Accurate High Speed Imaging of Single Protein Diffusion within the Live Cell Membrane

Abstract

The diffusive motion of proteins and lipids within the cellular membrane is complex and nuanced owing to the diverse and heterogeneous nature of its composition and structure. Insight into the diffusive motion of such proteins yields important information on their behavior and biological function. Conventional means to investigate diffusion uses a fluorescent label, but the intrinsic limitations of fluorescence such as photobleaching and saturation ultimately restrict what we may observe. These limitations strongly restrict both the timescale over which the protein may be observed and the accuracy to which they can be localized in space. Fluorescence microscopy additionally suffers from poor axial resolution and so provides almost no information as to the out-of-plane movement of the protein.Free from these constraints, interferometric imaging of scattered light (iSCAT microscopy) provides three-dimensional information on the motion of a single protein labeled with a gold nanoparticle. Here, we demonstrate single particle tracking of individual epidermal growth factor receptor (EGFR) proteins within the live HeLa cell membrane. We localize the diffusing protein to sub-nanometer accuracy with microsecond temporal resolution. The interferometric nature of the detection additionally provides information on the subtle out-of-plane movement of the protein to nanometer accuracy. We are therefore able to track with high spatio-temporal resolution the diffusion of single EGFR proteins in three-dimensions as they navigate a live HeLa cell membrane. This yields unheralded information about the minutia of diffusion within the cell membrane and demonstrates the potential of iSCAT microscopy in cell membrane imaging.