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

Area of Science:

• Neuroscience
• Microscopy
• Biophysics

Background:

• Neural activity recordings in behaving animals are crucial for understanding decision-making.
• Brain motion during behavior introduces artifacts in optical recordings, particularly with two-photon microscopy.
• Existing methods require rigid brain-microscope coupling, limiting in vivo studies.

Purpose of the Study:

• To develop a two-photon microscopy system capable of tracking and imaging neural activity in freely moving animals.
• To overcome motion artifacts that compromise neural recordings during behavior.

Main Methods:

• Development of a two-photon tracking microscope with hardware-implemented, low-latency feedback (360 μs).
• The microscope achieves stable focus on neurons with high velocities (3 mm/s) and accelerations (1 m/s²), both in-plane and axially.
• Application in unrestrained, freely behaving fruit fly larvae for calcium imaging and voltage indicator measurements.

Main Results:

• Continuous focus maintained on moving neurons, enabling artifact-free recordings.
• Successful correlation of neural activity (calcium dynamics) with stimuli and behavior in fruit fly larvae.
• Demonstrated measurement of neural depolarization in a moving animal using a voltage indicator.

Conclusions:

• The developed two-photon tracking microscope significantly reduces motion artifacts in neural recordings.
• This technology allows for stable optical imaging of neural circuits in freely moving subjects.
• The technique is adaptable for stabilizing recordings in various moving biological substrates.