Dataset to "Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters"

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.