Optimisation of fluorescent DNA labels for two-photon microscopy

We have investigated a series of mono-, bis- and trisvinyl-pyridinium TP derivatives (TP-Py) which exhibit interesting properties : good water solubility, reduced size, high photostability, large two-photon absorption cross-sections (delta up to 700 GM), red emission (lambda(em)=660-680nm). Most importantly, TP-Py happen to be poorly fluorescent in water whereas their fluorescence is strongly enhanced when binding to various forms of DNA. We showed that this property originates in the spontaneous immobilisation of the dye inside the double-stranded DNA helix. Due to moderate fluorescence quantum yield efficiencies (eta), the brightness of TP-Py appears however to be improvable (eta delta=19). For this purpose, a new generation of specifically engineered TP derivatives have been designed and extensively characterized. We showed that switching from a classical TP core to a more electron-rich trinaphtyl (TN) core enables to increase the molecular brightness although reducing drastically the dye-DNA specific interactions.