Simultaneous tracking of autophagy and oxidative stress during stroke with an ICT-TBET integrated ratiometric two-photon platform

Over recent years, fluorescent probes exhibiting simultaneous responses to multiple targets have been developed for in situ, real-time monitoring of cellular metabolism using two photon fluorescence sensing techniques due to numerous advantages including ease of operation, rapid reporting, high resolution, long visualization time and being non-invasive. However, due to interference from different fluorescence channels during simultaneous monitoring of multiple targets and the lack of ratiometric capability amongst the available probes, the accuracy in tracing metabolic processes has been restricted. With this research, using a through-bond energy transfer (TBET) mechanism, we designed a viscosity and peroxynitrite (ONOO-) mitochondria-targeting two-photon ratiometric fluorescent probe Mito-ONOO. Our results indicated that with decreasing levels of mitochondrial viscosity and increasing levels of ONOO-, the maximum of the emission wavelength of the probe shifted from 621 nm to 495 nm under 810 nm two-photon excitation. The baselines for the two emission peaks were significantly separated (Δλ = 126 nm), improving the resolution and reliability of bioimaging. Moreover, by ratiometric analysis during oxygen-glucose deprivation/reoxygenation (OGD/R, commonly used to simulate cell ischemia/reperfusion injury), the real-time visualization of the metabolic processes of autophagy and oxidative stress was possible. Our research indicated that during cellular oxygen-glucose deprivation/reoxygenation, cells produce ONOO-, causing cellular oxidative stress and cellular autophagy after 15 min, as such Mito-ONOO exhibits the potential for the monitoring and diagnosis of stroke, as well as providing insight into potential treatments, and drug design.