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Summary: Exploring tissue heterogeneity on a single-cell level by imaging mass cytometry (IMC) remains challenging because of its limiting resolution. We previously demonstrated that combining higher resolution fluorescence with IMC data in the analysis pipeline resulted in high-quality single-cell segmentation. Here, we provide a step-by-step workflow of this MATISSE pipeline, including instructions regarding the staining procedure, and the analysis route to generate single-cell data.For complete details on the use and execution of this protocol, please refer to Baars et al., 2021.