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Phoenix Fluor 555: Ultra-photostable fluorescent molecule reveals hidden dynamics of intracellular proteins

A research team at POSTECH (Pohang University of Science and Technology) has successfully developed a super-photostable organic dye after two years of dedicated research. Their study is in the journal Nature Methods.

Single-molecule imaging, a technique that uses fluorescent markers to track proteins with precision, plays a crucial role in cell biology, biochemistry, molecular biology, and drug discovery.

However, conventional organic fluorophores have been hindered by their low photostability. The issue of photobleaching—the loss of fluorescence upon prolonged light exposure—has made it difficult to track proteins inside cells or monitor intricate biological processes over extended periods.

Professor Sung Ho Ryu's research team at POSTECH made a serendipitous discovery while conducting single-molecule imaging: an ultra-photostable fluorescent molecule that emerged as a result of the photoblueing phenomenon. In collaboration with Professor Young-Tae Chang's team, they identified its structure using mass spectrometry and nuclear magnetic resonance analysis, naming it Phoenix Fluor 555 (PF555).

PF555 offers significantly greater photostability than existing fluorescent dyes, making it highly effective for tracking both individual proteins at the single-molecule level and multiple proteins simultaneously at a bulk level. Notably, PF555 remains unaffected by oxygen concentration and has a long photobleaching lifetime.

Using PF555, the research team was able to observe biological processes that were previously untraceable, including endocytosis and protein interactions. Their findings revealed that epidermal growth factor receptor (EGFR)—a key regulator of cell growth and differentiation—exists in two distinct states: one in which it remains trapped within Clathrin-Coated Structures (CCS) on the cell membrane, and another where it moves around its surroundings.

This suggests that EGFR actively navigates its environment, potentially to detect external signals or facilitate molecular interactions. Thanks to PF555's unparalleled photostability, researchers were able to track the complete process of EGFR's endocytosis and recycling, something that had been challenging with conventional fluorescent dyes.

Professor Sung Ho Ryu said, "PF555 is an ultra-stable organic fluorophore unlike any previously reported. It will allow researchers to observe biological phenomena that were once restricted by time limitations."

Professor Young-Tae Chang added, "The extraordinary stability of PF555 sets a new benchmark for organic fluorophores," emphasizing its broad applications in drug development, disease diagnostics, and cellular imaging.