Nano-Devices for Probing Single Molecules

Abstract

By confining molecules to 2-dimensional sheets or zero-dimensional wells, with nanometer-scale dimensions, one can observe individual fluorescently labeled molecules for long times, at high concentrations, and without surface attachment. We present two simple nano-devices that achieve these goals. We use these devices to obtain previously inaccessible information on molecular size, composition, and dynamics.2-dimensional confinement is achieved near the point of contact between a convex lens and a planar coverslip. The lens-coverslip spacing varies smoothly from zero to many microns as the radial distance from the point of contact is increased. Commercial fused silica optics have surface roughness of approximately 1 nm, so one can select a vertical confinement with nanometer accuracy simply by imaging at a given radius from the point of contact. The lens-coverslip system allows: a) fluorescence imaging of immobilized single molecules in the presence of a micromolar concentration of diffusing fluorophores; b) long-time observations of freely diffusing single molecules in dilute solution, which further allows determination of diffusion coefficients, brightness, and spectral dynamics molecule-by-molecule; and c) direct mechanical measurement of the size distribution in a population of fluorescently labeled molecules.Zero-dimensional confinement is achieved in nanometer-scale wells in a fused silica coverslip. A solution of fluorophores is washed over the wells, and then the bulk solution is replaced with a fluorinated oil. At most one molecule, or molecular complex, is immobilized in a nanoscale water droplet in each well. As with the lens-coverslip system, the dimple machine allows long-time observations of individual molecules, without surface attachment and in the presence of a high fluorescence background. By counting the number of photobleaching steps in each of several thousand chambers, we determine the distribution of stoichiometries in multimeric complexes.