Recording Neural Activity in Unrestrained Animals with Three-Dimensional Tracking Two-Photon Microscopy

Doycho Karagyozov, Mirna Mihovilovic Skanata, Amanda Lesar, Marc Gershow

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360 μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates. Optically measuring neural activity in behaving animals is complicated by motion artifacts. Rigidly fixing the microscope to the animal perturbs behavior and does not work for small model organisms. Karagyozov et al. demonstrate a two-photon tracking microscope with real-time feedback to record from neurons moving rapidly in three dimensions.

Original languageEnglish (US)
Pages (from-to)1371-1383.e10
JournalCell Reports
Issue number5
StatePublished - Oct 30 2018
Externally publishedYes


  • Drosophila, 3D tracking system
  • calcium imaging
  • freely behaving larval Drosophila
  • neuroscience
  • tracking microscopy
  • two photon microscopy
  • voltage imaging

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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