Precise elucidation of dynamic chemical events at the cell membrane interface is vital for understanding fundamental biological processes. However, current fluorescent tools often fail to confine signal generation strictly to the membrane, leading to background interference that compromises imaging fidelity. To address this, we propose an "Interfacial Activation" strategy that transforms the cell membrane from a passive anchoring point to an active signal trigger. This is realized using NACP, an amphiphilic two-photon fluorophore that remains completely nonemissive in aqueous media via aggregation-caused quenching (ACQ) but displays dramatic fluorescence enhancement upon specific insertion into the lipid bilayer. Leveraging this mechanism, we engineered a dual-gated probe, NACP-DBS, for the high-fidelity detection of transmembrane hydrogen sulfide (H2S). The probe features precise outer-membrane localization, spatially isolating it from intracellular thiol interference. NACP-DBS exhibits exceptional analytical performance, including near-infrared emission (643 nm), a large two-photon absorption cross-section (287 GM), and nanomolar sensitivity (LOD = 6.7 nM). We successfully applied the probe to monitor endogenous H2S dynamics in living cells, mouse inflammation models, and deep tissue slices, achieving high-contrast, wash-free imaging. The "Interfacial Activation" strategy establishes a design paradigm for next-generation high-fidelity membrane sensing tools, facilitating in-depth investigations of transmembrane signal transduction.
He et al. (Wed,) studied this question.