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.
Highlights
Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure
We reasoned that using both the temperature and the composition of the mixture interchangeably would deliver the most precise calibration graph possible, Fig. 1. From these data we were able to draw the following conclusions: firstly, the fluorescence lifetime of BODIPY molecular rotor follows the Forster–Hoffmann equation between 10 cP and 4500 cP, since a good linear correlation is observed between ln t and ln Z in this range, Fig. 1
In view of the fact that our rotor can be used as a probe for live biological cells[7,8] we focused on aspects of membrane biophysics that are thought to accompany many important membrane associated processes, such as changes in the level of cholesterol, in saturated versus non-saturated lipid ratios and, importantly, the lipid phase
Summary
Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. 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. Since viscosity is the major parameter which controls the speed of diffusion in condensed media, including that of lipids and proteins in membranes,[12] the molecular rotor approach can provide valuable complementary information on diffusion in biological systems, which is unavailable when using any other currently existing spectroscopic techniques. In molecular rotors the non-radiative deactivation rate responds strongly to the viscosity of their immediate environment.[9,11] This typically occurs as a result of a viscosity-dependent intramolecular structural change, such as twisting or rotation, which is coupled to a population of the dark non-emissive electronic excited state(s). The calibration plot of fluorescence parameters versus viscosity can be created based on the Forster–Hoffmann equation:[13]
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