Revealing the Photodynamic Stress In Situ with a Dual-Mode Two-Photon ¹O₂ Fluorescent Probe

Singlet oxygen (¹O₂) plays significant physiological and pathological functions, especially in causing photodynamic stress in vivo. However, specific ¹O₂ monitoring is an immense challenge, owing to its short half-lives and high oxidizing ability. To address this, we engineered three photostable two-photon fluorescence probe NBs for highly efficient ¹O₂ monitoring based on bioinspired novel tryptophan derivatives, among which NB-MOT was the best one comprehensively. Upon being cracked with ¹O₂, NB-MOT rapidly (within 5 s) demonstrated a remarkable enhancement in fluorescence intensity (∼180 fold) and lifetime (∼18 fold). Taking these advantages into account, NB-MOT was applied to evaluate exogenous and endogenous ¹O₂ in diverse biosystems. We successfully tracked the intracellular ¹O₂ level during photodynamic therapy, and for the first time achieved ¹O₂ mapping in live cells with dual-mode imaging as well as revealed ciprofloxacin-induced photodynamic stress in mice. NB-MOT was thus believed to be of instructive significance for studying the ¹O₂-mediated stress in wider biological milieus.