Section F of the American Association for the Advancement of Science

Rue, George R. La

doi:10.1126/science.78.2025.357.b

Cited by 1 publication

(1 citation statement)

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“…Techniques that work well in other models - light sheet, lightfield, and confocal microscopy, or various tethering schemes - have not translated to the larva (supplemental discussion 10). Workarounds, including interpreting motor neuron activity in dissected or immobilized animals [61, 65–67] and photoswitchable activity markers [68, 69], have ameliorated but not eliminated this difficulty. CRASH2p allows functional two photon imaging with ratiometric correction in unrestrained larvae, further advancing the utility of this model organism.…”

Section: Discussionmentioning

confidence: 99%

CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals

McNulty,

Wu,

Yamaguchi

et al. 2024

Preprint

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Direct measurement of neural activity in freely moving animals is essential for understanding how the brain controls and represents behaviors. Genetically encoded calcium indicators report neural activity as changes in fluorescence intensity, but brain motion confounds quantitative measurement of fluorescence. Translation, rotation, and deformation of the brain and the movements of intervening scattering or auto-fluorescent tissue all alter the amount of fluorescent light captured by a microscope. Compared to single-photon approaches, two photon microscopy is less sensitive to scattering and off-target fluorescence, but more sensitive to motion, and two photon imaging has always required anchoring the microscope to the brain. We developed a closed-loop resonant axial-scanning high-speed two photon (CRASH2p) microscope for real-time 3D motion correction in unrestrained animals, without implantation of reference markers. We complemented CRASH2p with a novel scanning strategy and a multi-stage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely movingDrosophilalarvae and discovered previously unknown neural correlates of behavior.

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