Tunable fluorogenic DNA probes drive fast and high-resolution single-molecule fluorescence imaging.

A main limitation of single-molecule fluorescence (SMF) measurements is the 'high concentration barrier', describing the maximum concentration of fluorescent species tolerable for sufficient signal-to-noise ratio. To address this barrier in several SMF applications, we design fluorogenic probes based on short single-stranded DNAs, fluorescing only upon hybridizing to their complementary target sequence. We engineer the quenching efficiency and fluorescence enhancement upon duplex formation through screening several fluorophore-quencher combinations, label lengths, and sequence motifs, which we utilize as tuning screws to adapt our labels to different experimental designs. Using these fluorogenic probes, we can perform SMF experiments at concentrations of 10 μM fluorescent labels; this concentration is 100-fold higher than the operational limit for standard TIRF experiments. We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6-nt-long imager; through the faster acquisition of binding events, the imaging of viral genome segments could be sped up significantly to achieve extraction of 20-nm structural features with only ∼150 s of imaging. The exceptional tunability of our probe design will overcome concentration barriers in SMF experiments and unlock new possibilities in super-resolution imaging, molecular tracking, and single-molecule fluorescence energy transfer (smFRET).