Highly photostable two-photon NIR AlEgens with tunable organelle specificity and deep tissue penetration

Photostability is a particularly important parameter for fluorescence imaging especially long-term dynamic tracking in live samples. However, many organic fluorophores show poor photostability under one-photon and two-photon continuous irradiation. In addition, these traditional fluorophores also suffer from aggregation caused quenching (ACQ) in aggregate state in insolvable water environment. Therefore, it remains challenging to develop photostable and ACQ-free fluorophores for biological imaging. In this work, we developed two highly photostable aggregation-induced emission luminogens (AlEgens) based on the cyanostilbene core for in vitro and ex vivo bioimaging. These AlEgens named CS-Py+ SO3- and CS-Py+ exhibit near-infrared solid-state emission, large Stokes shift (> 180 nm), high fluorescence quantum yield (12.8%-13.7%) and good two-photon absorption cross section (up to 88 GM). CS-Py+ SO3- and CS-Py+ show specific organelle staining with high biocompatibility in membrane and mitochondria in live cells, respectively. In addition, selective two-photon mitochondria visualization in live rat skeletal muscle tissues with deep-tissue penetration (about 100 tun) is successfully realized by using CS-Py+. Furthermore, these AlEgens especially CS-Py+ exhibit remarkably high resistance to photobleaching under one-photon and two-photon continuous irradiation. These highly photostable AlEgens could be potentially utilized in visualizing and tracking specific organelle-associated dynamic changes in live systems.