Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking

Abstract

MINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with more relaxed 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, and over typical time scales ({micro}s-10ms), sample and excitation conditions used in MINFLUX imaging. By subsequent simulations of representative MINFLUX localization procedures, we found that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. However, these localization errors 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.