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.
