The Molecular\nMechanism of Fluorescence Lifetime of\nFluorescent Probes in Cell Membranes

Fluorescence probes play crucial roles in unraveling\nthe structure\nand dynamics of cell membranes including membrane fluidity, polarity,\nand lipid molecule ordering. The fluorescence lifetime of probes describes\nthe average duration of time that a fluorescent molecule remains in\nan excited state before returning to the ground state, which is sensitive\nto environmental changes. However, the molecular mechanism and inherent\nproperties to determine the fluorescence lifetimes remain unexplored\nand inadequately studied. Furthermore, the effects of the probe on\nthe membrane are also unclear. In this study, we investigated the\ninteractions between probes and lipids, as well as the structural\nproperties of probes within the outer and inner membrane of <i>Mycobacterium smegmatis</i> (<i>Msm</i>) by combining\nmolecular dynamics (MD) simulations, enhanced sampling methods, fluorescence\nlifetime imaging microscopy (FLIM), and time-correlated single photon\ncounting (TCSPC). The results show that even though the probes have\nvery little effect on the membrane lipids, different membrane environments\nsignificantly affect the fluorescence lifetime of the probes. The\nanalysis based on the all-atom simulations shows a strong correlation\nbetween the probe’s immersion depth within the membrane and\nits fluorescence lifetime. Specifically, probes buried in the membrane\nenvironment shielded from rapid water molecule collisions exhibit\nlonger fluorescence lifetimes. The molecular basis of the fluorescence\nlifetime of probes in cell membranes revealed in this work would enhance\nthe comprehension of fluorescence probes and facilitate the rational\ndesign of novel efficient probes.