Molecular Design Strategies of Fluorescence Probes with Long-Term Membrane Retention for Plasma Membrane Dynamics...

The plasma membrane is central to numerous cellular processes, yet long-term fluorescence imaging has been limited by insufficient probe retention. Here, we report a rational design of naphthalimide-based, phospholipid-mimicking probes that combine hydrophobic alkyl chains with ionic head groups to achieve strong membrane affinity. Among the series, a zwitterionic derivative, Betaine-DNap, exhibited selective and prolonged plasma membrane staining, maintaining localization for extended time-lapse imaging. Structural analyses revealed that molecular length comparable to phospholipids, zwitterionic electrostatics, and balanced hydrophobicity are key parameters for stable retention. Using Betaine-DNap, we visualized plasma membrane dynamics including endocytosis and oxidative stress-induced vesicle release, processes previously difficult to monitor due to probe instability. This work provides a generalizable design principle for long-term plasma membrane probes and establishes Betaine-DNap as a practical tool for studying membrane physiology in living cells.