Datensatz

Zusammenfassung

DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled "imager" and complementary target-bound "docking" strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA PAINT, as the blinking rate of probes is limited by an upper bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.