zebrafish mounting, positioning, and image acquisition 

Im lebenden Organismus Ganzhirn-Bildgebung von Zebrafischlarven

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.

Autor im Rampenlicht: In vivo Ganzhirnbildgebung von Zebrafischlarven mittels dreidimensionaler Fluoreszenzmikroskopie  

Im lebenden Organismus Ganzhirnbildgebung von Zebrafischlarven mittels dreidimensionaler Fluoreszenzmikroskopie

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   

Montieren, Positionieren und Aufnehmen von Zebrafischen 

Bereiten Sie zunächst das Experiment und das Material vor, das für die Befestigung und Positionierung von Zebrafischen erforderlich ist. Beobachten Sie die gelähmte Zebrafischlarve unter einem Stereomikroskop, um sicherzustellen, dass ihre Bewegung aufgehört hat. Beurteilen Sie die Gesundheit der Larven visuell, indem Sie ihren Herzschlag überprüfen.Geben Sie dann mit einer Transferpipette eine einzelne Zebrafischlarve in das 1,5-Milliliter-Mikrozentrifugenröhrchen mit Agarosegel. Gießen Sie das Agarose-Gel in die Petrischale, um eine ein bis zwei Millimeter dicke Schicht zu erhalten. Übertragen Sie die Larve mit einer Transferpipette aus dem Mikrozentrifugenröhrchen in die Petrischale.Achten Sie darauf, die Larve in die Mitte der Schale zu legen. Positionieren Sie die Larve mit einer Pinzette in der gewünschten Ausrichtung, so dass Kopf und Schwanz flach sind. Drehen Sie dann die Larve mit einer Pinzette, um beide Augen auszurichten.Warten Sie nach Abschluss des Alignments, bis sich das Agarosegel verfestigt hat, bevor Sie die Petrischale mit Eiwasser füllen. Stellen Sie die Schale mit der eingebetteten Larve zur Bildaufnahme auf den Mikroskoptisch. Schalten Sie das Mikroskop ein und lokalisieren Sie die Larve in der Mitte des Sichtfeldes.Stellen Sie mit der Bilderfassungssoftware die Bildgebungsparameter ein. Wenn das Bild gesättigt ist, reduzieren Sie die Laserleistung. Finden Sie als Nächstes das Gehirn der Larve, indem Sie den Tisch bewegen, und bestimmen Sie seine Dicke im Live-View-Modus in der Software, indem Sie die Fokusebenen manuell nach oben und unten ändern.Legen Sie die untere und obere Grenze der Lautstärke fest. Fahren Sie dann mit der Bildaufnahme für das eingestellte Sichtfeld fort. Für die volumetrische Strukturbildgebung erfassen Sie ein 3D-Bild des gesamten Gehirns, indem Sie nacheinander 2D-Bilder jeder Z-Ebene erhalten.Für die funktionelle Bildgebung einer einzelnen Z-Ebene werden Zeitreihenbilder der neuronalen Aktivität des Gehirns in einer bestimmten Tiefe aufgenommen.

zebrafish-mounting-positioning-and-image-acquisition

Research

JoVE Journal

Neuroscience

Zebrafish Mounting, Positioning, and Image Acquisition 

740 Aufrufe.  KAIST. Bereiten Sie zunächst das Experiment und das Material vor, das für die Befestigung und Positionierung von Zebrafischen erforderlich ist. Beobachten Sie die gelähmte Zebrafischlarve unter einem Stereomikroskop, um sicherzustellen, dass ihre Bewegung aufgehört hat. Beurteilen Sie die Gesundheit der Larven visuell, indem Sie ihren Herzschlag überprüfen.Geben Sie dann mit einer Transferpipette eine einzelne Zebrafischlarve in das 1,5-Milliliter-Mikrozentrifugenröhrchen mit Agarosege...

Serie: 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 ...

Forschung

Neurowissenschaften

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.