<em>In Vivo</em> Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

Eun-Seo Cho; Seungjae Han; Gyuri Kim; Minho Eom; Kang-Han Lee; Cheol-Hee Kim; Young-Gyu Yoon

doi:10.3791/65218

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

インビボ 3次元蛍光顕微鏡を用いたゼブラフィッシュ幼生の全脳イメージング

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06:27 min

April 28, 2023

DOI: 10.3791/65218-v

Eun-Seo Cho¹, Seungjae Han¹, Gyuri Kim¹, Minho Eom¹, Kang-Han Lee², Cheol-Hee Kim², Young-Gyu Yoon^1,3

¹School of Electrical Engineering,KAIST, ²Department of Biology,Chungnam National University, ³KAIST Institute for Health Science and Technology

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Overview

This article presents a detailed protocol for in vivo whole-brain imaging of larval zebrafish utilizing three-dimensional fluorescence microscopy. The zebrafish model is favored due to its optical transparency and genetic accessibility to study neural computation and activity. The protocol addresses common issues such as motion artifacts and agarose gel aberrations, ensuring high-quality image data acquisition.

Key Study Components

Area of Science

Neuroscience
Biophysics
Imaging Techniques

Background

Larval zebrafish are a vital model for neuroactivity studies due to their transparency.
Significant challenges include aberrations from agarose gel and motion artifacts during imaging.
Current protocols lack detailed steps for effective sample mounting and positioning.
High-resolution imaging is critical for understanding neural computation.

Purpose of Study

To develop a reproducible protocol for high-quality imaging of zebrafish brain activity.
To reduce noise and motion artifacts during imaging sessions.
To facilitate the study of neural computation over long periods.

Methods Used

The main platform used is three-dimensional fluorescence microscopy.
The biological model involves larval zebrafish, prepared and immobilized for imaging.
An optimized experimental workflow is instituted to ensure minimal artifacts.
Critical steps include precise positioning of the zebrafish and correct imaging parameter adjustments.
Visualization of acquired data is achieved using software tools like napari.

Main Results

The imaging protocol allows for comprehensive visualization of brain areas, including neuronal structures in the forebrain, midbrain, and hindbrain.
Neuronal activity can be captured through time-series imaging, revealing significant insights into neural functions.
Clear visibility of all brain regions indicates the protocol's effectiveness.

Conclusions

This study demonstrates a novel imaging approach that enhances the understanding of zebrafish neuroactivity.
The protocol not only sets a standard for zebrafish imaging but also has implications for broader neural research.
Further development of imaging pipelines is anticipated to improve brain mapping and neural computation studies.

Frequently Asked Questions

What are the advantages of using larval zebrafish?

Larval zebrafish are advantageous due to their optical transparency, which allows for clear imaging of neural structures and activities using fluorescence microscopy.

How is the zebrafish prepared for imaging?

Zebrafish are paralyzed and positioned in a solidified agarose gel within a Petri dish to ensure stability during imaging.

What type of data is obtained from this imaging protocol?

The protocol facilitates the acquisition of volumetric structural images and time-series functional imaging of neuronal activity in the zebrafish brain.

How can this method be adapted for other applications?

While focused on brain imaging, the protocol can be adapted for visualizing other organs in larval zebrafish and potentially other transparent models.

What are some limitations of this imaging approach?

Limitations include potential artifacts from the agarose gel and any inevitable movement of the zebrafish during the imaging process.

How does the imaging method improve upon previous protocols?

This method provides a comprehensive, detailed workflow for zebrafish preparation and imaging, addressing gaps in existing protocols that often overlook critical steps.

ここでは、3次元蛍光顕微鏡を用いたゼブラフィッシュ仔魚の in vivo 全脳イメージングのプロトコルを紹介します。実験手順には、サンプル調製、画像取得、および視覚化が含まれます。

脳全体の神経活動を高い空間分解能と時間分解能で記録・解析するための光学免疫系や計算アルゴリズムを開発しています。実験用魚は研究に理想的なモデル動物です。それぞれの光透過性と多様な遺伝的ツールの利用可能性のおかげで、サンプルマウントに使用されるアガロースゲルによってもたらされる収差により光学画質が低下し、記録中に魚が移動して画像にモーションアーチファクトが発生したり、画像からの正確な信号抽出が妨げられたりする可能性があります。

公開されているプロトコルは、実験手順の簡単な概要、アガロース凝固の正確な取り付け技術、簡単な位置決めなど、詳細のかなりの部分を示しています。したがって、高品質の画像データを取得するには、効果的で再現性のあるプロトコルが必要です。最小限のノイズと動きで。

当社のプロトコルは、最適化され、再現性のある実験手順を提供します。このプロトコルは、長期間にわたるin vivo全脳イメージングと、取得したイメージングデータの視覚化を可能にします。ワークフローは全脳イメージングに焦点を当てていましたが、多くのゼブラフィッシュの他の臓器のイメージングにも簡単に適用できます。

私たちの目標は、神経計算の根底にある原理を解明することです。そのために、神経活動や構造の大規模イメージングや、体系的な脳マッピングのための計算解析を含むパイプラインに引き続き取り組んでいきます。