Toxicity assessment and long-term three-photon fluorescence imaging of bright aggregation-induced emission nanodots in zebrafish

Aggregation-induced emission (AIE) luminogen displays bright fluorescence and has photobleaching resistance in its aggregation state. It is an ideal fluorescent contrast agent for bioimaging. Multiphoton microscopy is an important tool for bioimaging since it possesses the ability to penetrate deep into biological tissues. Herein, we used AIE luminogen together with multiphoton microscopy for long-term imaging of zebrafish. A typical AIE luminogen, 2, 3-bis(4-(phenyl(4- (1, 2, 2-triphenylvinyl) phenyl)amino)phenyl) fumaronitrile (TPE-TPA-FN or TTF), was encapsulated with 1, 2-distearoyl-sn-glycero-3-phosphoethanola-mine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG₂₀₀₀) to form nanodots that exhibited bright three-photon fluorescence under 1, 560 nm-femtosecond (fs) laser excitation. The TTF-nanodots were chemically stable in a wide range of pH values and showed no in vivo toxicity in zebrafish according to a series of biological tests. The TTF-nanodots were microinjected into zebrafish embryos, and the different growth stages of the labeled embryos were monitored with a three-photon fluorescence microscope. TTF-nanodots could be traced inside the zebrafish body for as long as 120 hours. In addition, the TTF-nanodots were utilized to target the blood vessel of zebrafish, and three-photon fluorescence angiogram was performed. More importantly, these nanodots were highly resistant to photobleaching under 1, 560 nm-fs excitation, allowing long-term imaging of zebrafish.