Next, we introduced a second auxiliary dye. Due to the highest fluorescence turn-on, the first auxiliary dye was placed adjacent to QB. In C5-2AF568-QB, the two AF568 dyes were placed as 3' and 5' next neighbours of the QB base surrogate. This probe showed characteristics of aggregation-induced quenching (Fig. ). Quantum yields were low in both single and double stranded state (ss = 0.02, ds = 0.04). Also in absence of the QB dye in C5-2AF568, fluorescence quantum yields were much lower than observed for single AF568-labelled probes (ds(C5-2AF568) = 0.26 vs. ds(C1-AF568) = 0.92). This and the absorption spectra (peak at 540 nm, Fig. ) suggest that proximity allows the AF568 dyes to readily form an H-dimer. In stark contrast, probes C6-2AF568-QB and C7-2AF568-QB (Fig. ) showed exceptional brightness of up to 177,000 M -1 •cm -1 (Table ). These probes contained the second auxiliary dye in 9 or 7 nt distance to the first AF568 dye. Importantly, in the single stranded state, the single QB dye was still able to quench emission of the two AF568 dyes, enabling 14-to 17fold enhancements in fluorescence upon hybridization (Fig. , Fig. ). In J1-2AF568-QB, which is specific for the TCR CDR3 region expressed by Jurkat cells, we again placed the first AF568 near the QB dye, and the second AF568 in a larger distance to prevent aggregation-induced self-quenching. In this sequence context too, introduction of a QB base surrogate and two light harvesting AF568 dyes provided the desired features of high brightness in the target-bound state while fluorescing poorly in the absence of target (Fig. ).