We developed a system for single molecule detection of DNA-protein interactions by observing the motion of a metal nano-bead in three-dimensions (3D). The method is based on tethered particle motion (TPM) and total internal reflection microscopy (TIRM). 80 nm gold nano particles bound to the tail of a tethered DNA and used as markers. There position can be detected with spatial precision of ~10nm.

The distribution of the end-to-end distance of a DNA molecule that is bound at one end to surface is not symmetric for the three spatial axes, due to the bounding surface. The theoretical probability distribution function (PDF) of the X or Y axes is Gaussian, and was measured experimentally before. On the other hand, the PDF of the Z axis was never measured although it is known theoretically for many years.

Using this system we measured for the first time the PDF for all the three axes and we show a good agreement to the theory.