Time‐Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time‐resolved fluorescence anisotropy imaging (TR‐FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non‐equivalent viscosity‐related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip‐and‐rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano‐environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano‐environments in complex, biological systems. Fluorescent molecular rotors (FMRs, viscosity‐dependent fluorescence lifetime probes) are combined with time‐resolved fluorescence anisotropy imaging for novel multiplex viscosity imaging in model and live cell lipid droplets. All‐atom molecular dynamics simulations identify two FMR tilt states which are connected to extracted viscosity parameters, namely FMR lifetime and rotational correlation time.