Monitoring Cell Membrane Hydration Using a Fluorescent Probe Sensitive to Trace Water

The interaction between the cell membrane and water plays an important role in maintaining membrane structure and function. However, direct observation of cell membrane hydration has remained challenging due to the lack of specific and sensitive fluorescent probes for trace water in the cell membrane. Here, we present RMDR (rhodamine-derivatized membrane deep red), a membrane-localized fluorescent probe that reports the membrane hydration through polarity-dependent changes in fluorescence lifetime. RMDR displays clear lifetime decreases with increasing local water content in the range of 0%-1%. Using RMDR, we visualized the increase of cell membrane hydration upon hypotonic stimulation and identified a spatial-temporal gradient change from the periphery to the center in cell clusters. In addition, the increased membrane hydration was also observed during cuproptosis and other lipid peroxidation processes. These findings demonstrate that RMDR enables the examination of membrane hydration dynamics in living cells and may facilitate investigations of water-associated processes in physiological and stress-related contexts.