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Biophys. J. BioFAST: First Published May 13, 2005. doi:10.1529/biophysj.104.053470
© 2005 by the Biophysical Society.

A more recent version of this article appeared on August 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Measurement of Single Macromolecule Orientation by Total Internal Reflection Fluorescence Polarization Microscopy

Joseph N Forkey 1, Margot E Quinlan 2 and Yale E Goldman 3*

1 Precision Optics Corporation
2 University of California San Francisco
3 Univ. of Pennsylvania School of Med.

* To whom correspondence should be addressed. E-mail: goldmany{at}mail.med.upenn.edu.

Submitted on September 23, 2004
Revised on December 20, 2004
Accepted on 27 April 2005


  Abstract
A new approach is presented for measuring the three dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled filamentous actin are compared to ensemble averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10° at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the new technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the larger of two values of the torsional rigidity of F-actin previously reported.

Key Words: actin, fluorescence, microscopy, polarization, single molecule, total internal reflection




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Copyright © 2005 by the Biophysical Society.