Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules

We give an overview of using fluorescence correlation spectroscopy (FCS) for measuring rotational diffusion of macromolecules, and present a new experimental scheme, pulsed-interleaved excitation or PIE-FCS, which allows for measuring all conceivable correlation curves of a polarization-sensitive FCS experiment. After giving a brief review of the theoretical foundations, we systematically study the impact of different experimentally relevant parameters such as depolarization by the objective, or non-collinearity between absorption and emission dipole of the fluorescent label. We also discuss the possibility to extract information about anisotropic rotational diffusion, and exemplify that by determining the size and shape of the large protein aldolase.