ConfoCor 2 overcomes this limitation by incorporating two avalange photo detectors (APDs) allowing for the simultaneous detection of two fluorescence signals. This permits analysis of two interaction partners labelled with different fluorescent dyes. In this setting, the APD-pair now receives a threefold signal from both free ligands and the ligand complex. Thus, in contrast to the classical FCS approach with one fluorescent binding partner, the doubly-labelled complex submits an autonomous fluorescence signal that reaches both APDs.
Clearly, the software used for the processing of the raw experimental data must now cope with an additional task: the discrimination of signals emanating from free ligands versus the complex. The new ConfoCor 2 software performs this so-called cross-correlation leading to signal deconvolution. With other words, ConfoCor 2 no longer necessarily relies on the evaluation of changes in a ligand's diffusion properties: by use of the two-channel principle and the new software, ConfoCor 2 can ignore the single-labelled free binding partners and directly track the appearence of the bimolecular complex providing a signal which is directly proportional to the extent of complex formation.
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