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In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.


John F Beausang, Yujie Sun, Margot E Quinlan, Joseph N Forkey, Yale E Goldman. Orientation and rotational motions of single molecules by polarized total internal reflection fluorescence microscopy (polTIRFM). Cold Spring Harbor protocols. 2012 May;2012(5)

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PMID: 22550303

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