zebrafish mounting, positioning, and image acquisition 

インビボ ゼブラフィッシュ幼生の全脳イメージング

Zebrafish (Danio rerio) has been widely adopted as a model vertebrate animal in neuroscience, owing to its optical transparency at the larval stage, its fast development, its low maintenance cost, and the availability of diverse genetic tools1,2,3,4. In particular, the optical transparency of larvae combined with genetically encoded fluorescent reporters of biological events5,6,7,8,9 provides a unique opportunity to image both neuronal activity and structure at the whole-brain level10,11,12,13,14. However, even with a microscope that supports cellular resolution, the acquired images do not necessarily retain the information at the single-cell level; the optical image quality may be degraded due to the aberration introduced by the agarose gel used for sample mounting, the fish may be mounted at an angle, thus the regions of interest are not fully contained within the field of view of the microscope, and the fish may move during the recording, causing motion artifacts in the images or impeding accurate signal extraction from the images.
Thus, an effective and reproducible protocol is needed to acquire high-quality image data with minimal noise and motion. Unfortunately, publicly available protocols for imaging a whole brain of larval zebrafish in vivo15,16,17,18,19 only briefly describe the procedure, leaving substantial parts of the details, such as agarose solidification, precise mounting techniques, and sample positioning using forceps, up to each experimentalist. In addition, inconsistencies in agarose concentration and immobilization methods10,11,14,15,16,17,18,19 can lead to challenges arising from fish movement during the imaging process.
Here, a detailed protocol for whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy is provided. Figure 1 provides a graphical overview of the protocol: sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The structural and functional imaging of the larval zebrafish brain in vivo is demonstrated using a commercial confocal microscope and a custom-designed fluorescence microscope. This protocol can be tailored by practitioners for brain imaging with certain sensory stimuli or behavior contexts depending on the experimental needs and design.

著者スポットライト:三次元蛍光顕微鏡を用いたゼブラフィッシュ幼生のin vivo全脳イメージング  

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

We are developing optical immune systems and computational algorithms to record and analyze the neuroactivity of the entire brain with a high spatial and temporal resolution. Laboratory fish is an ideal model animal for research. Thanks to each optical transparency and the availability of diverse genetic tools The optical image quality may be degraded due to the aberration introduced by the Agarose gel used for sample mounting, and the fish may move during the recording causing motion artifacts in the images or impeding accuracy signal extraction from the images.Publicly available protocols provide only a brief overview of the experimental procedure, living substantial parts of the details, such as agarose solidification precise mounting techniques, and simple positioning. Therefore, an effective and reproducible protocol is needed to acquire high quality image data. With minimal noise and motion.Our protocol provides an optimized and reproducible experimental procedure. This protocol enables in vivo whole brain imaging over an extended period and visualizations of the acquired imaging data. The workflow focused on whole brain imaging but it can be easily applied to imaging other organs of lot of our zebra fish.Our goal is to unravel the underlying principles of neural computation. For that we'll continue to work on a pipeline that involves large scale imaging of neural activity and structure and computational analysis of such for systematic brain mapping.

Watch this Scientific Journal Video about Zebrafish Mounting, Positioning, and Image Acquisition at JoVE.com

Zebrafish Mounting, Positioning, and Image Acquisition   

ゼブラフィッシュのマウント、位置決め、画像取得 

はじめに、ゼブラフィッシュの取り付けと位置決めに必要な実験と材料を準備します。実体顕微鏡で麻痺したゼブラフィッシュの幼虫を観察し、その動きが止まったことを確認します。幼虫の心拍をチェックして視覚的に健康状態を評価します。次に、トランスファーピペットを使用して、単一の仔魚ゼブラフィッシュをアガロースゲルを含む1.5ミリリットルのマイクロ遠心チューブに入れます。アガロースゲルをペトリ皿に注ぎ、1〜2ミリメートルのコートを作ります。トランスファーピペットを使用して、幼虫をマイクロ遠心チューブからペトリ皿に移します。幼虫を皿の中央に置くようにしてください。鉗子を使用して、頭と尾が平らになるように幼虫を希望の方向に配置します。次に、鉗子を使用して幼虫を回転させて両目を水平にします。アライメントが完了したら、アガロースゲルが固まるまで待ってから、ペトリ皿に卵水を入れます。幼虫が埋め込まれた皿を顕微鏡ステージに置いて画像を取得します。顕微鏡の電源を入れ、視野の中心に幼虫を配置します。画像取得ソフトウェアを使用して、イメージングパラメータを設定します。画像が飽和している場合は、レーザー出力を下げます。次に、ステージを動かして幼虫の脳を見つけ、ソフトウェアのライブビューモードを使用して、焦点面を手動で上下に変更して厚さを決定します。音量の下限と上限を設定します。次に、設定した視野の画像取得に進みます。体積構造イメージングでは、各Z面の2D画像を順次取得することで、脳全体の3D画像を取得します。単一のZ平面の機能イメージングのために、特定の深さにおける脳の神経活動の時系列画像を取得します。

zebrafish-mounting-positioning-and-image-acquisition

Research

JoVE Journal

Neuroscience

Zebrafish Mounting, Positioning, and Image Acquisition 

740 ビュー.  KAIST. はじめに、ゼブラフィッシュの取り付けと位置決めに必要な実験と材料を準備します。実体顕微鏡で麻痺したゼブラフィッシュの幼虫を観察し、その動きが止まったことを確認します。幼虫の心拍をチェックして視覚的に健康状態を評価します。次に、トランスファーピペットを使用して、単一の仔魚ゼブラフィッシュをアガロースゲルを含む1.5ミリリットルのマ?...

ビデオ: Zebrafish Mounting, Positioning, and Image Acquisition   

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

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, 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.

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

神経科学

To begin, prepare the experiment and material required for zebrafish mounting and positioning. Under a stereo microscope, observe the paralyzed zebrafish larva to verify its movement has stopped. Evaluate the larval health visually by checking its heartbeat.Then, using a transfer pipette, place a single larval zebrafish into the 1.5-milliliter micro centrifuge tube containing agarose gel. Pour the agarose gel into the Petri dish to make a one-to two-millimeter coat. Transfer the larva from the micro centrifuge tube to the Petri dish using a transfer pipette.Ensure to place the larva in the center of the dish. Using forceps, position the larva in the desired orientation so that the head and tail are flat. Then, using forceps, rotate the larva to level both eyes.After the alignment is complete, wait until the agarose gel has solidified before filling the Petri dish with egg water. Place the dish with the embedded larva on the microscope stage for image acquisition. Turn on the microscope and locate the larva at the center of the field of view.Using the image acquisition software, set the imaging parameters. If the image is saturated, reduce the laser power. Next, find the brain of the larva by moving the stage, and determine its thickness using Live View mode in the software by changing the focal planes manually up and down.Set the lower and upper limits of the volume. Then proceed with the image acquisition for the set field of view. For volumetric structural imaging, acquire a 3D image of the entire brain by obtaining 2D images of each Z-plane sequentially.For functional imaging of a single Z-plane, acquire time series images of the neuronal activity of the brain at a certain depth.

Visualizing Zebrafish Brain Structures Using Napari

After installing Napari and Napari Animation, create a new Jupyter notebook file. To visualize images and create movies of the rendered zebrafish brain, load the 3D image and open the Napari window. Then connect the Napari animation plugin.Next, set the parameters such as voxel size, color map, and contrast limits in the layer controls. Adjust the viewer settings in the canvas. Then to capture the rendered image, press the capture button in the animation wizard.To generate movies of the rendered volume, modify the viewer settings and add key frames. After adding key frames, set the number of frames between key frames in the animation wizard. Finally, save the rendered animation.The volumetric field of view of a larval zebrafish brain expressing pan neuronal GCaMP 7A covered the brain regions of the forebrain, midbrain, and hindbrain. Neuronal cell bodies of the medulla oblongata, cerebellar plate, optic tectum, hebenula, and dorsal telencephalon were clearly visible in the whole brain of a larval zebrafish. In 2D functional imaging, neuronal cell bodies were clearly visible in both the background and the superimposed neuronal activity.