study aims for a better understanding of underlying mechanisms allowing T.atroviride to actually sense a pathogen. For this we developed an agar-based micro assay to study especially the formation of secondary metabolites (SMs) that play a crucial role in fungal sensing and interaction. Applying MALDI MSI on an 7T FT-ICR instrument we monitor the spatial distribution of known and unknown substances in an interaction zone (almost) not harboring fungal hyphae. By this we aim to identify highly mobile SMs migrating towards the interaction partner resulting in a response in the interaction partners T.atroviride and B.cinerea. Methods Pre-cultures grown at 25°C in petri dishes were transferred on opposite ends of an agar medium deposited on glass or ITO slides. Incubation was performed until growth fronts were 1cm apart (confrontation zone). After quenching (liquid nitrogen) and vacuum drying, matrix deposition was done with a home-built sublimation unit or a TM sprayer (HTX). Extractions of the confrontation zone were done with acidified water/acetonitrile. MALDI MSI of the confrontation zone and direct-infusion ESI was performed on a 7T scimaX FT-ICR-MRMS equipped with an ESI/MALDI dual source (Bruker). Data analysis was done with SCiLS and DataAnalysis 5.0 (Bruker). Preliminary data (results) A confrontation assay of standardized measures was developed for regular glass or ITO slides. This standardization in preparation makes parameters like fungal growth highly reproducible. The miniaturization on commercially available glass slides also allows for samples measurement by MALDI directly after quenching, drying and matrix application using commercial sample holders. The distribution of peptaibols and other metabolites formed by T. atroviride as well as potentially novel metabolites from B.cinerea and T. atroviride were visualized by MALDI MSI in the confrontation zone. The agar is producing high chemical backgrounds for every MSI experiment. To overcome this limitation of our approach we tested different matrices (e.g. 2,5-DHB, CHCA, 1,5-DAN, 3-Hydroxycoumarin, 6-Aza-2-Thiothymine and mixtures thereof) for efficiency and most comprehensive analysis of the analyte signals we can detect. Furthermore, sublimation and spraying for matrix deposition were studied in more detail for agar samples and experiments for derivatization of functional groups were conducted to improve the signal-to-noise ratio of interesting target compounds. To confirm our findings, the agar from the confrontation zone was extracted with a mixture of 50:50 (v:v) ACN:H2O acidified with 0.1% Formic acid or 0.1% Acetic acid. Direct-infusion ESI FT-ICR MS analyses allowed further evaluation of ESI versus MALDI ionization efficiencies of metabolites to assess the potential for detecting metabolites in the presented MALDI MSI setup. Our putative results were then compared with metabolomics outcomes from experiments using isotopically-labeled standards in the same assay to verify the fungal origin of the metabolites.