Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (F?rsterresonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairsof short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating differentFRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strandsresults in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINTby designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origamistructures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, howeverensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor andacceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. Thecombination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction withdistance-sensitive readout in the 1 to 10 nm range.
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