Tethered particle motion (TPM) has been extensively used for single molecules studies during the last two decades. In TPM, one end of a target molecule (DNA or RNA) is bound to a surface, while a marker is attached to its other end. In solution, the marker is moving in a Brownian motion that is constrained by the tethered molecule. By studying the statistics of the marker position, one can extract the properties of the target molecule.

The distribution in the lateral (xy) plane is well known and was measured before. In the axial (z) direction on the other hand, the theoretical distribution in known for decades, but it has never been measured before.

Our setup enables measuring the 3D distribution, using Total Internal Reflection (TIR) illumination. We also compared it to computer simulations and found small deviations from the theory that can be explained by the dependence of the tangent-tangent correlations along the DNA on its length.