title: Time-resolved, confocal single-molecule tracking of individual organic dyes and fluorescent proteins in three dimensions.
abstract: We demonstrate following individual fluorescent protein constructs and individual organic dyes as they diffuse in 3-D in solution at rates up to 1 μm(2)/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 μm(2)/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 μm(2)/s) are comparable to or faster than many intracellular transport processes.
authors.affiliation: Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Han
authors.forename: Csaba
authors.initials: C
authors.lastname: Kiss
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
authors.forename: James H
authors.initials: JH
authors.lastname: Werner
mesh_terms: D000465:Algorithms; D005456:Fluorescent Dyes / Q000032:analysis*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D009930:Organic Chemicals / Q000032:analysis; D010363:Pattern Recognition, Automated / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals