Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: Application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells

A statistical method for the analysis of fluorescence fluctuation amplitude s including bright spikes is presented. This situation arises e. g. when fl uorescent ligands interact with receptors carrying multiple binding sites, the technique gives information an the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscop y analysis, which cannot be applied in this situation. Two simple statistic al tests are proposed that can discriminate between fluorescence intensitie s originating from free ligands or complexes. The performance of the two te sts is evaluated and compared on mixtures of a fluorophore and fluorophore- coated beads that mimic the behaviour of multi-liganded complexes. An appli cation to ligand binding to the serotonin receptor, expressed on Escherichi a coli cells, is also provided. Specific binding of a fluorophore to this r eceptor, as well as competition with several ligands, is assessed.