Single-molecule anisotropy imaging

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 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D-lat = 3.5 x 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-beta 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.