To enable the fine tuning of FRET design, a simple green-red dyad was synthesized, in which the respective fluorophores are connected by a short linker (scheme 3). Scheme 3. Synthesis of basic red/green dyad. Reagents and conditions: a) 1,4-hydroxybenzaldehyde, TFA, CH2Cl2, 3 h followed by DDQ, 1 h followed by BF3•Et2O, Et3N, 3 h; b) K2CO3, KI, acetone, reflux, 12 h. Initial experiments were done to establish the best parameters for FRET measurements, utilizing the simple dyad 12 and related compounds with the respective green and red fluorophores 11 and 4. First the most useful excitation wavelength for the green-red dyad was determined. Ideally, the excitation of the green donor fluorophore does not lead to excitation of the red fluorophore (cross-talk). However, in real systems the excitation of the green fluorophore inevitably leads to some excitation of the red fluorophore, since the absorbance of the red dye in the 500 nm region is small, but different from zero (fig. ). The absorption spectrum of dyad 12 is a nearly perfect superposition of the individual spectra of the green and the red Bodipy 11 and 4 suggesting no interaction between the two fluorophores in the respective ground states. In dyad 12 (scheme 3) the excitation energy is transferred from the green donor fluorophore to the red acceptor fluorophore. Consequently, the excitation of the dyad at 505 nm leads to insignificant emission at 535 nm, and full emission at 648 nm. This is indicative of virtually complete energy transfer from the green to the red fluorophore, which is not surprising with a view to the short distance between the green and the red fluorophore.