Single-molecule imaging has revolutionized our ability to visualize biological systems at an unprecedented scale, offering a detailed glimpse into protein dynamics that were once beyond reach. Traditionally, organic fluorescent molecules, quantum dots, and fluorescent proteins have been employed to tag proteins for imaging. However, the quest for an organic fluorophore with high photostability—a property more commonly attributed to quantum dots—has remained elusive. This challenge is precisely what propelled our research team to discover Phoenix Fluor 555 (PF555), a new member of the cyanine family dubbed “oxo-cyanine” for its distinctive 3-oxo-quinoline ring expansion.

Our breakthrough occurred when an unexpected ultralight-stable organic fluorophore emerged through a photoreaction known as photoblueing. Intrigued by this phenomenon, we purified several milligrams of the new compound and determined its structure via mass spectrometry and nuclear magnetic resonance. We christened the molecule PF555, referencing the mythical bird that is reborn from ashes—a perfect metaphor for how this dye arose from a seemingly routine imaging process.

PF555 stands apart for its consistent photobleaching lifetime, whether oxygen is present or absent, rendering it extraordinarily robust for both in vitro and cellular experiments. By applying PF555 to observe membrane proteins such as the epidermal growth factor receptor (EGFR), we discovered diverse single-protein pathways—including a “scanning state” and the ability to monitor the complete receptor internalization and recycling cycle. Where previous fluorophores faltered, PF555 excelled, bridging the gap between fleeting molecular events and our desire to track protein trajectories continuously over time.

The inspiring story behind this discovery, however, goes beyond the molecule itself. Only one student, then a graduate student named Sunhyuk Lee, embraced the arduous challenge of isolating PF555 in pure form, investing over two years of tireless work with five lasers to accumulate the milligrams necessary for structural identification. The process required the same unwavering dedication exemplified by Madame Curie, who famously extracted a mere 0.1 grams of radium from eight tons of ore. Like Madame Curie’s monumental achievement, the emergence of PF555 was made possible by a relentless pursuit of knowledge against significant odds. This spirit of perseverance, fueled by a vision that transcended immediate practicality, ultimately redefined what is possible in the realm of single-molecule imaging.

In recalling the parallel to Madame Curie, our team hopes that this discovery will encourage investigators everywhere to pursue bold ideas that may initially appear distant or impossible. The unprecedented photostability of PF555 not only sets a new benchmark for organic fluorescent materials but also opens opportunities to unveil hidden molecular events, expand our understanding of fundamental processes, and spark breakthroughs in scientific research. Just as Madame Curie’s grit led to transformative insights in physics and chemistry, so too can a fearless approach to innovative, seemingly insurmountable goals pave the way for the next generation of discoveries in molecular biology.