Recording neural activity in unrestrained animals with 3D tracking two photon microscopy

Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but such recordings are compromised by brain motion that often accompanies behavior. Two-photon point scanning microscopy is especially sensitive to motion artifacts, and to date, two-photon recording of activity has required rigid mechanical coupling between the brain and microscope. To overcome these difficulties, we developed a two-photon tracking microscope capable of tracking neurons moving with velocities of 3 mm/s and accelerations of 1 m/s² both in-plane and axially. We maintained continuous focus on targeted neurons allowing high-bandwidth measurements with modest excitation power. We recorded from motor- and inter- neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentation and behavioral outputs. Our technique can be extended to stabilize recordings in a variety of moving substrates.