Three Dimensional Orientation Measurements of Single Fluorescent Molecules by Newly Developed Polarized Fluorescence Microscopy

Abstract

Single molecule fluorescence techniques are increasingly important to observe the dynamic properties of single molecules. One such important dynamic property is the single molecule's orientation. In order to observe three dimensional motions of proteins in solution, it is necessary to measure three dimensional orientations of proteins. We developed new microscopy for determining three dimensional orientations based on the principal of polarization analysis proposed by Fourkas. This method requires only that one collect fluorescence counts from a single molecule at three different polarizations followed by a simple mathematical calculation to yield the three dimensional orientations. In this method, the relatively small numbers of photons are sufficient for a reliable orientation measurement and this should decrease the time scale needed to determine the orientation of any given fluorophore. Here, we demonstrate axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface by polarization measurement of individual rhodamine phalloidin fluorophores sparsely bound to filaments. This new microscopy will be available for investigating the wide range of dynamic processes through single molecule orientation dynamics in various biophysical studies.