Using Dark Dyes for Fast Super-Resolution Imaging: A Proof-of-Concept Study.

DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) exploits the transient hybridization of a short fluorescent DNA imager strand to a complementary docking strand to achieve super-resolution imaging. However, its application is hampered by the background noise caused by unbound imager strands and the subsequent slow image acquisition. Several variants of DNA-PAINT have already been developed to successfully overcome these limitations. Here we design and validate a new alternative, based on intermolecular dark resonance energy transfer (DRET) and the use of a fluorenyl nucleobase (X) substitute incorporated into the imager strand, acting as a dark donor. Upon annealing, the fluorescence of the bright acceptor (ATTO 647N) bound to the docking strand is turned on via DRET with X. Single-molecule experiments showed that an 11 nt X-labeled imager strand provides appropriate hybridization rates with the ATTO 647N-labeled docking strand to perform DNA-PAINT. Thanks to their low background, high concentrations of imager strands can be used, facilitating the acquisition of super-resolved images of cellular microtubules within 30 s. This study provides the first proof-of-concept for this novel route to fast DNA-PAINT nanoscopy.