Ultra-wide-field, deep, adaptive two-photon microscopy for multi-scale neuronal imaging

Abstract Observing the activity patterns of large neural populations throughout the brain is essential for understanding brain function. However, capturing neural interactions across widely distributed brain regions from both superficial and deep cortical layers remains challenging with existing microscopy technologies. Here, we introduce a state-of-the-art two-photon microscopy system, ULTRA, capable of single-cell resolution imaging across an ultra-large field of view (FOV) exceeding 50 mm², enabling deep and wide field in vivo imaging. To demonstrate its capabilities, we conducted a series of experiments under multiple imaging conditions, successfully visualizing brain structures and neuronal activities spanning a spatial range of over 7 mm from superficial layers to depths of up to 900 μm, while covering a volume of 45.24 mm 3 in the mouse brain. This versatile imaging platform overcomes traditional spatial constraints, providing a powerful tool for comprehensive exploration of neuronal circuitry over extensive spatial scales with cellular resolution.