Label-Free Third-Harmonic Generation (THG) Imaging for Cerebellar Research: Layer-Specific Structural Imaging and Long-Term Pathological Tracking

The cerebellum possesses a unique and delicate cytoarchitecture that is essential for motor coordination and cognitive processing. However, persistent technical limitations hinder precise, deep, and longitudinal in vivo imaging of the cerebellar structure and function by using endogenous signals. In this work, we present third-harmonic-generation (THG) imaging as a robust platform for long-term cerebellar observation. By employing low-power second near-infrared (NIR-II) 1300 nm excitation, we achieved real-time in vivo THG imaging of the mouse cerebellar gray matter, revealing its three-layer structure and histological morphology. Then, we combined the THG signal with fluorescence signal to identify the unique structure signals from erythrocytes and myelin sheath in different layers. Furthermore, we successfully longitudinally quantitatively monitored pathological progression in cuprizone-induced demyelination over 9 weeks and tracked postischemic recovery dynamics in photothrombosis models. In summary, our work affirms that the THG signal could demonstrate the physiological and pathological changes of regional cerebellar structures, a finding which provides a potential longitudinal tracking and label-free in vivo means for exploring the mechanism of cerebellar development and disease in three layers and white matter (WM) within the deep region.