Time-Resolved Three Dimensional Molecular Tracking in Live Cells

J. Werner
Los Alamos National Lab, US

Keywords: Molecular Tracking

Summary:

Our lab has developed a method to follow the fast, 3D motion of individual fluorescently labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 microns demonstrated here), substantially lower excitation powers (15 microWatts used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual quantum dot (QD)-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (950 nm/sec) vesicular transit through the cell. In addition to tracking QDs, we will also demonstrate 3D tracking of individual dyes and individual green fluorescent proteins diffusing at rates comparable to intracellular transport/diffusion rates.