Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH₂, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO₂ for detecting nitroreductase (NTR). Upon incubated with NTR for 15min, CM-NO₂ displayed a ~90-fold fluorescence enhancement at 505nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO₂ to image NTR in tissues was demonstrated.