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.