A two-photon fluorescent probe for endogenous superoxide anion radical detection and imaging in living cells and tissues

Superoxide anion radical (O2−), the “primary” reactive oxygen species (ROS) in living systems, is linked to a variety of physiological and pathological processes. Therefore, developing an effective strategy to monitor the fluctuation of O2− in biological systems is of great importance. This paper describes a new turn-on two-photon fluorescent probe for endogenous O2− detection and imaging, which was rationally designed and synthesized via a non-redox strategy. In the presence of O2−, the probe exhibited notable fluorescence enhancement (∼235-fold) with a low detection limit down to 1 nM, indicating a high signal-to-background ratio and excellent sensitivity. In addition, short response time, good biocompatibility, low cytotoxicity, long-term stability against light illumination, specificity to O2− over general reductants, and pH stability were demonstrated, indicating that the requirements for cellular O2− determination are met. Furthermore, the probe was successfully applied in two-photon fluorescence imaging of endogenous O2− in living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ∼150 μm, illustrating the promising potential for practical applications in complex biosystems and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of O2−.