Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores

Quantitative fluorescence analysis is often used to derive chemical properties, including stoichiometries, of biomolecular complexes. One fundamental underlying assumption in the analysis of fluorescence data─whether it be the determination of protein complex stoichiometry by super-resolution, or step-counting by photobleaching, or the determination of RNA counts in diffraction-limited spots in RNA fluorescence in situ hybridization (RNA-FISH) experiments─is that fluorophores behave identically and do not interact. However, recent experiments on fluorophore-labeled DNA origami structures such as fluorocubes have shed light on the nature of the interactions between identical fluorophores as these are brought closer together, thereby raising questions on the validity of the modeling assumption that fluorophores do not interact. Here, we analyze photon arrival data under pulsed illumination from fluorocubes where distances between dyes range from 2 to 10 nm. We discuss the implications of non-additivity of brightness on quantitative fluorescence analysis.