Separation of translational and rotational contributions in solution studies using fluorescence photobleaching recovery

Although fluorescence photobleaching recovery (FPR) experiments are usually interpreted in terms of the translational motions of a fluorescently labeled species, rotational motions can also modulate recovery through the cosine-squared laws for dipolar absorption and emission processes. In a complex interacting system, translational and rotational contributions may both be simultaneously present. We show how these contributions can be separated in solution studies using an FPR setup in which (a) the linear polarization of the low-intensity observation beam and the high-intensity photobleaching pulse can be varied independently, and (b) all emitted fluorescent photons are counted equally. The fluorescence recovery signal obtained with the observation beam polarized at the magic angle, 54.7 degrees, from the bleach polarization direction is independent of label orientation, whereas the anisotropy function formed from a combination of parallel and perpendicular polarizations isolates the orientational recovery. The anisotropy function is identical to that in fluorescence correlation spectroscopy and, for rigid-body rotational diffusion, can be expressed as a sum of five exponential terms