The photophysics of a Rhodamine head labeled phospholipid in the identification and characterization of membrane lipid phases

► Rhod-DOPE fluorescence properties are strongly sensitive to distinct lipid phases. ► Rhod-DOPE forms probe-rich clusters in gel and lo phases. ► Probe-rich clusters result in efficient fluorescence self-quenching processes. ► lo unique environment is underneath Rhod-DOPE enhanced fluorescence self-quenching. The organization of lipids and proteins into domains in cell membranes is currently an established subject within biomembrane research. Fluorescent probes have been used to detect and characterize these membrane lateral heterogeneities. However, a comprehensive understanding of the link between the probes’ fluorescence features and membrane lateral organization can only be achieved if their photophysical properties are thoroughly defined. In this work, a systematic characterization of N-(lyssamine Rhodamine B sulfonyl)-1,2-dioleoyl-sn-3-phosphatidylehanolamine (Rhod-DOPE) absorption and fluorescence behavior in gel, liquid-ordered (lo) and liquid-disordered (ld) model membranes was performed. In agreement with a previous study, it was found that Rhod-DOPE fluorescence lifetimes present a strong sensitivity to lipid phases, becoming significantly shorter in lo membranes as the probe membrane concentration increases. The sensitivity of Rhod-DOPE absorption and fluorescence properties to the membrane phase was further explored. In particular, the fluorescence lifetime sensitivity was shown to be a consequence of the enhanced Rhod-DOPE fluorescence dynamic self-quenching, due to the formation of probe-rich membrane domains in these condensed phases that cannot be considered as typical probe aggregates, as excitonic interaction is not observed. The highly efficient dynamic self-quenching was shown to be specific to lo phases, pointing to an important effect of membrane dipole potential in this process. Altogether, this work establishes how to use Rhod-DOPE fluorescence properties in the study of membrane lipid lateral heterogeneities, in particular cholesterol-enriched lipid rafts.