Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking

Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of D(FCS) ~ 3 μm(2) · s(−1) and D(SPT) ~ 2 μm(2) · s(−1), respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D ≳ 1 μm(2) · s(−1) for FCS and D ≲ 5 μm(2) · s(−1) for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes.