Three-dimensional orientational colocalization of individual donor-acceptor pairs

The three-dimensional orientation of the donor and acceptor transition dipoles in fluorescence resonance energy transfer (FRET) pairs was discussed. The orientation of the dipoles was demonstrated by means of scanning optical microscopy with annular illumination. In this model system perylenediimide as the the donor and terryelenediimide as the acceptor were coupled via a stiff p-terphenyl linker. The results show that the application of annular illumination in conjuction with selective excitation of the donor and acceptor absorption transition dipoles in individual spFRET pairs allows for determination of their manual orientation.