All Probes Plasmids (APPs) for multicolor and long-term tracking of single-mRNA translation dynamics.

Live-cell single-mRNA imaging of translation is inherently challenging, demanding precise optimization of multiple imaging components. To simplify these experiments, we developed All Probes Plasmids (APPs)-a panel of plasmids encoding all the necessary probes for imaging at optimized relative expression levels. APPs incorporate widely used translation tags, fluorescent proteins, and mRNA labeling systems, streamlining both multiplexed imaging and reporter immobilization. By cotransfecting just two plasmids-a reporter and an APP-individual translation sites can be visualized in living cells with high signal-to-noise. We demonstrate how APPs facilitate high-fidelity multicolor translation imaging, long-term single-mRNA tracking, and fluorescence correlation spectroscopy to quantify ribosome kinetics. By lowering technical barriers and enhancing experimental flexibility, APPs provide a versatile platform for advancing single-mRNA translation research in living cells.