Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores

TL;DR

This paper investigates how neighboring identical fluorophores interact and affect fluorescence measurements, challenging the common assumption that fluorophores behave independently, which has implications for quantitative biological imaging.

Contribution

It introduces an analysis of non-additive effects in fluorophore brightness, providing new insights into fluorophore interactions at nanometer distances.

Findings

Neighboring fluorophores exhibit non-additive brightness effects.

Interactions occur at distances of 2-10 nm between dyes.

Implications for improving quantitative fluorescence analysis methods.

Abstract

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 {\it 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 2-10 nm. We discuss the implications of non-additivity of brightness on quantitative fluorescence analysis.