Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three Dimensions

Jason J. Han †, Csaba Kiss ‡, Andrew R. M. Bradbury‡, and James H. Werner †*

^†Center for Integrated Nanotechnologies and^‡Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States

ACS Nano, Article ASAP

DOI: 10.1021/nn302912j

Publication Date (Web): September 8, 2012

Copyright © 2012 American Chemical Society

We demonstrate following individual fluorescent protein constructs and individual organic dyes as they diffuse in 3-D in solution at rates up to 1 μm²/s over distances of several micrometers in X, Y, and Z. Our 3-D tracking method is essentially a stage scanning confocal microscope that uses a unique spatial filter geometry and active feedback 200 times/s to follow fast 3-D motion. Here we detail simulations used to find optimal feedback parameters for following individual fluorescent proteins in 3-D and show that a wide range of parameters are capable of following individual proteins diffusing at 1 μm²/s rates. In addition, we experimentally show that through 3-D single-molecule tracking of a protein oligomer series (monomer, dimer, and tetramer) of the fluorescent protein Azami Green one can determine the protein oligomerization state. We also perform time-resolved spectroscopy (photon pair correlation measurements) during the measured 3-D trajectories. The photon pair correlation measurements show clear fluorescence photon antibunching, demonstrating that the trajectories are of single fluorescent molecules. We note that the rates of single-molecule diffusive motion we follow (approximately 1 μm²/s) are comparable to or faster than many intracellular transport processes.