Investigations into neurodegenerative progression have traditionally relied on pathological sectioning and intergroup analysis, with outcomes heavily dependent on sample size. Emerging in vivo imaging techniques now enable dynamic, long-term tracking of the same subjects, enhancing comparability and reducing variability. In this study, we use two-photon microscopy to visualize neurons and microglia through fluorescent markers introduced by adeno-associated virus (AAV) labeling and transgenic mice. Additionally, AAV-driven tau pathology induces neurodegeneration in the brain. A head ring is attached to the mouse’s skull for reversible mounting on a head-fixed treadmill. This setup allows us to image neurodegeneration progression without anesthesia, quantifying changes in the number, morphology, and spatial positioning of tau-overexpressing neurons and microglia. This approach provides spatiotemporal insights into neurodegeneration previously unattainable.