Lifting the Spectral Crosstalk in Multifluorophore Assemblies

A general strategy for measuring the energy-transfer efficiencies in multifluorophore assemblies is demonstrated. The present method is based on spectral shaping of the excitation light with molecular solutions representing donor and acceptor fluorophores, which causes a suppressed excitation of the respective donor and acceptor molecules in the sample. The changes in the acceptor emission resulting from spectral shaping of the excitation light are then used to determine the energy-transfer efficiencies (Ex) associated with all participating donor–acceptor pairs. Here, the technique is demonstrated through energy-transfer (ET) measurements in a 4-fluorophore construct featuring a DNA supported assembly of three donor/donor-relay (Cy3, Cy3.5, and Cy5) and one acceptor (Cy5.5) molecules. The resulting Ex were validated using the standard photoluminescence (PL) quenching approach as well as measurements of partial 2- and 3-dye assemblies. The present work highlights general benefits of the spectrally shaped excitation approach to measuring donor–acceptor energetics, including the ability to resolve the spectral cross talk between multiple fluorophores and to exclude charge transfer contributions into donor PL quenching.