DOI: 10.5281/zenodo.5569507
title: Dataset to submitted manuscript "Imaging fluorescence blinking of a mitochondrial localization probe - cellular localization probes turned into multi-functional sensors"

description: <strong>This folder contains all raw data underlying the results presented in a manuscript, submitted to </strong><strong>PNAS</strong><strong>, and entitled:</strong> <strong><em>Imaging fluorescence blinking of a mitochondrial localization probe - cellular localization probes turned into multi-functional sensors</em></strong> <strong>Authored by:</strong> Zhixue Du<sup>a</sup><em>, </em>Joachim Piguet<sup>a,+</sup>, Gleb Baryshnikov<sup>b,+</sup>, Johan Tornmalm<sup>a</sup>, Baris Demirbay<sup>a</sup>, Hans Ågren<sup>b</sup>, Jerker Widengren<sup>a,*</sup> <sup>a</sup> Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova Univ Center 106 91 Stockholm, Sweden <sup>b</sup> Royal Institute of Technology (KTH), Dept Theroretical Chemistry and Biology, Albanova Univ Center 106 91 Stockholm, Sweden <sup>+</sup> Contributed equally * Corresponding author: Email: jwideng@kth.se, Phone: +46-8-7907813 <strong>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. </strong> <strong>ABSTRACT</strong> Mitochondrial membranes and their microenvironments directly influence and reflect cellular metabolic states, but are difficult to probe on site, in live cells. Here, we demonstrate a strategy, showing how the widely used mitochondrial membrane localization fluorophore 10-Nonyl Acridine Orange (NAO) can be transformed into a multi-functional probe of membrane microenvironments, by monitoring its blinking kinetics. By transient state (TRAST) studies of NAO in small unilamellar vesicles (SUVs), together with 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. Here, not only cardiolipin concentration, but also the cardiolipin acyl chain composition was found to strongly influence the NAO blinking kinetics. The blinking kinetics also reflect hydroxyl ion dependent transitions to and from the fluorophore doublet radical, closely coupled to the proton transfer events in the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Following the SUV studies we show by TRAST imaging that the fluorescence blinking properties of NAO can be imaged in live cells, in a spatially resolved manner. Generally, the demonstrated blinking imaging strategy can transform existing fluorophore markers into multi-parametric sensors reflecting conditions of large biological relevance, which are difficult to retrieve by other means. This can significantly extend the possibilities for fundamental membrane studies in lipid vesicles and live cells.