Differentiating Lipid Phase Domains in Cells using Fluorescence Lifetime of DiI

Abstract

Dynamic heterogeneous distribution of lipid phases is thought to be an important means by which lipids modulate cellular biology. However, information about these domains is not accessible using conventional optical microscopy because their size (10-100nm) is well below the diffraction limit. Fluorescence lifetime (FL) of variable chain length di-alkyl carbocyanine dyes (DiIs) has been proposed to reflect lipid phase, but verification that fluorescence readouts reflect lipid phase in cells is lacking. Thus, we used time-correlated single photon counting techniques to test the fidelity with which photophysical properties of DiI-C12 and DiI-C18 report lipid order in cells. DiI FL increases with increased solution viscosity (1 to 70 cP) were independent of acyl chain length, demonstrating that lifetime reports chromophore headgroup local viscosity. Sensitivity of FL of DiI to membrane order was evaluated in giant unilamellar vesicles (GUVs) composed of DOPC (liquid-disordered), DOPC:Chol (liquid ordered), DPPC:Chol (liquid ordered), and DPPC (gel phase). FL of DiI increased significantly with increase in membrane order, and correlated well with a decrease in diffusion coefficient. Phase partitioning of DiI-C12 and DiI-C18 was characterized in phase-coexistent GUVs composed of DOPC:DPPC:Chol (2:2:1). Short chain DiI-C12 and long chain DiI-C18 partitioned to liquid-disordered and liquid-ordered membrane phases, respectively. FL of DiI-C18 stained cells (1.47 ± 0.49 ns) was higher compared to DiI-C12 stained cells (1.26 ± 0.12 ns), indicating that DiI-C18 and DiI-C12 partitioned into liquid-ordered and liquid-disordered phases respectively. In conclusion, FL of DiI is a sensitive indicator of membrane fluidity and different chain length DiI's partition to different membrane phases both in model and cell plasma membranes.