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

Abstract

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.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization

“…Methanol solutions containing various amounts of glycerol (0-30%) were prepared. Solution viscosities at room temperature (22 °C) were established previously using a rheometric expansion system rheometer (ARES) (Kuimova et al, 2008) (Steinmark et al, 2020). Fluorescein (10 μM) was added to the solutions and fluorescence was measured at room temperature to extract the rotational correlation times as described above.…”

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization

“…Methanol solutions containing various amounts of glycerol (0-40%) were prepared. Solution viscosities at room temperature (22 C) were established previously using a rheometric expansion system rheometer (ARES) (Kuimova et al, 2008;Steinmark et al, 2020). Fluorescein (10 mM) was added to the solutions and fluorescence was measured at room temperature to extract the rotational correlation times as described above.…”

Multichannel Fluorescence Microscopy: Advantages of Going beyond a Single Emission

“…[ 127 ] More recently, FPA has been used to obtain a more accurate description of nanoregions within nonisotropic, heterogeneous systems environment, such as lipid droplet membranes, going beyond the concept of macroscopic viscosity. [ 128 ]…”