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 similar to 3 mu m 2 . s-1 and D SPT similar to 2 mu m 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 greater than or similar to 1 mu m 2 s 1 for FCS and D. 5 less than or similar to mm(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