Dataset to "Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters" doi-10-5281-zenodo-8006451-0 https://doi.org/10.5281/zenodo.8006451 Swedish National Data Service Svensk nationell datatjänst Landing page Dataset to "Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters" doi-10-5281-zenodo-8006451-0 https://doi.org/10.5281/zenodo.8006451 Baris Elin Joachim Jerker Swedish National Data Service Svensk nationell datatjänst Landing page This folder contains all raw data underlying the results presented in a manuscript, submitted for publication to Journal of Physical Chemistry B, and entitled: Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters Authored by: B. Demirbay‡, E. Sandberg‡, J. Piguet, J. Widengren* Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova University 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 Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, is a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level, as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept, and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time-resolution and is thus broadly applicable on different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional, useful strategy for fluorescence-based multiplexing, implementable on a broad range of both stationary and moving samples. Access to data through an external actor. Access to data is restricted. Åtkomst till data via extern aktör. Tillgång till data är begränsad. restrictedAccess