Lagerholm, B. Christoffer5; Clausen, Mathias P.5; Christensen, Eva Arnspang6
1 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU2 Faculty of Science, SDU3 MEMPHYS Center, Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU4 Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, SDU5 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU6 Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, SDU
Ultra high-speed single particle tracking (image frame rates 40-50,000 Hz) 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 with an image integration time of ~0.5 msec. 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 further show that an artificial lipid, biotin-cap-DPPE, inserted in a mouse embryo fibroblast (MEF), labeled with sAv-QD655, and imaged as described reveals examples of three types of motion, 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 sAv-QD655s that had a hydrodynamic radius (RH) of 12.3 +/- 0.4 nm as determined by fluorescence correlation spectroscopy (FCS). While these QDs are very large compared to fluorescent dyes, they are 40% smaller in radius and are four fold smaller in volume than the 40 nm diameter gold particles used in ultra high speed SPT. Nevertheless further studies are required to fully understand the role that the QD label has in these results.