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Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Instructor Prep
concepts
Student Protocol
JoVE Journal
Biology
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JoVE Journal Biology
Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

1. Zebrafish Husbandry, Embryo Generation, and Treatment

  1. Conduct the following zebrafish protocols under the guidance of an institutional animal care and use committee (IACUC) and within animal care guidelines of the NIH or other regulatory bodies/guidelines.
  2. Zebrafish strains that express fluorescent proteins in specific tissues, cells, or organs are available from the Zebrafish International Resource Center (ZIRC). For example, Tg (kdr:EGPF)s843 express EGFP in vascular endothelial cells6, which can be used to produce complete 3D vascular structures as shown in this protocol. Other lines of transgenic zebrafish are available from ZIRC.
  3. House adult zebrafish in an appropriate aquaculture system that monitors pH, salinity, temperature, dissolved oxygen, light, and other environmental factors7. The zebrafish shown here were housed in an Aquaneering Inc. system (San Diego, CA) at 28.5 °C with a 14 hour light/10 hour dark cycle. Feed adult zebrafish a balanced diet of brine shrimp and NRD 4/6 Fish food (Brine Shrimp Direct, Ogden, Utah).
  4. Adult male and female zebrafish of breeding age should be housed separately to increase mating successful.
  5. Stimulate egg laying and fertilization by placing females (2-4) and males (4-6) together in a mating container that has a mesh bottom with holes large enough for the eggs to fall through, but too small for the zebrafish adults to pass. Set up matings the evening before; eggs are laid near dawn, usually while the daytime light cycle slowly increases in intensity (dawn). Check for eggs at the bottom of the mating container every 15-30 minutes.
  6. Collect eggs using a mesh strainer and clean with E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Transfer the eggs to 100 mm culture plates filled with E3 buffer and store in a 28 °C incubator.
  7. To study chemicals that alter cerebrovascular branching add desired chemical concentrations. For example neovasular branching can be induced with γ-secretase inhibitor (GSI IX/DAPT/N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butyl ester) solubilized in DMSO4, beginning 24 hours post fertilization (hpf). At that time-point embryos are still within the chorion, and many chemicals can pass through it8. If a treatment condition has neural or motor effects that may impair the ability of embryos to break free of the chorion, then embryos should be de-chorionated between 24 and 48 hpf, which can be done with either forceps of pronase2. Embryos shown in the images provided were dechorionated by gently grabbing the chorion with two sharpened forceps and tearing it open.
  8. If needed/desired, pigment formation can be inhibited by adding 0.003% N-Phenylthiourea (PTU) to the E3 buffer at 24 hpf.

2. Confocal Imaging of Cerebrovascular Structures in Fixed Zebrafish Embryos

  1. Sacrifice embryos in 250 mg/L Tricaine methanesulfonate, and then fix them by immersion in 2 – 4% paraformaldehyde overnight at 4 °C. Containers with fluorescent embryos should be wrapped in foil. Once fixed, embryos should be stored in PBS at 4 °C until imaged – EGFP fluorescence intensity becomes less resolved after about a week, but morphology (as viewed in bright field) is preserved much longer.
  2. Prepare to mount the embryo by first removing one eye using a sharpened tungsten needle. Cut around the tissue connecting the eye first then cut the muscles, and finally cut the optic nerve to displace the eye. (Note: If imaging both sides is desired remove both eyes.)
  3. Once the eye is removed, mount the embryo on a coverglass using a drop of 3% methylcellulose. Orient the embryo so that the side with the removed eye is facing the coverglass and is as close to the glass as possible. Cover the entire embryo with methylcellulose to prevent desiccation during imaging.
  4. Image the mounted embryo immediately using an inverted confocal microscope equipped with a high-quality 20x Plan Apo objective (numerical aperture = 0.75 or better). This configuration is preferable to a non-inverted microscope that would require sandwiching the embryo between two glass planes, and pressing the embryo against the top glass.
  5. Collect optical slices in 1 µm increments using a medium or large aperture setting. Larger steps of 2.5 µm can also be used, but it may be more difficult to determine the spatial order of smaller objects. Small apertures produce sharper detail, but the longer scans required may bleach EGFP and also limit the depth of imaging into the embryo that can be achieved. A confocal microscope allows for imaging about half-way through an embryo.
  6. If imaging of the entire fish is desired, remove both eyes at the outset (see Note in step 3.2), rotate the embryo after imaging one side and repeat steps 2.3 – 2.5 for the opposite side.

3. 3D Reconstruction of Embryonic Zebrafish Cerebrovasculature

  1. Use the Fiji distribution3 of open source ImageJ (http://fiji.sc), which is optimized for 3D renderings, free of charge, and compatible with PC, Mac and Linux computers.
  2. Import confocal stacks to Fiji by going to Plugins>LOCI>BioFormats Importer 9 then select the confocal file, e.g. name.ids for Nikon stacks (Figure 2A). Select View stack with Hyperstack, Color mode: grayscale, check autoscale, check split channels.
  3. These selections will open four separate channel panels; for EGFP only one will be needed, usually the third one down. Close the other three panels (red, blue and alpha) leaving the 16-bit image with a long name followed by C=1 (Figure 2A).
  4. Adjust threshold by scanning though the image using the scroll tab along the bottom to find a slice that has the region or structure of interest, then go to Image>Adjust>Threshold (Figure 2C).
  5. In the panel that comes up, slide the top bar (black level) to the left so that the structure can be seen quite well over background, and leave the bottom slide (white level) where it is (Figure 2D). Select B&W, select Dark background. Do not select calculate Threshold for each image unless different adjustments are required for each slice, select Black background. This process creates a new 8-bit image.
  6. WARNING: Threshold cannot be undone or saved in ImageJ, so the confocal stack will have to be reloaded every time a change is needed. Write down the numbers and try different settings until the desired result is achieved.
  7. Go to Plugins>3D Viewer>Threshold 0, Resampling factor 1 (best) or 2 (good), deselect the red and blue color boxes to make a green 3D output – otherwise it will produce a white rendering – select Apply (not Auto) (Figure 2E).
  8. Rotate, spin and zoom the 3D image using the mouse and keyboard controls (Figure 2F).
  9. Save still images at any point by using the capture option in the menu. The size of the image box on the screen dictates pixel dimensions of the image produced so if a high resolution image is desired make the box larger by dragging the bottom right corner.
  10. Create a spinning 3D movie by selecting View>Record 360 deg rotation (Figure 2G). Rotation defaults to 2 degrees per step, but it can be changed to 5 degrees for example, which will create much smaller file sizes, but may add jerkiness to the animation.
  11. Save the file in one of several available formats for later viewing with media players, uploading to the Internet, or using in PowerPoint presentations. The open source media player, VLC (http://www.videolan.org/vlc/index.html), is free of charge and handles these videos very well.

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Learning Objectives

3D reconstruction of vascular structures provides a comprehensive and visually interesting perspective of zebrafish development. Figures 1 and 2 show methods as they typically done. Figure 3 shows several angles of vascular structures in a 6 dpf zebrafish embryo that expressed EGFP in endothelial cells. With a solid green or white color it can be difficult to appreciate signal intensity; pseudo-coloring provide image intensity from a look-up-table and allows better depth perception when structures overlap. An example of a pseudo-colored 3D image of the vasculature in a 6 dpf zebrafish is provided in Figure 4. Fluorescence imaging of live embryos can be used to study physiological characteristics that include eye and body movement, and cardiac activity. Figures 3 and 4 show representative results obtained with these methods, using the transgenic zebrafish line described. Imaging resolution depends on microscope characteristics, but the brightness of the EGFP signal is sufficient for good image quality with most commercial systems. Reconstruction and rendering of 3D representations is consistent and options within this open-source software provide consistently good results.

Figure 1
Figure 1. Eye removal. A) A fixed 3 dpf embryo with a tungsten need positioned next to the eye. Tissue is cut around the eye from this position. B) The eye is falls out and the underlying ocular muscles and optic nerve are cut. The empty eye socket is indicated with dashed circle. C) The same embryo is turned over and mounted with methyl-cellulose, with the intact eye facing up. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Step-by-step 3D reconstruction of a confocal image stack. A) Open file (4104.1.ids) loaded within Fiji using Plugins>LOCI>Bioformat to select. B) After finding a slice with the region of interest, threshold adjustment is selected as shown. C) Threshold is adjusted to 214 using the top slider and apply is selected. D) 3D viewer is called as shown. E) The 3D reconstruction is shown of a zebrafish with the eye intact, for orientation. F) The image has been zoomed and rotated. G) A 360 degree rotation movie is made as shown. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Perspectives from 3D reconstruction. A) Medial perspective of 6 dpf embryo imaged with 10x objective, mouth is on the right, not gills inside mouth. B) Lateral of the same embryo, note fin is a loop in the middle. C) Same embryo imaged with 20x objective, medial perspective, note gill resolution. D) Lateral perspective of 20x objective imaging. The fin is on the right edge of the panel. E) Antero-medial view of 20x objective imaging, note gills inside mouth. F) Abdomen of the same embryo imaged with a 20x objective, head it to the right. Note vasculature on the yolk sac at the bottom right. Please click here to view a larger version of this figure.

Figure 4
Figure 4. Intensity differences in 6 dpf embryo. Image of a 6 dpf reconstruction using a pseudocolor look-up-table for signal intensity. Mouth, brain, gills and yolk sac are labeled for orientation. Please click here to view a larger version of this figure.

Figure 5
Figure 5. Movie of reconstructed vascular system in a 4 dpf zebrafish. The fish was imaged at 2.5 μm. The images were from imaging one half of the embryo. Compare vascular structures with structures in a GSI-treated zebrafish provided in Figure 6. Note the lower density of blood vessels in the head and larger gills. (See the “Zfish_spin.avi” supplemental file under Downloads)

Figure 6
Figure 6. 3D Movie of vascular system in a GSI-treated embryo at 4 dpf. The fish was imaged at 2.5 μm through from lateral to midline. Compare vascular structures with the control 4 dpf fish shown in Figure 5. The arched back and smaller size are typical in embryos treated with this chemical. (See the “GSI-treated_4dpf_fish.avi” supplemental file under Downloads)

List of Materials

N – Phenylthiourea  Alfa Aesar, catalog #41972 0.2 M in E3 buffer, kept at 4oC
E3 buffer Sigma 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4
Confocal microscope  Nikon D-EclipseC1 on a Nikon TE-2000U
Glass bottom dishes Mat-Tek
GSI IX/DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butyl ester EMD Biosciences
24 well plates  Becton-Dickinson, cat# 351147 BD Falcon
Transfer pipettes  VWR,  cat #414004-001 VWR disposable transfer pipets
Methyl-cellulose  Alfa Aesar, cat#43146 3% in E3 buffer
NRD 4/6 Fish food  Brine Shrimp Direct Dried
Brine shrimp Brine Shrimp Direct Live
Tungsten wire  Small Parts # TW-016-60 0.016” OD
Tricaine VWR # 101107-950 Tricaine methanesulfonate 250 mg/L in E3 buffer

Lab Prep

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Procedure

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

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