Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

Many biophysical techniques, such as single-molecule fluorescence correlation spectroscopy, Förster resonance energy transfer, and fluorescence anisotropy, measure the translation and rotation of biomolecules to quantify molecular processes at the nanoscale. These methods often simplify data analysis by assuming isotropic rotational diffusion, e.g., that molecules wobble within a circular cone. This simplification ignores the anisotropy present in many biological contexts that may cause molecules to exhibit different degrees of diffusion in different directions. Here, we loosen this assumption and establish a theoretical framework for describing and measuring anisotropic rotational diffusion using fluorescence imaging. We show that anisotropic wobble is directly quantified by the eigenvalues of a 3-by-3 positive-semidefinite Hermitian matrix M consisting of the second-order moments of a molecule’s transition dipole μ. This formalism enables us to model the influence of unavoidable shot noise using a Hermitian perturbation matrix E; the eigenvalues of E directly bound errors in measurements of wobble via Weyl’s inequality. Quantifying various perturbations E reveals that anisotropic wobble measurements are generally more sensitive to errors compared to quantifying isotropic wobble. Moreover, severe shot noise can induce negative eigenvalues in estimates of M, thereby causing the anisotropic wobble measurement to fail. Our analysis, using Fisher information, shows that techniques with worse orientation measurement sensitivity experience stronger perturbations E and require larger signal to background ratios to measure anisotropic rotational diffusion accurately. Our work provides deep insights for improving the state of the art in imaging the orientations and anisotropic rotational diffusion of single molecules.