Closed-loop two-photon functional imaging in a freely moving animal

Area of Science:

• Neuroscience
• Biophysics
• Microscopy

Background:

• Direct measurement of neural activity in freely moving animals is crucial for understanding brain function.
• Brain motion, scattering, and autofluorescence complicate quantitative fluorescence measurements.
• Existing two-photon microscopy requires brain anchoring, limiting applications in unrestrained subjects.

Purpose of the Study:

• To develop a novel microscopy technique for real-time 3D motion correction in unrestrained, freely moving animals.
• To enable quantitative functional imaging of neural activity despite significant brain motion.
• To discover novel neural correlates of behavior in Drosophila larvae.

Main Methods:

• Development of a closed-loop resonant axial-scanning high-speed two-photon (CRASH2p) microscope.
• Implementation of a 'Pong' scanning strategy and a multi-stage registration pipeline for motion correction.
• Volumetric ratiometrically corrected functional imaging in the central nervous system (CNS) of Drosophila larvae.

Main Results:

• Successfully performed real-time 3D motion correction in unrestrained Drosophila larvae without reference markers.
• Achieved quantitative, volumetric, ratiometrically corrected functional imaging of neural activity.
• Discovered previously unknown neural activity patterns associated with larval behavior.

Conclusions:

• The CRASH2p microscope effectively overcomes motion artifacts in in vivo neural imaging.
• This technology provides a powerful tool for studying neural circuits controlling behavior in freely moving organisms.
• The findings open new avenues for investigating brain function and neural representations of behavior.