These techniques have not transferred to the Drosophila larva, whose peristaltic crawling induces 3D rotations, translations, and deformations of the brain that are out of sync with the animal's external movement (Sun and Heckscher, 2016). elegans lacks a visual system, maintains rigidity through osmotic pressure, crawls smoothly on its side in a nearly planar path, and has been successfully studied using a range of fluorescence microscopy approaches (Clark et al., 2007 Faumont et al., 2011 Hendricks et al., 2012 Schrödel et al., 2013 Prevedel et al., 2014 Kato et al., 2015 Abrahamsson et al., 2016 Nguyen et al., 2016, 2017 Venkatachalam et al., 2016 Voleti et al., 2019 Nejatbakhsh et al., 2020). These challenges are exacerbated in animals lacking a rigid skull-like enclosure, due to extensive motion-induced deformations. Although many 2P methods have been developed to record neural population activity (Lecoq et al., 2019 Grienberger et al., 2022) in immobilized animals, it has been particularly challenging to track and record activities from individual neurons during free behavior. Two-photon (2P) imaging techniques image deeper tissues with less light scattering compared to methods that utilize linear (one photon) absorption processes (Helmchen and Denk, 2005). This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning.Ĭalcium imaging is a versatile tool to monitor population neural activity with single-cell resolution imaging in moving animals allows the study of the correlation between neural activity and behavior (Yang and Yuste, 2017). With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D random access scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. A previously demonstrated two-photon tracking microscope recorded from individual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals.
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