Lipid tracking at kilohertz sampling rates on live cell membranes through Interferometric Scattering microscopy

Abstract

AbstractThe lateral dynamics of lipids on the cellular membranes are one of the most challenging topics to study in membrane biophysics, needing simultaneously high spatial and temporal resolution. In this study, we have employed Interferometric scattering Microscopy (ISCAT) to explore the dynamics of a biotinylated lipid analogue labelled with streptavidin-coated gold nanoparticles (20 and 40nm in diameter) at 2kHz sampling rate. We developed a statistics-driven analysis pipeline to analyse both ensemble average and single trajectory Mean Squared Displacements from each dataset, and to discern the most likely diffusion mode. We found that the use of larger tags slows down the target motion without affecting the diffusion mode. Moreover, we determined from our statistical analysis that the prevalent diffusion mode of the tracked gold-labelled lipids is compartmentalized diffusion. This model describes the motion of particles diffusing on a corralled surface, with a certain probability of changing compartment. This is compatible with the picket-fence model of membrane structure, already observed by similar studies. Through our analysis, we could determine significant physical parameters, such as average compartment size, dynamic localization uncertainty, and the intra- and inter-compartmental diffusion rates. We then simulated diffusion in an environment compatible with the experimentally-derived parameters and model. The closeness of the results from the analysis of experimental and simulated trajectories validates our analysis and the proposed description of the cell membrane. Finally, we introduce the confinement strength metric to compare diffusivity measurements across techniques and experimental conditions, which we used to successfully compare the present results with other related studies.