Location, dynamics and solvent relaxation of a nile red-based phase-sensitive fluorescent membrane probe

•The fluorophore in NR12S is localized at the membrane interface.•NR12S displays phase-sensitive REES in membranes.•Fluorescence properties of NR12S are dependent on the membrane phase state.•NR12S appears to be a promising probe to explore membrane organization in model and biological membranes.

Fluorescent membrane probes offer the advantage of high sensitivity, suitable time resolution, and multiplicity of measurable parameters, and provide useful information on model and cell membranes. In this paper, we have explored the location, dynamics, and solvent relaxation characteristics of a novel Nile Red-based phase-sensitive probe (NR12S). Unlike Nile Red, NR12S enjoys unique orientation and location in the membrane, and is localized exclusively in the outer leaflet of the membrane bilayer. By analysis of membrane depth using the parallax approach, we show that the fluorescent group in NR12S is localized at the membrane interface, a region characterized by slow solvent relaxation. Our results show that NR12S exhibits REES (red edge excitation shift), consistent with its interfacial localization. More interestingly, REES of NR12S displays sensitivity to the membrane phase. In addition, fluorescence emission maximum, anisotropy, and lifetime of NR12S are dependent on the membrane phase. We envision that NR12S may prove to be a useful probe in future studies of complex natural membranes.