Rational design of a nitroreductase-activatable two-photon fluorescent probe for hypoxia imaging in cell and in vivo

•We developed a two-photon fluorescent probe, TP-NO2, with simple synthesis steps and high yield.•TP-NO2 could determination of nitroreductase in a wide range (0−20 μg/mL) with a low detection limit of 26 ng/mL.•TP-NO2 could be applied for nitroreductase detection in hypoxia cells and tumor-bearing mice models.•TP-NO2 is a promising tool for clinical surgery pre-evaluation during tumor treatment via nitroreductase detection.