Abstract
MINimal photon FLUXes (MINFLUX) offers nanometer localization precision, with lower fluorophore requirements than for other super-resolution microscopy (SRM) techniques. Nonetheless, low localization probabilities hamper its application, and use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. Here, we devised strategies overcoming these limitations. We systematically studied the blinking properties of far-red and NIR cyanine fluorophores, followed by simulations of MINFLUX localizations, over typical time scales (microsecond to 10 milliseconds), sample and excitation conditions for MINFLUX imaging. We identified fluorophore blinking via photoisomerization and photoreduction as the main cause of localization errors, and that use of balanced redox buffers and repetitive excitation beam scans can suppress such errors. Implementing these strategies, we could demonstrate NIR-MINFLUX imaging with nanometer localization precision, thereby also presenting an overall strategy to design optimal sample and excitation conditions, for MINFLUX imaging and for SRM in general.