Isaac T. S. Li

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.