A Mitochondria-Localized Iridium(III) Complex for Simultaneous Two-Photon Phosphorescence Lifetime Imaging of Downstream Products N2O3 and ONOO– of Endogenous Nitric Oxide

Nitric oxide (NO) serves as a ubiquitous and fundamental signaling molecule involved in intricate effects on both physiological and pathological processes. NO, biosynthesized by nitric oxide synthase (NOS) or generated from nitrite, can form nitrosation reagent N₂O₃ (4NO + O₂ = 2N₂O₃) through its oxidation or quickly produce peroxynitrite anion ONOO^- (NO + •O₂^- = ONOO^-) by reacting with superoxide anion (•O₂^-). However, most of the existing luminescent probes for NO just focus on specificity and utilize only a single signal to distinguish products N₂O₃ or ONOO^-. In most of the present work, they differentiate one product from another simply by fluorescence signal or fluorescence intensity, which is not enough to distinguish accurately the behavior of NO in living cells. Herein, a new mitochondria-targeted and two-photon near-infrared (NIR) phosphorescent iridium(III) complex, known as Ir-NBD, has been designed for accurate detection and simultaneous imaging of two downstream products of endogenous NO, i.e., N₂O₃ and ONOO^-. Ir-NBD exhibits a rapid response to N₂O₃ and ONOO^- in enhanced phosphorescence intensity, increased phosphorescence lifetime, and an exceptionally high two-photon cross-section, reaching values of 78 and 85 GM, respectively, after the reaction. Furthermore, we employed multiple imaging methods, phosphorescence intensity imaging, and phosphorescence lifetime imaging together to image even distinguish N₂O₃ and ONOO^- by probe Ir-NBD. Thus, coupled with its excellent photometrics, Ir-NBD enabled the detection of the basal level of intracellular NO accurately by responding to N₂O₃ and ONOO^- in the lipopolysaccharide-stimulated macrophage model in virtue of fluorescence signal and phosphorescence lifetime imaging, revealing precisely the endogenous mitochondrial NO distribution during inflammation in a cell environment.