Fast Imaging of Mitochondrial Thioredoxin Reductase Using a Styrylpyridinium-Based Two-Photon Ratiometric Fluorescent Probe

Thioredoxin reductase (TrxR) is a pivotal antioxidant enzyme, but there remains a challenge for its fast imaging. This work describes the combination of a hydroxyl styrylpyridinium scaffold as the push–pull fluorophore with a carbonate-bridged 1,2-dithiolane unit as the reaction site to develop a fast mitochondrial TrxR2 probe, DSMP. It manifested a plethora of excellent properties including a rapid specific response (12 min), large Stokes shift (170 nm), ratiometric two-photon imaging, favorable binding with TrxR (Km = 12.5 ± 0.2 μM), and the ability to cross the blood–brain barrier. With the aid of DSMP, we visualized the increased mitochondrial TrxR2 activity in cancer cells compared to normal cells. This offers the direct imaging evidence of the connection between the increased TrxR2 activity and the development of cancer. Additionally, the probe allowed the visualization of the loss in TrxR2 activity in a cellular Parkinson's disease model and, more importantly, in mouse brain tissues of a middle cerebral artery occlusion model for ischemic stroke.