The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.
Harwardt, M. L. I. E., Dietz, M. S., Heilemann, M., & Wohland, T. (2018). SPT and Imaging FCS Provide Complementary Information on the Dynamics of Plasma Membrane Molecules. Biophysical Journal, 114(10), 2432–2443. https://doi.org/10.1016/j.bpj.2018.03.013