Label-free characterization of ischemic cerebral injury using intravital two-photon excitation fluorescence lifetime imaging microscopy

Characterizing an ischemic brain injury at its early stage is critical to biological research and the clinical diagnosis of cerebral ischemia-related diseases. However, approaches with intravital, label-free, and real-time characterization capabilities are scarce. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) can detect variations in energy metabolism based on the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH). Using this unique feature, we proposed a novel approach for cerebral ischemia characterization. From investigating cell and animal models, the cerebral NADH fluorescence lifetime was observed to be sensitive to metabolic changes caused by ischemia and consistent with ischemic time. A comparison with standard blood flow imaging and neuronal injury assessment further suggests that, the two-photon FLIM, using NADH as an indicator, can characterize degrees of cerebral ischemia and related injuries, particularly at the early stage. These findings demonstrate that NADH FLIM is promising for providing intravital, label-free, and real-time assessment of cerebral ischemia and ischemic brain injury that will be significant to the study and diagnosis of related diseases.