Fluorescence tracking live-cell plasma membranes via polarity ultra-sensitive non-ionic probes

Small-molecule fluorescent probes provide simple and convenient tools to visualize the cell plasma membrane, offering essential information for cellular-level studies. Most existing plasma membrane probes rely on the incorporation of highly charged moieties and long hydrophobic alkyl chains, and there has been scarce exploration of non-ionic probes that lack highly hydrophobic alkyl substituents. In this work, we exploited (2H)-benzofuran-3-one (3-coumaranone), a scaffold that combines an electron-withdrawing moiety with an active methylene unit, to construct eight D-π-A type fluorescent probes (CMO-1 ∼ CMO-8) with typical intramolecular charge transfer (ICT) characteristics via straightforward condensation and alkylation reactions. Experimental measurements and theoretical calculations consistently show that both the fluorescence intensity and peak position of these probes respond with ultra-high sensitivity to the changes of environmental polarity. Importantly, the representative probe CMO-2 enables wash-free and long-term fluorescence tracking of the plasma membrane and exhibits superior membrane-anchoring compared with the commercial probe DiD. Unlike the most reported membrane probes, this class of probes is non-ionic at physiological pHs, providing useful alternatives for wash-free live-cell plasma membrane imaging.