Single Molecule Motion Perpendicular to the Focal Plane of a Microscope: Application to Splicing Factor Dynamics within the Cell Nucleus

We present a method to obtain diffusion coefficients from video-microscopic sequences of single molecules moving in three-dimensionally extended samples. The approach is based on the number of fluorescent molecules within the detection slice near the focal plane. The number of initially present, mobile molecules decreases with increasing observation time due to diffusion. Obviously the decay kinetics contains information on molecular mobility along the optical axis of the microscope. For certain limiting conditions this kinetics can be described analytically. Somewhat surprisingly one finds that the decay rate depends on the time resolution, with which the process is observed. From experimental data the diffusion coefficient can only be determined by a comparison with the results of Monte-Carlo simulations. The simple counting algorithm has the advantage to be independent of the lateral localization precision, the signal-to-noise- ratio or the actual spatial signal shape in the image plane. The new approach yields a diffusion coefficient of D=3 μm2/s for the mobility of single splicing factor molecules U1 snRNP within the nuclei of semi-intact cells.