Rational Design of an Amphiphilic Membrane-Anchored Fluorescent Probe for Weakly Alkaline pH Bioimaging

Intracellular membrane structures establish highly heterogeneous microenvironments that are essential for regulating organelle function and intracellular signaling. Among the physicochemical parameters governing these processes, weakly alkaline pH microdomains have remained difficult to interrogate owing to the lack of fluorescent probes that combine high sensitivity, selectivity, and compatibility with biological imaging. Here, we report CAlkM, a membrane-anchored fluorescent probe engineered for imaging weakly alkaline intracellular environments. Through rational modulation of the coumarin scaffold, CAlkM exhibits a pK_a of 8.24 and shows a sensitive fluorescence response across the biologically relevant weakly alkaline pH range of 7.0-9.0. The probe enables selective detection of weakly alkaline conditions in aqueous systems and can be readily integrated into gel- and paper-based platforms for portable pH sensing. In living cells, stimulated emission depletion (STED) microscopy and molecular docking analysis indicate that CAlkM can localize in intracellular membrane-associated regions through a membrane-anchoring mode, including organelle-associated membranous and vesicle-like structures, enabling high-resolution in situ visualization of membrane-related local weakly alkaline pH changes. Furthermore, CAlkM displays clear pH-dependent fluorescence responses in zebrafish larvae, demonstrating its applicability for pH-responsive fluorescence imaging in living organisms. Together, these results establish a molecular design strategy for membrane-anchored weakly alkaline pH sensing and provide a versatile platform for investigating pH-regulated physiological and pathological processes across complex biological systems.