Three-photon population imaging of subcortical brain regions

Monitoring large-scale neuronal activity in deep brain circuits is crucial for understanding the principles of brain function. However, existing large-scale cellular-resolution imaging techniques are limited in imaging depth and cannot monitor activity in areas deeper than the cortex in the intact brain. To address this challenge, we developed a Large Imaging Field of view Three-photon (LIFT) microscope with a FOV of > 3 mm2. LIFT combines high-numerical-aperture optimized sampling, using a custom scanning module, with deep learning-based denoising, to enable population imaging in subcortical brain regions of mice and deep cortical regions of rats. We demonstrate calcium imaging in the intact mouse brain from more than 1500 cells across CA1, the surrounding white matter, and adjacent deep layers of the cortex, and show population imaging with high SNR at a depth of 1.2 mm in the rat cortex. The LIFT microscope offers versatility in imaging scale and rate and was built entirely from off-the-shelf components. Imaging large cell populations below the cortex in the intact brain is key for understanding brain function. LIFT, a multiscale three-photon microscope, tracks activity in over 1,500 cells across the hippocampus and adjacent regions in behaving mice.