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Mass spectrometry imaging (MSI) by laser ablation electrospray ionization (LAESI) enables the lateral mapping of molecular distributions in untreated biological tissues. However, direct sampling and ionization by LAESI–MSI limits the differentiation of isobaric ions (e.g., structural isomers) in a complex sample. Ion mobility separation (IMS) of LAESI-generated species is sufficiently fast to be integrated with the MSI experiments. Here, we present an imaging technique based on a novel combination of LAESI–MSI with IMS that enables in vivo and in situ imaging with enhanced coverage for small metabolites. Ionized molecules produced at each pixel on the tissue were separated by a traveling wave IMS and analyzed by a high performance quadrupole time-of-flight mass spectrometer. Plant (Pelargonium peltatum leaves) and animal tissues (frozen mouse brain sections) were imaged under atmospheric pressure. In …