Super-Resolution Tension PAINT Imaging with a Molecular Beacon

DNA-PAINT enabled super-resolution imaging through the transient binding of fluorescently-labelled single-stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB-PAINT provides localization precision similar to traditional linear imager DNA-PAINT. We also show that MB-PAINT is ideally suited for fast super-resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free-diffusing imagers in traditional DNA-PAINT at the cell-substrate interface.

Automated summary

DNA-PAINT achieves super-resolution imaging by transiently binding fluorescently-labelled single-stranded DNA imagers to target ssDNA. However, background fluorescence from imagers limits its performance, resulting in longer image acquisition and potential artifacts. To overcome this, we designed a molecular beacon (MB) as the PAINT imager, which reduces background fluorescence when unbound and becomes fluorogenic upon binding to target DNA. We demonstrated that MB-PAINT achieves similar localization precision as traditional linear imager DNA-PAINT and is ideal for fast super-resolution imaging of molecular tension probes in living cells, eliminating potential artifacts from free-diffusing imagers at the cell-substrate interface.