Grand Luminy Technopole 1 article published in JoVE Biology Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT) Vincent Rouger1,2,3, Nicolas Bertaux4,5,6, Tomasz Trombik1,2,3, Sébastien Mailfert1,2,3, Cyrille Billaudeau1,2,3, Didier Marguet1,2,3, Arnauld Sergé1,2,3 1Institut National de la Santé et de la Recherche Médicale, UMR 631, Parc scientifique de Luminy, 2Centre National de la Recherche Scientifique, UMR 6102, Parc scientifique de Luminy, 3Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, 4École Centrale Marseille, Technopôle de Château-Gombert, 5Institut Fresnel, Aix-Marseille University, 6Centre National de la Recherche Scientifique, UMR 6133, Aix-Marseille University Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.