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CRASH2p: Closed-loop Two Photon Imaging in a Freely Moving Animal.

Paul McNulty¹, Rui Wu¹, Akihiro Yamaguchi¹

¹Department of Physics,New York University, New York, USA.

Biorxiv : the Preprint Server for Biology

|June 3, 2024

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We developed a new microscope (CRASH2p) to accurately measure neural activity in freely moving animals by correcting for brain motion. This technique reveals new insights into neural activity and behavior.

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.

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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.