LONGITUDINAL THREE-PHOTON IMAGING OF NEURAL STEM CELL DYNAMICS IN THE INTACT ​MOUSE BRAIN

Multiphoton imaging is essential for studying cellular plasticity; however, accessing deep structures like the hippocampal dentate gyrus (DG) typically requires invasive surgical removal of overlying cortex, potentially altering brain physiology. Here, we optimized a three-photon (3P) microscopy approach to achieve high-resolution, longitudinal imaging of the DG in the completely intact mouse brain at unprecedented depths of up to 1800 µm. By utilizing a ~1650 nm excitation wavelength and a reduced pulse repetition rate (250 kHz), we achieved high pulse energy for deep penetration while maintaining low average power to ensure thermal safety. Using this platform, we performed chronic lineage tracing of neural stem cells (NSCs) and their progeny in the adult DG for over 80 days. Comparative analysis showed that while NSC division and survival rates were consistent with previous two-photon (2P) datasets, our 3P approach preserved cortical integrity and showed no signs of inflammatory or thermal damage. Furthermore, we successfully applied this method to the early postnatal brain (P5–8), capturing NSC structures in the intact brain previously inaccessible to chronic imaging. To facilitate post-hoc lineage tracing, we integrated deep learning-based denoising (Noise2Void) to enhance image contrast at extreme depths. Our findings establish optimized 3P imaging as a non-invasive standard for studying structural plasticity in deep brain regions. This approach opens novel avenues for investigating cellular dynamics across the lifespan in the truly intact mammalian brain.