Regioisomer‐Tuned Rhodamine Spirolactone‐Zwitterion Equilibrium for Fluorogenic Probe Design and Super‐Resolution Plasma Membrane Imaging

ABSTRACT Super‐resolution imaging relies on fluorogenic probes with a high signal‐to‐noise ratio (SNR) to deliver accurate fluorescence signals. Rhodamines offer an effective fluorogenic switching mechanism based on a spirolactone‐zwitterion transition, which is regulated by interactions between the meso‐phenyl ring and the xanthene core. Although the 5‐ and 6‐positions of the meso‐phenyl ring are critical for attaching targeting groups and tuning fluorogenicity, the corresponding regioisomers are difficult to separate and have not been systematically studied as pure compounds. To address this, we introduced alkyl chains of varying lengths at these positions, which not only enabled chromatographic separation of the isomers but also enhanced their affinity for the plasma membrane. In aqueous solution, both isomers formed non‐fluorescent aggregates. The 6‐isomer existed predominantly in the zwitterionic form, whereas the 5‐isomer adopted a mixture of zwitterion and non‐fluorescent spirolactone. The spirolactone form in the 5‐isomer contributed to exceptionally low background fluorescence. Upon binding to membranes, the aggregates disassemble, and the probes insert via their alkyl chains, yielding high‐SNR super‐resolution images of plasma membranes in bacteria, fungi, mammalian cells, and live zebrafish. Leveraging this mechanism, the hexadecyl‐substituted 5‐isomer ( R16‐5 ) delivered the highest SNR and enabled nanoscale visualization of membrane dynamics, including filopodia extension and vesicle transport.