Single-Molecule Fluorescence Microscopy and Tracking of Lipids in Mitochondrial-Like Supported Lipid Bilayers

Abstract

The mitochondrial membrane has a lipid and protein composition that results in unique physicochemical properties. These properties are important for many processes taking place at the mitochondrial membranes, such as the process of mitochondrial outer membrane permeabilization during apoptosis. In this study, we investigated the mobility of fluorescently-labeled lipids in supported lipid bilayers with mitochondrial-like lipid composition. We used and compared two different fluorescence methods: an ensemble method (image correlation spectroscopy) and a single particle method (single molecule tracking). Ensemble methods measure observables of the entire system. We use image correlation spectroscopy (ICS) on confocal microscopy images, in which the autocorrelation function for scanned lines is calculated to obtain information on particle densities and dynamics of the entire system. Single-molecule fluorescence microscopy methods image proteins, enzymes or other molecules of interest as diffraction-limited spots, which can be individually located and tracked using suitable algorithms. Instead of averaging over the entire system, tracks are analyzed for each particle. We investigate the dynamics of supported lipid bilayers with a composition that aims to mimic that found in mitochondrial membranes. The bilayers were labeled with the lipophilic dye DiD and formed via vesicle fusion on a mica solid support. Diffusion coefficients are obtained by line correlation analysis on the confocal images and mean square displacement analysis as well as displacement distribution analysis at constant time steps on Total Internal Reflection Fluorescence Microscopy data. Both methods show the presence of mobile as well as immobile particles. The average diffusion coefficients of the mobile population obtained with either acquisition method are in agreement. This provides the foundation for further work concerning the interaction of membrane proteins with the lipid bilayer.