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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 DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * 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 sup or approximatively 1um2 * s-1 for FCS and D inf or approximatively 5 um2 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.

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