This folder contains all raw data underlying the results presented in a manuscript, submitted to Small, and entitled: Imaging Fluorescence Blinking of a Mitochondrial Localization Probe – a Strategy Combining Site-Specificity with Multi-Parametric Sensing of Cellular Microenvironments Authored by: Zhixue Dua, Joachim Pigueta,+, Gleb Baryshnikovb,+, Johan Tornmalma, Baris Demirbaya, Hans Ågrenb, Jerker Widengrena,* a Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova Univ Center 106 91 Stockholm, Sweden b Royal Institute of Technology (KTH), Dept Theroretical Chemistry and Biology, Albanova Univ Center 106 91 Stockholm, Sweden + Contributed equally * Corresponding author: Email: jwideng@kth.se, Phone: +46-8-7907813 The data files are grouped into the different techniques used to generate them, and refer to the figures/tables in the manuscript where the extracted results are presented. ABSTRACT The local microenvironment of mitochondrial membranes directly influences cellular metabolic states but are difficult to follow locally. Here, we demonstrate a robust and straightforward strategy, transforming the widely used mitochondrial membrane localization fluorophore 10-Nonyl Acridine Orange (NAO) into a multi-functional probe of membrane microenvironments. By monitoring the blinking kinetics of NAO in small unilamellar vesicles, and by computational simulations, we found that NAO exhibits prominent reversible singlet-triplet state transitions and can act as a light-induced Lewis acid forming a red-emissive doublet radical. The resulting blinking kinetics are highly environment sensitive, specifically reflecting local membrane oxygen concentrations, redox conditions, membrane charge, fluidity and lipid compositions, and can also be imaged in live cells, in a spatially resolved manner. They also reflect hydroxyl ion dependent transitions to and from the fluorophore doublet radical, closely coupled to proton transfer events in the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Generally, by the demonstrated blinking imaging strategy existing fluorophore markers can be transformed into multi-parametric microenvironmental sensors. The strategy makes it possible to image local cellular conditions highly relevant to cancer, metabolic and infectious diseases, thereby providing a basis for cellular diagnostics and for fundamental membrane studies.