Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells

Significance Multichannel single-molecule tracking is a powerful tool to probe molecular interactions with high spatial-temporal resolution. Current methods have at least two major limitations: the lack of high-performance intracellular single-molecule fluorescence probes and the difficulty of multispecies single-molecule labelling. Here, we employ a nanopore-electroporation technique to deliver organic dye-labelled proteins into living cells to achieve multi-color single molecule tracking. These organic dyes have better optical and chemical properties than the probes currently used. Therefore, we significantly expand the selection of intracellular single-molecule probes to widely available dyes. We demonstrate the application of this technique by revealing detailed spatial resolution and dynamics of membrane EGFR signaling transductions, which will shed light on the understanding of its fundamental mechanism.