The Molecular Mechanism of Fluorescence Lifetime of Fluorescent Probes in Cell Membranes.

Fluorescence probes play crucial roles in unraveling the structure and dynamics of cell membranes including membrane fluidity, polarity, and lipid molecule ordering. The fluorescence lifetime of probes describes the average duration of time that a fluorescent molecule remains in an excited state before returning to the ground state, which is sensitive to environmental changes. However, the molecular mechanism and inherent properties to determine the fluorescence lifetimes remain unexplored and inadequately studied. Furthermore, the effects of the probe on the membrane are also unclear. In this study, we investigated the interactions between probes and lipids, as well as the structural properties of probes within the outer and inner membrane of () by combining molecular dynamics (MD) simulations, enhanced sampling methods, fluorescence lifetime imaging microscopy (FLIM), and time-correlated single photon counting (TCSPC). The results show that even though the probes have very little effect on the membrane lipids, different membrane environments significantly affect the fluorescence lifetime of the probes. The analysis based on the all-atom simulations shows a strong correlation between the probe's immersion depth within the membrane and its fluorescence lifetime. Specifically, probes buried in the membrane environment shielded from rapid water molecule collisions exhibit longer fluorescence lifetimes. The molecular basis of the fluorescence lifetime of probes in cell membranes revealed in this work would enhance the comprehension of fluorescence probes and facilitate the rational design of novel efficient probes.