Elke De Zitter 1 , Daniel Th é di é 2 , Viola Mönkemöller 1 , Siewert Hugelier 1 , Joël Beaudouin 2 , Virgile Adam 2 , Martin Byrdin 2 , Luc Van Meervelt 1 , Peter Dedecker* 1 , Dominique Bourgeois* 2 1 – KU Leuven, Belgium, 2 – Institut de Biologie Structurale, CNRS, Universite Grenoble Alpes, CEA, IBS, 38044 Grenoble, France; Corresponding authors: dominique.bourgeois@ibs.fr, peter.dedecker@kuleuven.be Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort. Main text Fluorescent proteins (FPs) and in particular green-to-red photoconvertible fluorescent proteins (PCFPs) have become indispensable tools for advanced imaging such as single- molecule localization microscopy (SMLM) or single-particle tracking photoactivated localization microscopy (sptPALM). Both techniques are however limited by blinking, a process in which the fluorophores stochastically enter reversible dark states. PCFPs display blinking on multiple timescales, arising from different underlying photochemical processes. 1 Fluorescence intermittencies shorter than the typical exposure times used in these imaging methodologies (~tens of milliseconds), such as caused by intersystem crossing to the triplet state, reduce the apparent brightness of the label. Intermittencies longer than the exposure time, in contrast, can cause severe complications such as multiple counting of target molecules in quantitative SMLM and interruptions of single-molecule tracks in sptPALM. 2 However, the mechanistic origin of long-lived dark states in PCFPs has remained unclear and there is presently no strategy to eliminate these. We set out to investigate the nature of long-lived fluorescence intermittencies in photoconverted (red) mEos4b, 3 one of the latest probes in a series of highly popular green- to-red PCFPs. Individual molecules of mEos4b were immobilized in a polyacrylamide (PAA) matrix, converted to the red emissive state using 405-nm illumination, and the fluorescence emission visualized in time using a sensitive widefield microscope. The single-molecule fluorescence traces (Fig. 1A) displayed reversible and long-lived intermittencies, which we identified as the blinking giving rise to interpretation difficulties in SMLM and sptPALM. Histograms of the intermittency duration (Supplementary Fig. 1) revealed the presence of at least two dark states, as previously reported in mEos2 or Dendra2 4,5 (Supplementary Note 1). While the shorter-lived dark state was insensitive to the intensity of the employed 561-nm illumination (Supplementary Figure 2), the rate at which the longer-lived dark state returned to the emissive state increased with the illumination intensity, reaching a saturation regime at ~0.8 s -1 above a power density of ~ 1.5 kW/cm² (Fig. 1B), indicating a sensitivity to light. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted November 21, 2018. . https://doi.org/10.1101/475939 doi: bioRxiv preprint