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Spatially resolved (2D) FCS Imaging

One of the drawbacks of FCS/FCCS to study protein-protein interactions is that measurements are conducted using a parked beam inside one specific volume element of a cell. This causes that each measurement is associated with a significant error that results from heterogeneity of the cellular interior that cannot be compensated for. As a consequence, statistical significant numbers of measurements must be conducted, which provides reliable averages but at the same time prevents single cell studies.

Protein interactions (KD’s) among components of multimeric complexes are dependent on the concentration of the constituents and predicted to be influenced by competitive or cooperative modes of protein-protein interactions, as we have shown by theoretical considerations (Maeder et al., 2007). In reverse, this means that reliable KD measurements as a function of the concentration of the binding partners would allow one to obtain information about the principles of protein-complex formation for given complexes. This, and the desire to conduct FCS using an imaging type of approach that would provide spatially resolved protein mobility and interaction data led to the construction of a combination of a single plane illumination microscope suitable for camera based FCS/FCCS measurements. In collaboration with Malte Wachsmuth (>) we developed a new implementation of this microscope that enables now the acquisition of fluorescence fluctuation information form within cells that provide a time resolution sufficient for autocorrelation analysis of the fluorophore mobilities. In a first series of proof-of-principle experiments we used this instrument to characterize GFP diffusion in animal cells and in tissue samples (wing discs from Drosophila) and we investigated the nuclear mobility of heterochromatin binding protein 1 alpha (HP-1alpha) known to be involved in various types of dynamic interactions with chromatin in regions of eu- and heterochromtin. This study revealed the first time a spatial patterning of this chromatin with respect to low- and high-affinity regions (Capoulade et al., 2011). This instrument is currently being set up for dual-color FCS/FCCS.