Counterion-Driven Long-Term Imaging of Cell Membranes and Migrasomes Using Dicationic NIR AIEgens

Tracking migrasome formation and dynamics is challenging due to the limited specificity and stability of existing membrane dyes for long-term imaging. Here, we develop a series of aggregation-induced emission (AIE) probes featuring a dicationic donor-π-acceptor structure (CDPP-2X) with NIR emission for selective long-term membrane and migrasome labeling. Among the synthesized (Z)-4-(4-(1-cyano-2-(4-(diphenylamino)phenyl)vinyl)phenyl)pyridine-1-ium (CDPP) derivatives, CDPP-2PF6 exhibited superior performance, with the PF6 counterion enhancing lipophilicity for dual-mode membrane anchoring via electrostatic and hydrophobic interactions. This mechanism, combined with restricted intramolecular motion, enables strong fluorescence enhancement and up to 3 h of membrane retention. CDPP-2PF6 selectively accumulates in phosphoinositide-rich migrasomes within 5 min, showing a preferential and strong affinity for phosphatidylinositol 4,5-bisphosphate (PIP2), a key lipid involved in migrasome biogenesis and cell migration. Comparative analyses with wheat germ agglutinin (WGA) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) confirm CDPP-2PF6's superior specificity and phospholipid-dependent localization. Pharmacological studies with blebbistatin and methyl-β-cyclodextrin (MβCD) reveal that actin polymerization is essential for migrasome formation. At the same time, lipid microdomain integrity is crucial for both biogenesis and probe localization, as evidenced by the complete loss of fluorescence upon cholesterol depletion. Confocal fluorescence lifetime imaging captures a distinctive fluorescence lifetime shift from 1.8 to 0.9 ns during migrasome formation, enabling real-time membrane remodeling tracking. CDPP-2PF6's sensitivity to phospholipid composition provides insights into lipid remodeling during migrasome formation and cell migration. These features establish CDPP-2PF6 as a robust platform for long-term membrane and migrasome imaging, offering new opportunities to investigate lipid remodeling, cellular signaling, and phospholipid-rich organelles in physiological and pathological processes.