Excited state Relaxation Activation Energy (ESRAct) of Di-4-ANEPPDHQ Maps Nanoscale Molecular Organization in Biomembranes

Live cell plasma membranes show spatially heterogeneous liquid-ordered (Lo)-like and liquid-disordered (Ld)-like regions similar to the co-existing Lo/Ld phases observed in lipid vesicles. The Lo-like regions are relatively less hydrated and less polar due to tight packing of the membrane components compared to the Ld-like regions. The steady-state fluorescence spectra of Di-4-ANEPPDHQ (Di-4), a widely used polarity-sensitive probe, is blue or red shifted when solvated in less polar (Ld- like) or more polar (Lo-like) regions respectively. However, quantification of Di-4 fluorescence in blue and red channels for the evaluation of membrane phase state suffers from the lack of specific wavelength choice for these two channels and Di-4s relatively higher concentration in Ld phase (red channel) due to its partitioning preference. To address these issues, we employed fluorescence lifetime of Di-4, a concentration independent photophysical parameter, to understand membrane biophysical properties. The fluorescence lifetime of Di-4 in lipid vesicles exhibits Arrhenius-like temperature dependence. Centred around this energetic feature of Di-4 photophysics, we developed a novel analytical module, namely excited state relaxation activation energy (ESRAct), that serves as an intrinsic descriptor of the membrane nano-environment sensed by this probe. We show that the ESRAact value scales with increasing disorder in nanoscale phase separation (i.e., ESRAct of pure Ld > mixed Ld/Lo > pure Lo phase). We then extended its applications to giant plasma membrane vesicles (GPMVs) isolated from MCF-7 cells and found that these vesicles exhibit nanoscale Lo/Ld co-existing phase within 16-37{degrees}C. We envisage wide applications of ESRAct to delineate plasma membrane phase behavior as well as general photophysical studies on other newly designed polarity-sensitive probes.