Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful technique used to image the distribution of hundreds of small molecules directly from biological samples, without the need of extensive sample preparation. Operated at ambient temperature and in the open-air environment, DESI-MS employs a fine spray of charged droplets to rapidly extract molecules from the sample surface into the solvent droplets and to transfer the analytes to the mass spectrometer. When operated in the imaging mode, a two-dimensional map of the compounds within the sample surface is obtained with a typical spatial resolution of hundreds of micrometers or less. Here, we describe the imaging of complex lipids from a mouse brain tissue section using a histologically compatible solvent system. The imaging experiment described is performed in the negative ion mode using an Orbitrap mass spectrometer for mass analysis. Following DESI-MS imaging, the same tissue section is subjected to histochemistry allowing molecular information to be correlated to histological information.