Understanding the function of complex biomacromolecular assemblies requires detailed knowledge of the structure and dynamics of the individual molecular components as well as of their interactions within complexes. Fluorescence based methods offer the possibility to measure protein properties and interactions with a high sensitivity and selectivity. The advent of bright and more photo-stable fluorescent dyes and an enormous methodical and technical improvement of high resolution fluorescence spectroscopy and microscopy enabled studies on proteins even at a single molecule level. Due to the fact that single molecule techniques provide information on the distribution of parameters characterizing the biological macromolecule, these methods are often the approach of choice to clarify and better understand the structure and function of proteins.