A Ratiometric Two-Photon Fluorescent Cysteine Probe with Well-Resolved Dual Emissions Based on Intramolecular Charge Transfer-Mediated Two-Photon-FRET Integration Mechanism.

The development of an efficient ratiometric two-photon fluorescence imaging probe is crucial for in situ monitoring of biothiol cysteine (Cys) in biosystems, but the current reported intramolecular charge transfer (ICT)-based one suffers from serious overlap between the shifted emission bands. To address this issue, we herein for the first time constructed an ICT-mediated two-photon excited fluorescence resonance energy transfer (TP-FRET) system consisting of a two-photon fluorogen benzo[ h]chromene and a Cys-responsive benzoxadiazole-analogue dye. Different from a previous mechanism that utilized single two-photon fluorogen to acquire a ratiometric signal, ICT was used to switch on the TP-FRET process of the energy transfer dyad by eliciting an absorption shift of benzoxadiazole with Cys to modulate the spectral overlap level between benzo[ h]chromene emission and benzoxadiazole absorption, resulting in two well-separated emission signal changes with large emission wavelength shift (120 nm), fixed two-photon excitation maximum (750 nm), and significant variation in fluorescence ratio (over 36-fold). Therefore, it can be successfully employed to ratiometrically visualize Cys in HeLa cells and liver tissues. Importantly, this new ICT-mediated TP-FRET integration mechanism would be convenient for designing ratiometric two-photon fluorescent probes with two well-resolved emission spectra suitable for high resolution two-photon fluorescence bioimaging.