Cyanobacteria are among the most ancient forms of life, yet they are known to synthesize highly sophisticated defense molecules, such as the highly hepatotoxic cyclic peptides microcystins and nodularins produced by the genera Microcystis, Anabaena and Nodularia. These metabolites are released by cyanobacteria to water environments causing episodes of fatalities among animals and humans. To better understand the releasing of these metabolites, imaging mass spectrometry (IMS) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was herein applied to determine the spatial distribution of such toxins directly on agar-based cultures. Other key metabolites such as aeruginosin 602 and the siderophore anachelin were also mapped in mixed cyanobacterial cultures, showing the great potential of IMS to spatially monitor the biochemical details of cyanobacterial defense and interactions. Keywords: cyanobacteria, MALDI-TOF, mass spectrometry, imaging IntroductionCyanobacteria are believed to represent one of the most ancient yet highly sophisticated forms of life 1 and these gram-negative oxygenic photosynthetic autotrophic microorganisms are adapted to various ecological habitats such as fresh water, marine, brackish, glaciers, and terrestrial environments.2 Fresh and brackish water cyanobacteria are known to produce a diversity of highly elaborated secondary metabolites, with a truly fascinating variety of structures that exhibit a broad range of applications in the food, nutritional, cosmetic, pharmaceutical and nutraceutical industries. Bloom-forming cyanobacteria (Figure 1), for instance, such as Microcystis aeruginosa, Anabaena cylindrica and Nodularia harveyana, are known to produce a variety of bioactive metabolites.Among these metabolites, cyclic and linear peptides such as microcystin, nodularin, aeruginosin, anabaenopeptin, cyanopeptolin, microginin, cyclamides and microviridin Braz. Chem. Soc. 522 are found, which show activity against various proteases or protein phosphatases. 4 Most of these potent and "dangerous" peptides are released by the cyanobacteria cells into the environment and numerous cases of cyanobacteria poisoning in animals and humans have been reported worldwide.5 Some cyanobacteria also produce iron chelators known as siderophores such as anachelin, schizokinen and synechobactin. 6 There is an interesting behavior of these metabolites, among which the cyclic peptides microcystins and nodularins are such that they are retained inside the cell membrane, whereas anachelin finds its way out in the form of a siderophore, which passes through the membrane into and out of the cell.Mass spectrometry (MS) and mainly matrixassisted laser desorption ionization-mass spectrometry (MALDI-MS) Imaging mass spectrometry (IMS) that incorporates spatial distribution is an emerging and promising new technology for metabolomics MS analysis. This methodology was introduced in 1997 by Caprioli et al. 12 and mostly applied to create 2D images based on the spatial distribution of pe...