Super-photostable organic dye for long-term live-cell single-protein imaging

Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime than conventional organic dyes without the requirement of any anti-photobleaching additives. PF555 is an asymmetric cyanine structure in which, on one side, the indole in the conventional Cyanine-3 is substituted with 3-oxo-quinoline. PF555 provides a powerful tool for long-term live-cell single-molecule imaging, as demonstrated by the imaging of the dynamic single-molecule interactions of the epidermal growth factor receptor with clathrin-coated structures on the plasma membrane of a live cell under physiological conditions.