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In vivo whole-brain imaging of zebrafish larvae using three-dimensional fluorescence microscopy

Abstract

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor the whole-brain activity at cellular resolution. Here we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy that includes sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. Our protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at cellular resolution over an hour using confocal microscopy and custom-designed fluorescence microscopy. We discuss the critical steps in the protocol, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

This repository contains the source code we used for creating visualizations with Napari in Protocol Steps 5-7.

Installation

The installation time may depend on your network speed, but it generally takes less than 20 minutes.

  1. Clone the repository
git clone [email protected]:NICALab/Zebrafish-brain-visualization.git
  1. Navigate into the cloned folder
cd ./Zebrafish-brain-visualization
  1. Create the conda environment
conda env create -f env.yml
  1. Activate the conda environment
conda activate Zebrafish-brain-visualization

Getting Started

1. Download demo data

Demo data can be downloaded from Google Drive Link

2. (Optional) Decompose background and activity from data using BEAR

Run BEAR_v2_JoVE.m in the “BEAR_MATLAB_v2” folder.

3. Run demo notebook

Run Visualizing_structure.ipynb or Visualizing_neuronal_activity.ipynb.

Data availability

Demo data can be downloaded from Google Drive Link

Contributors

We are happy to help with any questions or requests. Please contact the following authors to get in touch!

Citation

Cho, E.-S. et al. Imaging whole-brain of larval zebrafish in vivo using three-dimensional fluorescence microscopy. in revision (2023).

@article {Cho2023,
	author = {Cho, Eun-Seo and Han, Seungjae and Kim, Gyuri and Eom, Minho and Lee, Kang-Han and Kim, Cheol-Hee and Yoon, Young-Gyu},
	title = {In vivo whole-brain imaging of zebrafish larvae using three-dimensional fluorescence microscopy},
	year = {2023},
	doi = {doi:10.3791/65218},
	publisher = {Journal of Visualized Experiments (JoVE)},
	URL = {https://www.jove.com/t/65218},
	eprint = {https://www.jove.com/kr/pdf/65218/jove-protocol-65218-in-vivo-whole-brain-imaging-zebrafish-larvae-using-three-dimensional},
	journal = {Journal of Visualized Experiments (JoVE)}
}