Abstract
Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here, we first developed a method for a time-resolved general polarization (GP) analysis with fluorescence-lifetime imaging microscopy that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity, whereas curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a supercritical angle fluorescence objective, to our knowledge the first time fluorescence-lifetime imaging microscopy images were generated with supercritical angle fluorescence. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes.
Highlights
It is recognized that the plasma membrane of mammalian cells is not a homogenous lipid bilayer but is diverse in composition, organization, and shape, giving rise to distinct membrane domains [1]
Time-resolved general polarization (GP) measurements by microscopy In time-resolved emission spectra (TRES) measurements, Laurdan lifetime is typically recorded at each emission wavelength across the entire emission spectra as shown in Fig. 1, a and b
Our approach could not track the shift in the emission peak as done in TRES measurements, the extent and rate of photon transition from the blue to the green channel contains information on the Laurdan solvent-relaxation process that can be analyzed in a quantitative manner
Summary
It is recognized that the plasma membrane of mammalian cells is not a homogenous lipid bilayer but is diverse in composition, organization, and shape, giving rise to distinct membrane domains [1]. Several membrane models have been proposed to account for the inhomogeneous nature of the plasma membrane such as the lipid raft and picket fence models. The lipid raft hypothesis, for example, suggests that densely packed lipid domains exist within the plasma membrane that are enriched in cholesterol and sphingomyelin and have different biophysical properties to the rest of the membrane [2]. Sensitive fluorescence techniques such as fluorescence correlation spectroscopy coupled to stimulated emission depletion have successfully revealed the heterogeneous diffusion of raft lipids such as sphingomyelin [3]. The picket fence model proposes that the plasma membrane is compartmentalized by a cortical actin network that can temporarily trap. Single-particle tracking in intact cells has provided evidence for the picket fence model by analyzing the diffusion trajectory of membrane lipids and proteins [5]. Methods that provide insights into the biophysical causes of membrane heterogeneity are highly desirable [8]
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