Three photon microscopy of mouse brain structure and function at 2 mm depth and beyond

High-resolution noninvasive imaging of neuronal activity at single-cell resolution deep within brain tissue is essential for understanding brain function and disease. Here we show that an improved 1300-nm three-photon microscope for maximal excitation and collection efficiency enables imaging up to the three-photon depth limit in the intact mouse brain. Our platform achieves structural imaging of brain vasculature at depths up to 2.5 mm and functional imaging of neural activities at depths up to 2 mm, reaching deep brain regions previously inaccessible by multiphoton imaging. These advances extend the frontier of deep-tissue functional imaging and open new possibilities for longitudinal and mechanistic studies in neuroscience and beyond.