NIR‐II Light Field Microscopy for Through‐Skull Hemodynamic Volumetric Imaging in Awake Mice

Non‐invasive, dynamic monitoring of cerebral blood flow in awake animals is essential to a comprehensive understanding of the hemodynamics associated with natural sensorimotor activities. This requires both high imaging speed and deep tissue penetration for microscopy. Current second near‐infrared window (NIR‐II, 1000–1700 nm) fluorescence microscopy techniques, including multi‐photon microscopy and light‐sheet microscopy, offer greater imaging depth due to low scattering properties, but are still limited by their imaging speed. In contrast, light‐field microscopy (LFM) enables 3D image reconstruction from a single‐shot, facilitating high‐speed volumetric imaging but with limited depth penetration. Here, a NIR‐II light field microscopy (NIR‐II LFM) system is developed for real‐time 3D monitoring of cerebral blood vessels in awake mice, combined with skull‐clearing treatment. With high speed and reduced light scattering, the system achieves volumetric vessel imaging, monitors blood flow dynamics, and performs 3D cerebral hemodynamic analysis. Additionally, in an acute pathology model, the system enables real‐time observation of cerebral thrombus formation in the cortex through the skull in awake mice. As a novel optical approach, NIR‐II LFM not only facilitates dynamic 3D imaging of cerebral vasculature in vivo but also demonstrates unique advantages in studying acute vascular pathologies and beyond.