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Abstract

Dual-color fluorescence cross-correlation analysis has proven to be a powerful tool to probe interactions of different molecular species in solution and living cells on a single molecule level. Probing the coordinated motion of molecules through the measurement volume, it is a much more selective and data-compressing alternative to co-localization analysis by dual-color imaging, and provides additional access to fast internal dynamics of the co-migrating molecules. However, cellular FCS applications often suffer from extremely low molecular mobility, introducing bleaching artifacts or entirely impeding fluctuation analyses of any kind. Thus, to meet the increasing demand for interaction measurements of nearly stationary molecules, such as receptor-ligand complexes on cell membranes, these limitations of conventional fluorescence correlation and cross-correlation analysis need to be overcome. This can be achieved by combining a piezo-driven stage scanning unit with the confocal FCS setup, minimizing the photodynamic strain imposed on immobile single molecules without compromising the relevant cross-correlation information. Different scanning patterns were chosen and the corresponding auto- and cross-correlation curves recorded for both in vitro and in vivo systems. Expectedly, the shape of the correlation curves depends crucially on the different modes of stage motion. Nevertheless, cross-correlation amplitudes clearly reflect on the presence or absence of linkages between the different molecular species. Marked differences between bound and unbound single molecules could be observed on immobilized proteins in PAA gels and on cell membranes.