The diffraction-limited width of a small object in conventional microscopy is approximately λ/2 (≈ 250 nm for green light). Under the proper conditions, the position of the object is determined by the center of the emission distribution, which can be determined to arbitrarily high precision, limited only by the total number of collected photons. Here we present fluorescence data showing the ability to localize single fluorescent dyes on a surface with a standard deviation of ≤ 1.5 nm with 0.5 seconds temporal resolution and a photostability that enables observation times of several minutes. We used total internal reflection microscopy and a centroid analysis — fitting the point-spread-function of a single fluorophore emission to a 2-D Gaussian. This accuracy enables us to see 8, 16, 30 nm steps on a model system of Cy3-DNA immobilized on a surface moved with a nanometric stage. This ability is expected to be very useful for analyzing molecular motor stepping characteristics.
We first applied this technique to myosin V, cargo transporter. Myosin V takes 37 nm step per ATP hydrolyzed. We have labeled Myosin V with a single fluorophore at different positions on the neck region and measured the step size as the Myosin V dimer moves on an actin filament. Depending on the exact position of the dye on the neck, hand over hand model predicts alternating steps like 37 nm -2x , 37 nm + 2x, 37 nm -2x, where x is the lateral distance between the fluorophore and the center of mass. Inchworm model predicts constant 37 nm steps regardless of the position of the dye on the molecule. We determined the steps either alternate between 74 nm and 0 nm, or between 42 nm and 33 nm, or between 52 nm and 23 nm. These results strongly support a hand-over-hand model of motility and are not consistent with an inchworm model.