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Simultaneous sensing of cellular microenvironment parameters and redox signaling molecules by a single fluorescent probe allows an efficient access to numerous applications ranging from convenient diagnostic assays to investigation on their pathophysiological roles and cross-link. Focusing on a lack of efficient molecule tools for simultaneous intravital imaging of mitochondrial viscosity and superoxide radical anion (O 2 •-), we bridged diethylaminobenzene with diphenylphosphinate-modified pyridinium moiety by an olefinic bond to construct a styrylpyridinium-based two-photon fluorescent probe V-OS. V-OS triggered a turn-on response either at 625 nm to viscosity which blocks intramolecular rotation, or at 530 nm to O 2 •- via nucleophilic attack of O 2 •- followed by 1,6-elimination. With the aid of the two-photon probe, we not only identified the cross-talk between viscosity and O 2 •- but successfully mapped the burst of O 2 •- and the increased viscosity during either cellular ferroptosis process or cerebral ischemia reperfusion injury of living mice. [Display omitted] • Simultaneous imaging of mitochondrial viscosity and O 2 •- by a single fluorescent probe V-OS. • Turn-on response of V-OS to viscosity and O 2 •- at well-separated red and green channels. • Identifying the cross-talk between viscosity and O 2 •-. • Visualizing the generation of O 2 •- and the increased viscosity during cellular ferroptosis process. • Mapping the burst of O 2 •- and the increased viscosity during cerebral ischemia reperfusion injury in living mice. [ABSTRACT FROM AUTHOR]

Boronate-based fluorescent probes are widely used for the imaging of hydrogen peroxide (H 2 O 2). However, their selectivity might be subjected to the interference of other reactive oxygen species, especially peroxynitrite (ONOO-), due to the reaction of boronates with ONOO- being several orders of magnitude faster than with H 2 O 2. This work highlights a doubly activated electrophilicity-increasing strategy to develop a boronate-based fluorescent probe THMP for selective imaging of H 2 O 2 over ONOO-, where a boronate-modified pyridiniumylacrylonitrile is grafted on the 2-(2′-hydroxy-3′-methoxyphenyl) benzothiazole scaffold. Specifically, the boronate oxidation of THMP by H 2 O 2 leads to the release of the free fluorophore THMP-N, triggering a ratiometric fluorescence response from red to green. The presence of a doubly activated electrophilic site on the carbon-carbon double bond of THMP, by both the strong electron-withdrawing cyano group and the pyridinium moiety, allows selective oxidative cleavage of the double bond by ONOO- to an aldehyde HMBT-CHO, thereby excluding the interference of ONOO- in monitoring H 2 O 2. With the aid of the probe, we successfully visualized increased levels of H 2 O 2 during ferroptosis of HepG2 cells, and burst of H 2 O 2 in brains of live mice during cerebral ischemia reperfusion injury. [Display omitted] • Highlighting a doubly activated electrophilicity-increasing strategy. • Developing a boronate-based fluorescent probe THMP based on the strategy. • Selective imaging of H 2 O 2 over ONOO- by THMP. • Detailed mechanistic investigation. • Visualizing H 2 O 2 in brains of live mice during cerebral ischemia reperfusion. [ABSTRACT FROM AUTHOR]