Importantly Seta-670-mono-NHS has a significant overlap integral between its absorption and emission spectra. Its Förster distance (R₀) for homo-FRET was calculated to be about 50 A, a distance comparable to the size of the antibody. The homo-FRET is expected to occur already when an antibody is labeled with only two fluorophores and increases quickly with the number of labels. Analysis of single molecule traces shows an interesting behavior where efficient non-radiative excitation energy transfer and self-quenching already manifests itself with only 2 or 3 labels. Detailed analyses of the overall and average residence times reveal that multiple labeling with fluorophores, such as Seta-670-mono-NHS, could be a good approach for increasing the number of available photons and extending the overall observation time to study/observe binding at the single antibody level. Intrinsically, the signal and lifetime of the individual, intermediate fluorescently labeled species strongly depend on the number of labels, but the average residence time for each single species is similar. Apparently due to energy transfer the change in fluorescence intensity is compensated by the change in average fluorescence lifetime. Contrary to ensemble measurements where over-labeling is commonly recognized as a problem, this approach appears to have significant advantages for single antibody (protein) studies.

Another class of dyes which promises excellent performance for single-molecule applications are Squaraine-rotaxanes (SeTau dyes). These combine high photostability and high sensitivity. They are available in hydrophobic and hydrophilic forms.

Our cyanine-based dyes are more photostable compared to the conventional Cy dyes and Seta 555 (K8-3335), Seta 650 (K8-5035) and Seta 750 (K8-7035) exhibit even higher photostability compared to Alexa dyes and are therefore well suited for single molecule measurements.