This letter demonstrates the use of infrared matrix-assisted laser desorption/ionization coupled with microscope mode mass spectrometry imaging. It is aimed to explore the use of intrinsic water in tissue as a matrix for imaging at spatial resolutions below the diffraction limit of the employed IR optics. Stigmatic ion optics with a magnification factor of ~70 were used to project the spatial distribution of produced ions onto a detector while separating ions with different mass-to-charge ratios using a time-of-flight mass spectrometer. A pixelated detector was used to simultaneously record arrival time and impact position. A previously described dried-droplet sample system of 2,5-dihydroxybenzoic acid (DHB) and 5 peptides covered by a copper grid for defined surface structure was used to benchmark the light- and ion-optical setup for spatial resolution and mass spectrometric performance. A spatial resolving power of 9.8 μm, well below the optical limit of diffraction (14 μm for the given setup), was established. After, frozen cryo-sections from a biological model system were measured by exploiting the endogenous water content as a matrix. Principal component analysis enabled a clear distinction between distinct tissue regions identified by both light microscopy and MS imaging.