Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors

Abstract

In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.

A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress

“…In this work, we utilized BODIPY‐C 10 17, 18 (Figure 1), a fluorophore that belongs to a group of dyes termed ‘molecular rotors’ that have viscosity‐dependent fluorescence quantum yields, lifetimes,19, 20 and depolarization 21, 22. When combined with fluorescence lifetime imaging microscopy (FLIM), molecular rotors can be used to obtain spatially resolved viscosity maps of microscopic objects,17, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 as well as to observe dynamic change in viscosity during relevant processes of interest 37, 39, 41, 42. Thus, we aimed to use BODIPY‐C 10 , which is known to completely embed into the fluid‐phase lipid bilayers40 to directly examine how photooxidation during PDT affects viscoelastic properties of model lipid membranes, with spatial‐ and time‐resolution.…”

“…When combined with fluorescence lifetime imaging microscopy (FLIM), molecular rotors can be used to obtain spatially resolved viscosity maps of microscopic objects,17, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 as well as to observe dynamic change in viscosity during relevant processes of interest 37, 39, 41, 42. Thus, we aimed to use BODIPY‐C 10 , which is known to completely embed into the fluid‐phase lipid bilayers40 to directly examine how photooxidation during PDT affects viscoelastic properties of model lipid membranes, with spatial‐ and time‐resolution. As a model system, we have employed giant unilamellar vesicles (GUVs) composed of an unsaturated lipid 1,2‐dioleoyl‐ sn ‐glycero‐3‐phosphocholine (DOPC), which is susceptible to oxidation by a variety of ROS.…”

White‐Fluorescent Dual‐Emission Mechanosensitive Membrane Probes that Function by Bending Rather than Twisting

“…Contributions from thermochromism,2 thermal fluctuations in viscosity16 and the transition from S o into L d membranes to this inversion were dissected using DOPC LUVs, which are in L d phase at both temperatures ( T m (DPPC)=41 °C, T m (DOPC)=−18 °C) 17. At 55 °C, the emission spectra of 1 in L d DPPC and DOPC membranes were superimposable (Figure 3 d, red).…”

Tuning the Sensitivity of Fluorescent Porphyrin Dimers to Viscosity and Temperature

“…This advantage led to wide‐spread use of molecular rotors for dynamic microviscosity measurements in live mammalian and bacterial cells,4, 11, 12 as well as in model biological systems 13, 14, 15, 16…”