Photostable Organic Two-photon Dyes with Ultrahigh Brightness for Long-term Fluorescence Imaging

Developing photostable two-photon dyes with high brightness and negligible photocytotoxicity is of key importance in fluorescence imaging but remains a huge challenge. Here, a two-photon dye with ultrahigh brightness and photostability is demonstrated for high-performance long-term two-photon fluorescence imaging. By terminated donor engineering, the designed DBD shows a higher two-photon absorption cross-section (δ, 418 GM vs 329 GM) and photoluminescence quantum yield (Φ_PL, 62.74% vs 54.63%) than its counterpart DBA. As a consequence, two-photon fluorescence brightness (δ×Φ_PL) of DBD exhibits a 10-folded enhancement (262 GM vs 19 GM) in comparison with typical Coumarin 307 dye. More importantly, DBD displays ultrahigh photostability and negligible photobleaching under 10 min femtosecond laser irradiation, which stands in marked contrast to Coumarin 307. Furthermore, femtosecond transient absorption spectroscopy ascribes this ultrahigh photostability and negligible photobleaching to the inefficient intersystem crossing. With these merits, DBD can be used long-term two-photon fluorescence imaging in vitro.