Simultaneous measurements of single molecule position and orientation are essential for deciphering molecular mechanisms that involve dynamic conformational changes.We developed a new single molecule fluorescence polarization method to resolve single fluorophore orientation with minimal dipolar degeneration,in addition to precise position tracking.The new approach is based on two orthogonally positioned imaging objectives which are used to collect polarized emission photons from single fluorophores.Compared with other polarization methods,our new method is capable of imaging multiple single molecules with both position and orientation information.We tested its performance by imaging fixed Calmodulin that were labeled with bifunctional Rhodamin.Its efficient use of emission photons ensures high spatial-temporal resolution,which is important for study of dynamic conformation changes.