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

Abstract

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 \(D_{\text{FCS}} \sim\) 3 \(μ\text{m}^2\cdot\text{s}^{-1}\) and \(D_{\text{SPT}} \sim\) 2 \( μ\text{m}^2\cdot\text{s}^{-1}\), 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: \(D \gtrsim\) 1 \(μ\text{m}^2\cdot\text{s}^{-1}\) for FCS and \(D \lesssim\) 5 \(μ\text{m}^2\cdot\text{s}^{-1}\) 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.

Keywords

• lipid
• bilayer
• membrane
• diffusion
• fluorescence
• confocal microscopy
• total internal reflection fluorescence microscopy (TIRF)
• FCS
• SPT