Simultaneous Observation of the Three-Dimensional Orientation and Position of a Single Fluorescent Probe

Abstract

We have developed a new microscopy system for simultaneously measuring the three-dimensional orientation and position of a fluorescent probe based on the principal of polarization analysis. This method requires only that one collect fluorescence counts from a single fluorescent probe at four different polarizations such that only relatively simple optics and mathematical equations are required to yield the orientation. Orientations can be determined with less than 10° accuracy at 33ms time resolution except for a situation where a probe is nearly parallel to optical axis. We used the newly developed microscopy and quantum rods as our fluorescent probes to simultaneously measure myosin V movement along an actin filament and rotation around its own axis. Myosin V was seen to rotate 90° around its own axis for each step. From this result, we suggest that in the two-headed bound state the necks are perpendicular to one another, while in the one-headed bound state the detached trailing head is biased forward in part by rotating its lever arm about its own axis. This new microscopy system should have particular applicability to biophysical studies that investigate single molecule orientation dynamics.