DNA-PAINT single-particle tracking (DNA-PAINT-SPT) enables extended single-molecule studies of membrane protein interactions

Abstract

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in-vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-colour DNA-PAINT-SPT for quantifying protein dimerisation and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

Long-term single molecule localization microscopy uncovers dynamic co-assembly of Lrp6 and Ror2 into Wnt-signalosomes

“…Strikingly, we achieved video-rate rbTALM imaging in living cells at up to three channels with no significant loss in labeling density and uncompromised tracking fidelity during acquisition at video rate over up to 90 min. The rbTALM imaging technique is conceptionally related to live-cell PAINT approaches based on transient labeling of peptide/protein-subdomain [52][53][54][55] or DNA/RNA [56][57][58] binding pairs. However, the binders used for rbTALM were engineered for longer lifetimes and higher affinity to achieve not only extended single-molecule trajectories, but also higher degree of labeling required for effective co-localization analyses.…”

The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

“…The K2 TIRF microscope can be used for a wide range of live-cell and single-molecule experiments [45]. Fig.…”