CRASH2p: Closed-loop Two Photon 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.
• Genetically encoded calcium indicators are widely used but susceptible to motion artifacts.
• Existing two-photon microscopy methods require stable brain anchoring, limiting applications in unrestrained subjects.

Purpose of the Study:

• To develop a novel two-photon microscopy system for real-time 3D motion correction in freely moving animals.
• To enable quantitative functional imaging without the need for implanted reference markers.
• To uncover neural correlates of behavior in unrestrained organisms.

Main Methods:

• Development of a closed-loop resonant axial-scanning high-speed two-photon (CRASH2p) microscope.
• Implementation of a novel 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:

• The CRASH2p microscope successfully performed real-time 3D motion correction in unrestrained *Drosophila* larvae.
• Quantitative functional imaging was achieved despite significant brain motion.
• Previously unknown neural activity patterns associated with specific behaviors were identified.

Conclusions:

• The CRASH2p microscope overcomes motion artifacts in two-photon imaging of neural activity.
• This technology enables unprecedented quantitative analysis of neural dynamics in behaving animals.
• The findings open new avenues for studying brain-behavior relationships in naturalistic settings.