Tracking single quantum dots in three dimensions: Following cell receptor traffic and membrane topology

By Jim Werner, LANL

Feb 10, 2009

CNLS Conference room.

We have constructed a new confocal fluorescence-microscope that uses active feed-back and a unique spatial filter geometry to follow individual fluorescent quantum dots as they diffuse throughout 3 dimensional space at rates faster than most intracellular transport processes (~microns/second). By using a pulsed excitation laser source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations (photon anti-bunching). Since the timing of individual photons are recorded with ~100 picoseconds resolution and 3D trajectories are recorded for periods up to minutes, this system can help bridge the enormous time-scale difference between conformational fluctuations and cellular signaling processes.

More important than the superior temporal resolution, this system can follow individual molecular motion over an extended X, Y, and Z range (tens of microns), enabling one to study the transport of individual fluorescently labeled biomolecules (proteins, DNA, or RNA) performing their functions inside living cells. Our preliminary investigations in this area are focused on following the spatial dynamics of the IgE receptor Fc{epsilon}R1 on rat mast cells, an important signaling molecule for the allergic response. We find the types of motion of this receptor on the surface are highly heterogeneous, with substantial and measurable excursions in all three spatial dimensions (X, Y, and Z).

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