Giant unilamellar vesicle (GUV) as model system in advanced 3d orientation determinations

One of the unique features of single molecule absorption/emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule’s 3d orientation. Therefore, several techniques have been proposed in order to determine the full three-dimensional orientation of dipole emitters on a single molecule level. We recently demonstrated a technique that combines emission distribution and polarization detection [1,2,3]. As the method is an intensity distribution technique and based on single photon detection in principle, one can extend the 3d orientation determination to fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements. This allows for the determination of the dynamics in 3d orientation of single molecules down to a nanosecond timescales. The 3d orientation is particularly interesting in non-isotropic environments. A lipid membrane is such a non-isotropic environment of enormous importance in biological systems. We therefore use giant unilamellar vesicle (GUV) labeled with dyes like DiO as a model system. Due to the defined curvature of such vesicles all possible dipole orientations can be achieved. This allows us to show the capabilities of our method on different timescales and to quantify the error in determination of 3d orientation dynamics in lipid membranes. Furthermore Monte Carlo simulations of rotational dynamics taking into account the excitation/emission characteristics of dipoles incorperated in the lipid membrane in conjunction with the 3D orientation determination complement our experiment.