Probing the influence of the particle in Single Particle Tracking measurements of lipid diffusion

We present a systematic study of the influence of the nanoparticle used as a probe in Single Particle Tracking experiments with a supported lipid bilayer as a model system. Quantum Dots, 40 nm and 200 nm diameter fluorescent latex spheres, and 40 nm diameter gold colloids were attached to headgroup-labeled lipid molecules in the bilayer by means of an antibody. The percentage of the particle's surface covered with antibody was also varied. We made the choice to extract the diffusion coefficient from the first two points of the mean square displacement of the particle after demonstrating that the accuracy of this method is equivalent to a more rigorous but less prevalent analysis based on the cumulated probability function. We show that, under optimal conditions, Quantum Dots and also 40 nm latex beads have a negligible influence on the lipid diffusion, whereas the diffusion coefficient of gold colloids and 200 nm diameter latex particles was systematically reduced 2- to 3-fold compared to the reference value obtained by Fluorescence Recovery After Photobleaching. Interestingly, the diffusion coefficient of Quantum Dots and 40 nm latex beads depended on the percentage of the surface of the nanoparticle covered with antibody in an unexpected manner: at very low percentage the perturbation to the lipid diffusion due to the particle increased. We speculate that in this regime subtle details of antibody binding to the nanoparticle become important.