We designed a head-mounted three-photon microscope for imaging deep cortical layer neuronal activity in a freely moving rat. Delivery of high-energy excitation pulses at 1,320 nm required both a hollow-core fiber whose transmission properties did not change with fiber movement and dispersion compensation. These developments enabled imaging at >1.1 mm below the cortical surface and stable imaging of layer 5 neuronal activity for >1 h in freely moving rats performing a range of behaviors.
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