Bioorthogonal Ligation‐Activated Fluorogenic FRET Dyads

An energy transfer‐based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue‐excitable coumarins to yellow/red emitting cyanine frames is presented. Such relay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes‐shifts allowing lower background fluorescence even in no‐wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells. These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three‐color imaging of intracellular targets using one single excitation source with separate emission windows. Sub‐diffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopy is also presented.