Accelerated Left-Handed DNA-PAINT Using Fluorogenic Probes.

Single-molecule localization microscopy (SMLM) techniques offer nanometer-scale resolution by stochastically localizing individual fluorescent molecules. Among these, DNA-mediated point accumulation for imaging in nanoscale topography (DNA-PAINT) stands out for its high multiplexing capability and excellent localization accuracy. In this work, we introduce a fast method using left-handed DNA (L-DNA) probes that show a lower background in DNA-PAINT experiments. The use of L-DNA reduces the background originating from unspecific imager and antibody binding. When fluorogenic probes are employed, emission from free binders is quenched, opening access to volumetric DNA-PAINT. Additionally, like fluorogenic right-handed (R-DNA-)PAINT, fluorogenic L-DNA-PAINT allows for lower integration times and faster accumulation of localizations. The binding kinetics, brightness, and localization precision of the fluorogenic R- and L-DNA probes are indistinguishable. Furthermore, fluorogenic left- and right-handed probes do not show cross-reactivity, thus expanding the range of available sequences for reliable multiplexed imaging. Integrating L-DNA-PAINT with a fluorogenic probe design significantly expands the experimental versatility of DNA-PAINT.