Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging

Abstract

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

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

“…Hence, the biexponential decays observed during Type I peroxidation can be either the result of two kinds of lipid environments created by peroxidation or the presence of aggregates 17. We first tested if biexponential decays of BODIPY‐C 10 are the result of the aggregation of BODIPY‐C 10 .…”

“…We first tested if biexponential decays of BODIPY‐C 10 are the result of the aggregation of BODIPY‐C 10 . The aggregates are known to cause quenching of the BODIPY‐C 10 monomeric species, with the long‐lived fluorescence of the aggregates appearing in the red region of the spectrum (>570 nm), overall leading to a biexponential decay 17. Hence, we recorded the fluorescence decay traces over two detection windows, 500–550 and 600–650 nm (Figure S6 in the Supporting Information).…”

“…Consequently, a wide range of fluorescent probes suitable for probing multiple properties of lipid membranes was developed,13 including probes for sensing membrane potential and fluidity,14 for detecting lipid order in the outer lipid leaflet of the lipid bilayer15 and for sensing changes in the membrane during apoptosis 16. 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.…”

“…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.…”

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…”