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The imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF−SIMS) and MALDI-MS sample preparation methods were combined. We used this method, named matrix-enhanced (ME) SIMS, for direct molecular imaging of nervous tissue at micrometer spatial resolution. Cryosections of the cerebral ganglia of the freshwater snail Lymnaea stagnalis were placed on indium−tin−oxide (ITO)-coated conductive glass slides and covered with a thin layer of 2,5-dihydroxybenzoic acid by electrospray deposition. High-resolution molecular ion maps of cholesterol and the neuropeptide APGWamide were constructed. APGWamide was predominantly localized in the cluster of neurons that regulate male copulation behavior of Lymnaea. ME-SIMS imaging allows direct molecule-specific imaging from tissue sections without labeling and opens a complementary mass window (<2500 Da) to MALDI imaging …