Imaging mass spectrometry is becoming a reference technique in the field of lipidomics, due to its ability to map the distribution of hundreds of species in a single run, along a tissue section. The next frontier is now achieving increasing resolution powers to offer cellular (or even sub-cellular) resolution. Thus, the new spectrometers are equipped with sophisticated optical systems to decrease the laser spot to <30 μm. Here, we demonstrate that by using the correct matrix (i.e., a matrix that maximizes ion detection and forms small crystals) and a careful preparation, it is possible to achieve resolutions of ∼5-10 μm, even with spectrometers equipped with non-optimal optics, which produces laser spots of 50 μm or even larger. As a proof of concept, we present images of distributions of lipids, both in positive and negative ion mode, over human colon endoscopic sections, recorded using 2-mercaptobenzothiazole for positive ion mode and 2,5-diaminonaphtalene for negative ion mode and an LTQ-Orbitrap XL, equipped with a matrix-assisted laser desorption ionization (MALDI) source that produces astigmatic laser spots. Graphical Abstract Imaging mass spectrometry is becoming an invaluable technique to complement traditional histology, but still higher resolutions are required. Here we deal with such issue.