NIR-emitting molecular-based nanoparticles as new two-photon absorbing nanotools for single particle tracking

In order to provide a green alternative to QDs for bioimaging purposes and aiming at designing bright nanoparticles combining both large one-and two-photon brightness, a bottom-up route based on the molecular engineering of dedicated red to NIR emitting dyes that spontaneously form fluorescent organic nanoparticles (FONs) has been implemented. These fully organic nanoparticles built from original quadrupolar dyes are prepared using a simple, expeditious and green protocol that yield very small molecular-based nanoparticles (radius similar to 7 nm) suspension in water showing a nice NIR emission (lambda(em)= 710 nm). These FONs typically have absorption coefficient more than two orders larger than popular NIR-emitting dyes (such as Alexa Fluor 700, Cy5.5 ... ) and much larger Stokes shift values (i.e. up to over 5500 cm(-1)). They also show very large two-photon absorption response in the 800-1050 nm region (up to about 10(6) GM) of major promise for two-photon excited fluorescence microscopy. Thanks to their brightness and enhanced photostability, these FONs could be imaged as isolated nanoparticles and tracked using wide-field imaging. As such, thanks to their size and composition (absence of heavy metals), they represent highly promising alternatives to NIR-emitting QDs for use in bioimaging and single particle tracking applications. Moreover, efficient FONs coating was achieved by using a polymeric additive built from a long hydrophobic (PPO) and a short hydrophilic (PEO) segment and having a cationic head group able to interact with the highly negative surface of FONs. This electrostatically-driven interaction promotes both photoluminescence and two-photon absorption enhancement leading to an increase of two-photon brightness of about one order of magnitude. This opens the way to wide-field single particle tracking under two-photon excitation