Reductively Caged, Photoactivatable DNA-PAINT for High-Throughput Super-resolution Microscopy

Abstract

In DNA points accumulation in nanoscale topography (DNA-PAINT), capable of single-molecule localization microscopy with sub-10-nm resolution, the high background stemming from the unbound fluorescent probes in solution limits the imaging speed and throughput. Herein, we reductively cage the fluorescent DNA probes conjugated with a cyanine dye to hydrocyanine, acting as a photoactivatable dark state. The additional dark state from caging lowered the fluorescent background while enabling optically selective activation by total internal reflection (TIR) illumination at 405 nm. These benefits from "reductive caging" helped to increase the localization density or the imaging speed while preserving the image quality. With the aid of high-density analysis, we could further increase the imaging speed of conventional DNA-PAINT by two orders of magnitude, making DNA-PAINT capable of high-throughput super-resolution imaging.

Keywords: DNA-PAINT; cyanine dyes; fluorescent probes; reductive caging; super-resolution microscopy.