Total internal reflection fluorescence (TIRF)-microscopy makes use of the formation of an exponentially decaying layer of intensity (evanescent field) in an aqueous solution when light is totally reflected at a glass/water interface. The intensity of the evanescent field decays over about 200-300nm to 1/e of its value right at the interface, and thus allows to limit fluorescence excitation to a layer of a few hundred nm next to the glass surface, e.g., of a cover slip. Limitation of fluorescence excitation to such a narrow layer suppresses background fluorescence to sufficiently low levels that individual fluorophores can be visualized. Fitting 2D-Gaussian functions to the individual spots of intensity allows to locate the position of individual fluorophores with precision of a few nm. With this approach the stepping of individual, processive myosin and kinesin molecules can be followed step by step. Labeling of the two head domains of e.g., myosin V with two different fluorophores allowed to demonstrate the hand over hand stepping of these molecules. With fluorescently labeled ATP as substrate, ATP-hydrolysis by, for example, an individual myosin molecule has been followed ATP- by ATP-molecule. At low protein densities several individual enzyme molecules can be characterized in parallel even within a mix of different isoforms or when wildtype and mutant forms of the protein are coexpressed. Other applications and further refinements will be demonstrated.