High-Speed Quantum Dot Tracking in Plasma Membranes Reveals Short-Lived Small-Size Corralled Diffusion

Abstract

In this work, we show that tracking of single quantum dots (QDs) is possible at image acquisition rates up to ∼2,000 Hz. We image with a spatial precision of ∼30 nm for several seconds using a standard wide-field fluorescence microscope and an EMCCD camera.Using this system, we show that membrane proteins and lipids, exogenously labeled with functionalized QDs, undergo corralled diffusion in the plasma membrane of live cells where the molecules move between corrals of ∼100 nm in diameter within a lifetime of ∼50 ms (figure). We further investigate the cholesterol and actin dependence of corralled diffusion.Observation of these dynamic features is dependent on three parameters; the diffusion rate of the investigated molecule, the corral size, and the image acquisition rate. If not imaged at sufficient speed, corralled diffusion appears as slow free diffusion. Therefore, we compare our experimental result with computer simulations to find the relation between these three parameters.The QDs used in this imaging system has a signal brightness and stability advantage compared to conventional fluorophores allowing for long-term fast imaging, and a size advantage to gold particles used for high-speed single particle tracking.View Large Image | View Hi-Res Image | Download PowerPoint Slide