Optical Windows for Transcranial Brain Imaging in Living Mice: Skull Thinning, Clearing, and Beyond

ABSTRACT Longitudinal noninvasive in vivo imaging is essential for studying brain physiology and pathological mechanisms. Advances in transcranial optical windows, including thinned‐skull and optical clearing techniques, have markedly improved imaging depth and resolution when combined with multiphoton microscopy. However, their optical performance often deteriorates rapidly, and quantitative studies on long‐term stability and the causes of image quality loss remain limited. In this work, current transcranial window approaches are systematically investigated using multiphoton excited fluorescence microscopy (MPEFM) and adaptive optics, examining longevity, imaging quality and optical aberrations. These results reveal that progressive skull regrowth is a fundamental limitation across all window types, leading to substantial declines in signal quality and resolution. To address these challenges, a localized glucocorticoid (GC) delivery strategy that significantly extends window performance for up to one month is developed. Furthermore, it is demonstrated that a GC‐loaded hydrogel sealing method effectively suppressed skull regrowth while preserving optimal optical properties, offering a potential and practical route to chronic, high‐fidelity transcranial imaging. These findings provide mechanistic insight into window degradation and establish a framework for sustained and, long‐term in vivo brain imaging.