Article

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: 3.97). 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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    • "Figure 15.2 illustrates the benefit of our improved analysis in the context of polTIRF studies of myosin V; see Section 2.1. In the figure, the dots represent orientations determined by the traditional method by binning the data into 80 ms intervals (Forkey et al., 2005). Although they generally cluster around the results of our method (horizontal lines), the latter is cleaner and eliminates the spurious outlier points that the traditional analysis generates close to changepoints (Section 1.2). "
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    • "With the current setup, the average orientation of the probe during each 80 ms detection cycle is resolved to within approximately 10°. Procedures for calibration and data collection can be found in Forkey et al., 2003; Quinlan et al., 2005; and Rosenberg et al., 2005. "
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