Probing rotation dynamics of biomolecules using polarization based fluorescence microscopy

Fluorescence polarization, particularly fluorescence anisotropy (FA) can be used to characterize the rotation dynamics and interactions of biomolecules. We report here fluorescence polarization microscopy based on a two-photon fluorescence microscope. Two-photon fluorescence excited by a linearly polarized fs laser beam was separated into components of parallel and perpendicular polarization and then recorded simultaneously by two detectors. From the images corresponding to different combinations of the polarization for the excitation and fluorescence photons, images of FA, or polarization difference, can be derived. It is demonstrated that FA microscopy is capable of probing rotational mobility of the fluorescent molecules and their interaction with the surroundings, but displays lower axial resolution than fluorescence intensity images. It is proved that the degraded axial resolution of FA imaging is intrinsic to the current experimental set-up. Artifacts in FA imaging of aligned molecules are also discussed.