High-Speed Single Quantum Dot Imaging of in Live Cells Reveal Hop Diffusion

Abstract

Ultra high-speed single particle tracking (image frame rates 40-50 kHz) experiments with 40 nm gold particles has indicated that lipids and proteins in the plasma membrane undergo hop-diffusion between nanometer sized compartments (Fujiwara et al. (2002) J Cell Biol. 157:1071-81). These findings have yet to be independently confirmed. In this work, we show that high-speed single particle tracking with quantum dots (QDs) and using a standard wide-field fluorescence microscope and an EMCCD is possible at image acquisition rates of up to ∼2000 Hz. The spatial precision in these experiments is ∼40 nm (as determined from the standard deviation of repeated position measurements of an immobile QD on a cell). Using this system, we show that membrane proteins and lipids, which have been exogenously labeled with functionalized QDs, show examples of three types of motion in the plasma membrane of live mouse embryo fibroblasts, 1) approximately free diffusion, 2) confined (immobile) diffusion, and 3) hop diffusion between compartments with a size of ∼100 nm diameter, and a lifetime of ∼100-200 milliseconds. In these experiments, we have used QDs that had a hydrodynamic radius (RH) of about 12-15 nm as determined by fluorescence correlation spectroscopy (FCS). While these QDs are very large compared to fluorescent dyes, they are 25-40% smaller in radius and are at least two fold smaller in volume than the 40 nm diameter gold particles used in ultra high speed SPT.