Near-Infrared in and out: Observation of Autophagy during Strokevia a Lysosome-Targeting Two-Photon Viscosity-Dependent Probe

Fluorescence imaging using probes with two-photon excitation andnear-infrared emission is currently the most popularin situmethod for monitoringbiological species or events, with a large imaging depth, low backgroundfluorescence,low optical damage, and high spatial and temporal resolution. Nevertheless, currentfluorescent dyes with near-infrared emission still have some disadvantages such as poorwater solubility, lowfluorescence quantum yield, and small two-photon absorptioncross sections. These drawbacks are mainly caused by the structural characteristics ofdyes with large conjugation surfaces but lacking strong and rigid structures. Herein, alysosome-targeted and viscosity-sensitive probe (NCIC-VIS) is designed andsynthesized. The protonation of morpholine not only helps anchor NCIC-VIS tothe lysosome but also significantly enhances its water solubility. More importantly, its viscosity can increase the rigid structure ofNCIC-VIS, which will improve thefluorescence quantum yield and the two-photon absorption cross section due to the imposedrestrictions on molecular torsion. Based on the abovementioned characteristics, the real-time imaging of cellular autophagy (couldincrease the viscosity of lysosomes) was realized using NCIC-VIS. The results demonstrated that the level of autophagy wassignificantly enhanced in mice during stroke, while the inhibition of oxidative stress significantly reduced the degree of autophagy.The study corroborates that oxidative stress induced by stroke can lead to the development of autophagy.