Single-Molecule Anisotropy Imaging

Abstract

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D rot = 7 × 10 7 rad 2/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D lat = 3.5 × 10 −8 cm 2/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L β gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D rot = 1.2 rad 2/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.