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

## TL;DR

This paper provides a detailed protocol for using two-photon calcium imaging to study brain activity in live fruit flies.

## Contribution

The paper introduces a step-by-step in vivo two-photon calcium imaging protocol for Drosophila brain research.

## Key findings

- The protocol allows real-time monitoring of neuronal activity in live fruit flies.
- It includes procedures for surgery, recording, and data analysis for functional brain mapping.
- The method supports optogenetics to study functional connectivity.

## 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

•Protocol for two-photon calcium imaging in live fruit flies•Preparation of the recording chamber and buffer solution•Guidance for fly brain surgery before recording•Imaging and analysis of calcium activities of target neurons

Protocol for two-photon calcium imaging in live fruit flies

Preparation of the recording chamber and buffer solution

Guidance for fly brain surgery before recording

Imaging and analysis of calcium activities of target neurons

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.