Long-Term Optical Access to an Estimated One Million Neurons in the Live Mouse Cortex

A major technological goal in neuroscience is to enable the interrogation of individual cells across the live brain. By creating a curved glass replacement to the dorsal cranium and surgical methods for its installation, we developed a chronic mouse preparation providing optical access to an estimated 800,000-1,100,000 individual neurons across the dorsal surface of neocortex. Post-surgical histological studies revealed comparable glial activation as in control mice. In behaving mice expressing a Ca²⁺ indicator in cortical pyramidal neurons, we performed Ca²⁺ imaging across neocortex using an epi-fluorescence macroscope and estimated that 25,000-50,000 individual neurons were accessible per mouse across multiple focal planes. Two-photon microscopy revealed dendritic morphologies throughout neocortex, allowed time-lapse imaging of individual cells, and yielded estimates of >1 million accessible neurons per mouse by serial tiling. This approach supports a variety of optical techniques and enables studies of cells across >30 neocortical areas in behaving mice.