Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking

This upload contains raw data and simulation software underlying the results presented in a manuscript submitted for peer-review, with the title:   Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking   Authored by: C Venugopal Srambickal1,*, H Esmaeeli1,*, J Piguet1, L Reinkensmeier2, R Siegmund2, M Bates2, A Egner2, J Widengren1,** 1 Experimental Biomolecular Physics, Bio-Opto-Nano Unit, Department of Applied Physics, Royal Institute of Technology, SE-10691 Stockholm, Sweden 2 Department of Optical Nanoscopy, Institute for Nanophotonics, D-37077 Göttingen, Germany * Contributed equally ** Corresponding author: jwideng@kth.se   ABSTRACT MINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with lower fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, over typical time scales (µs-10ms), sample and excitation conditions used in MINFLUX imaging. Subsequent simulations of representative MINFLUX localization procedures showed that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. These localization errors, however, could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies. Acknowledgements: This study was supported by the European Union's Horizon 2020 research and innovation program under grant agreement 101017180 (NanoVIB).   Files with raw data on which the manuscript is based are grouped into folders according to the figures/tables in the manuscript where the extracted results are presented. Additionally, software developed and used for the simulations are arranged into a separate folder.