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In-vivo and Ex-vivo Calcium Imaging of an Olfactory Circuit Neuron in the Third-Instar Drosophila melanogaster Larva
Published on: October 31, 2025
Protocol for in vivo two-photon calcium imaging of the Drosophila brain
Yue Tian1, Ruihan Jiang1, Fang Guo1
1Department of Neurobiology, Department of Neurology of Sir Run Run Shaw Hospital and School of Brain Science and Brain Medicine, Zhejiang University School of Medicine, Hangzhou 310058, China; MOE Frontier Science Center for Brain Research and Brain-Machine Integration, State Key Laboratory of Brain-Machine Intelligence, Zhejiang University, 1369 West Wenyi Road, Hangzhou 311121, China; NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, Hangzhou 310058, China.
Abstract:
Two-photon calcium imaging facilitates the real-time observation of neuronal activity. Here, we present a protocol for conducting in vivo two-photon calcium imaging of the Drosophila melanogaster brain. We describe steps for fly preparation, recording chamber construction, and preparation of the buffer solution. We then detail procedures for fly brain surgery, execution of the recording, and data analysis. This protocol enables the monitoring and assessment of neuronal responses to external stimuli and the mapping of functional connectivity coupled with optogenetics. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.
