1,3‐Dicarbonyl Rhodamines for Live‐Cell Single‐Molecule Super‐Resolution Imaging

Abstract

ABSTRACT 1,3‐Dicarbonyl compounds, with their diverse and intriguing chemical properties, have found broad applications in fields such as organic synthesis, pharmaceuticals, luminescent materials, and personal care products. Here, we integrated the 1,3‐dicarbonyl scaffold with rhodamine‐based fluorophores to construct a series of 1,3‐dicarbonyl–rhodamine derivatives. By exploiting the intrinsic thermodynamic and photochemical properties of 1,3‐dicarbonyl compounds, these rhodamine derivatives enable sparse localization, tunable emitter density, and a self‐triggered photooxidation cascade under single‐ or dual‐laser irradiation. These features render them suitable for single‐molecule localization microscopy (SMLM). Using click chemistry, HaloTag labeling, and phospholipid‐targeting groups, we achieved super‐resolution imaging and dynamic tracking of actin, the endoplasmic reticulum, mitochondria, and the plasma membrane in live cells. Notably, under low laser intensity, a rich repertoire of filopodia dynamics was resolved in live cells over an extended 20 min time course (encompassing 120,000 total frames). The imaging achieved a spatial localization accuracy of 22 nm and a temporal resolution of 20 s, enabling high‐fidelity tracking of dynamic cellular protrusions.