Closed-loop two-photon functional imaging in a freely moving animal
Abstract 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 measurem...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-60648-x |
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| author | Paul McNulty Rui Wu Akihiro Yamaguchi Ellie S. Heckscher Andrew Haas Amajindi Nwankpa Mirna Mihovilovic Skanata and Marc Gershow |
| author_facet | Paul McNulty Rui Wu Akihiro Yamaguchi Ellie S. Heckscher Andrew Haas Amajindi Nwankpa Mirna Mihovilovic Skanata and Marc Gershow |
| author_sort | Paul McNulty |
| collection | DOAJ |
| description | Abstract 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 autofluorescent 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 ‘Pong’ scanning strategy and a multi-stage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely moving Drosophila larvae and discovered previously unknown neural correlates of behavior. |
| format | Article |
| id | doaj-art-3d7694be18e04f208d827ab6bf007e37 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-3d7694be18e04f208d827ab6bf007e372025-08-20T04:01:35ZengNature PortfolioNature Communications2041-17232025-07-0116112210.1038/s41467-025-60648-xClosed-loop two-photon functional imaging in a freely moving animalPaul McNulty0Rui Wu1Akihiro Yamaguchi2Ellie S. Heckscher3Andrew Haas4Amajindi Nwankpa5Mirna Mihovilovic Skanata6and Marc Gershow7Department of Physics, New York UniversityDepartment of Physics, New York UniversityDepartment of Physics, New York UniversityDepartment of Molecular Genetics and Cell Biology, University of ChicagoDepartment of Physics, New York UniversityDepartment of Physics, New York UniversityDepartment of Physics, New York UniversityDepartment of Physics, New York UniversityAbstract 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 autofluorescent 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 ‘Pong’ scanning strategy and a multi-stage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely moving Drosophila larvae and discovered previously unknown neural correlates of behavior.https://doi.org/10.1038/s41467-025-60648-x |
| spellingShingle | Paul McNulty Rui Wu Akihiro Yamaguchi Ellie S. Heckscher Andrew Haas Amajindi Nwankpa Mirna Mihovilovic Skanata and Marc Gershow Closed-loop two-photon functional imaging in a freely moving animal Nature Communications |
| title | Closed-loop two-photon functional imaging in a freely moving animal |
| title_full | Closed-loop two-photon functional imaging in a freely moving animal |
| title_fullStr | Closed-loop two-photon functional imaging in a freely moving animal |
| title_full_unstemmed | Closed-loop two-photon functional imaging in a freely moving animal |
| title_short | Closed-loop two-photon functional imaging in a freely moving animal |
| title_sort | closed loop two photon functional imaging in a freely moving animal |
| url | https://doi.org/10.1038/s41467-025-60648-x |
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