Cytosolic delivery of cell-impermeable fluorescent probes by mixtures of cell-penetrating peptides for multicolor long-term live-cell nanoscopy

Because of their excellent photostability and brightness, organic fluorophores are becoming increasingly attractive for long-term live-cell superresolution imaging of subcellular structures but often suffer from low cell permeability. Here, we develop a general strategy to deliver cell-impermeable organic fluorescent probes, including various SNAP/Halo tag probes, into live cells with excellent delivery efficiency (up to 97% labeling) and almost 100% endo/lysosomal release by simple coincubation with a novel type of peptide vehicle, mixLPV, a mixture of two cell-penetrating peptides (CPPs). Using a mixLPV-mediated labeling system, we obtained multicolor live-cell superresolution images of diverse subcellular structures with high specificity, brightness, and photostability, which allowed us to track their dynamics for up to 1,000 time points and to reveal diverse dynamic interactions between them. Our results not only greatly expand the applications of nanoscopy in various live-cell dynamic studies but also provide a novel concept for the design of CPP-mediated delivery systems.