Protocol for in vivo two-photon calcium imaging of the Drosophila brain

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.