Microscopic imaging of single fluorescent molecules within cells provides a molecular, real-time view of physiological processes in vivo. Single fluorescent molecules produce diffraction-limited light spots in the image plane, which can be localised with a very high precision. In single-molecule fluorescence microscopy (SMF) the achievable localisation precision depends only on the signal-to-noise ratio (SNR) and the stability of the optical setup. Typically values between 20 and 40 nm can be achieved. Highly dynamic processes and Brownian motion characterised by diffusion coefficients <20 microm(2)/sec can be followed by high-speed imaging, hence the method is an ideal tool to study intranuclear protein or ribonucleoprotein particle mobility. In contrast to conventional techniques, different forms of mobility in a heterogeneous system may well be distinguished from each other. Furthermore, specific binding and bimolecular interaction events can be followed at the single molecule level. A prominent example of an application is the study of nucleocytoplasmic transport one molecule at a time. In this case, the high localisation precision allows to analyse the binding site distribution of single molecules at the nuclear pore complex, and the high time resolution allows determination of the binding duration of soluble receptors and transport substrates.