted laser desorption ionization (MALDI)-MSI, from ~9 μm practical laser spot size to a practical laser spot size of ~4 μm, thereby allowing for 5 μm resolution imaging without oversampling. This is accomplished through a combination of spatial filtering, beam expansion, and reduction of the final focal length. Most importantly, the new laser optics system allows for simple modification of the spot size solely through the interchanging of the beam expander component. Using 10×, 5×, and no beam expander, we could routinely change between ~4, ~7, and ~45 μm laser spot size, in less than 5 min. We applied this multi-resolution MALDI-MSI system to a single maize root tissue section with three different spatial resolutions of 5, 10, and 50 μm and compared the differences in imaging quality and signal sensitivity. We also demonstrated the difference in depth of focus between the optical systems with 10× and 5× beam expanders. Graphical Abstract ᅟ.