Details

Autor(en) / Beteiligte

• Han, Jason J
• Kiss, Csaba
• Bradbury, Andrew R. M
• Werner, James H

Titel

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

Ist Teil von

• ACS nano, 2012-10, Vol.6 (10), p.8922-8932

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2012

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.