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

Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania, Philadelphia, 19104-6083, USA.
Biophysical Journal (Impact Factor: 3.83). 09/2005; 89(2):1261-71. DOI: 10.1529/biophysj.104.053470
Source: PubMed

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 F-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 degrees 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 technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.

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Available from: Margot E Quinlan, Apr 23, 2014
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