Perspectives for confocal single molecule analysis in cells and membrane systems

Single molecule flourescence studies have come of age. While the detection and analysis of single biomolecules was still per se a difficult task to accomplish some years ago, and while some challenges still remain for in vitro applications, one of the greatest goals of modern biophysics is to follow single molecules in their native environment, i.e. the living cell or organism. Here, additional problems arise, such as the increased optical background, difficulties of labeling or controlling concentrations and environmental conditions, and, last but not least, the huge complexity of biological systems in general. To study protein interactions in situ, many precautions have to be taken to account for the increased complexity, and the employed methods such as Fluorescence Correlation Spectroscopy may have to be accommodated, e.g. to integrate complex stoichiometry or ternary interactions. Here we discuss the perspectives of FCS and related techniques as potential tools for single molecule based proteomics. Protein-protein interaction measurements with complex stoichiometry are introduced, as well as the use of controllable model systems for analyzing the dynamics of membrane bound proteins.