Super-resolution paint imaging with molecular beacon.

Abstract

DNA-PAINT offers simple super-resolution imaging through the transient binding of fluorescently-labelled DNA imagers to target molecules. It has many advantages, including super-multiplexing capability and robustness against photobleaching. However, the unbound fluorescent imagers in the solution can cause high fluorescence background and place constraints on the imaging conditions that limit the technique's potential, such as low imager concentration, thin TIRF illumination, and long exposure time. In this study, we developed a new strategy to improve PAINT imaging using a fluorogenic molecular beacon (MB) as the imager. MB-PAINT eliminates false localizations from nonspecific surface adsorption and fluorescence background at concentrations far exceeding conventional linear imagers. Furthermore, MB-PAINT achieves similar nanometer localization precision to conventional DNA-PAINT. Lastly, we demonstrate that MB-PAINT is ideally suited for super-resolution tension imaging in living cells even at high frame rates, suppressing the potential of artifacts from free-diffusing and spontaneous binding of imagers at the cell-substrate interface.