Thanks are due to Genia Brodsky for figure graphics; Vyacheslav Kalchenko, Douglas Lutz, and Leonid Roitman for technical advice and assistance with the two-photon uncaging; Maayan Tahor and Suliman Elsadin for technical assistance; Uwe Strahle for kindly providing the neurogenin1 reporter line and Amos Gutnick for comments on this manuscript. The research in the Levkowitz lab is supported by the German-Israeli Foundation (grant number 183/2007); Israel Science Foundation (grant number 928/08) and the Harriet&amp;Marcel Dekker Foundation. G.L. is an incumbent of the Tauro Career Development Chair in Biomedical Research.

We describe a protocol of cell labeling using caged fluorescein, however, other photo-activatable dyes and proteins can be similarly used.
1. Injection of Caged Fluorescein
<ol>
<li>Prepare 5% stock solution (5mg caged-fluorescein / 100 &mu;L 0.2M KCl) of Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic, Invitrogen, molecular probes, Carlsbad, CA, cat. no. D-3310). Aliquot and store in -20&deg;C. Note that DMNB-caged fluorescein is sensitive to light and should be kept in the dark.</li>
<li>Prepare an injection trough made of 1% agarose (Sigma, St. Louis, MO, cat. no. A9539) as described in "The zebrafish book"1.</li>
<li>Prepare 2 liters of E3 medium by diluting a 100 x E3 stock solution.</li>
<li>On the evening before injection, prepare mating cages separating the male(s) from the female(s) of the desired transgenic line expressing a visible fluorescent landmark (GFP, RFP etc.) used to localize the target cells for photoactivation.</li>
<li>Prepare capillary injection needles (Thin Wall Glass Capillaries with filament, World Precision Instruments, Sarasota, FL, cat. no. TW100F-6) using a micropipette puller (Sutter Instrument, Novato, CA, P-97).</li>
<li>Thaw an aliquot of 5% caged fluorescein on ice.</li>
<li>Dilute the 5% caged fluorescein stock solution in 0.2M KCl to a final concentration of 1%, and keep on ice. Avoid light exposure.</li>
<li>Setup mating crosses prepared in step 4.</li>
<li>Collect the fertilized eggs as soon as they are laid. Orient one-cell stage embryos in the agarose injection mold overlaid with E31.</li>
<li>Using a microloader tip (Eppendorf, Hamburg, Germany, cat. no. 5242 956.003) backfill an injection needle with ~1 &mu;L of 1% caged fluorescein solution.</li>
<li>Place loaded needle into a micromanipulator attached to a gas-powered microinjector (pneumatic picopump, World Precision Instruments, Sarasota, FL, PV820).</li>
<li>Adjust the injection volume to 2-3nl and inject the caged-fluorescein directly into the cell's cytoplasm. Inject a minimum of 50 embryos per experiment.</li>
<li>Incubate embryos in the dark at 28.5&deg;C in E3 to the desired developmental stage. If embryos older than 24 hours post fertilization are analyzed, add 0.1% phenylthiourea (PTU, Sigma, St. Louis, MO, cat. no. 22290-9) to inhibit pigmentation.</li>
</ol>
2. Embryo Mounting
<ol>
<li>Prepare fresh E2 solution (see solutions part; can be stored at 4&deg;C for a week).</li>
<li>Coat 60 mm Petri dishes with a thin layer of 1% agarose dissolved in E2 medium.</li>
<li>Prepare 2% low melting point agarose (Ultra Pure LMP agarose, Invitrogen, Carlsbad, CA, cat. no. 16520100) for embedding: dissolve the agarose in sterile double distilled water by microwave heating until boiling. Keep the tube's cap open during heating. Aliquot 1 mL into microcentrifuge tubes and keep in a heating block at 72&deg;C.</li>
<li>Remove the chorions of the embryos by gently pulling the chorion apart with sharp forceps (No. 5). Keep embryos in E2 solution on an agar-coated dish (see step 2). Dechorionated embryos should be handled with fire-polished glass Pasteur pipette as they may stick to polypropylene pipettes. Avoid exposing the dechorionized embryo to air as these embryos might be damaged by the liquid surface tension.</li>
<li>Collect the selected embryo in a small volume of E2 using a fire-polished glass Pasteur pipette, and place the embryo on 60 mm Petri dish. Mount a drop (~150 &mu;L) of 2% agarose (step 3) beside the embryo. Quickly mix the two drops to obtain a homogenous final ~1% agarose mixture and using a dissecting microscope orient the embryo with its dorsal side facing the objective lens.</li>
<li>Wait until the agarose has solidified and add E2 medium until the embedded embryo is submerged in liquid. It is preferred to obtain a single embryo per dish as it may be released from the agarose after the photoactivation process.</li>
</ol>
3. Photoactivation
<ol>
<li>We use a Zeiss LSM 510 META NLO two-photon microscope equipped with a broadband tunable (700-1020nm) Mai Tai- HP-femtosecond laser from Spectraphysics.</li>
<li>Place the mounted embryo on a plate holder under the microscope. The fluorescent landmark in the embryo is visualized using 40x/0.8W Achroplan water immersed objective. For the following steps we use non-descanned detector (NDD) with band pass filter (BP) of 500-550nm.</li>
<li>As a prerequisite to the photoactivation process, obtain spatial fluorescent information of the region of interest by acquiring image series from the most ventral to dorsal part of the detected fluorescent signal with two-photon laser wavelength of 860nm (for GFP detection). To this end, sufficient resolution can be obtained using a span of 6&mu;m between each acquired focal plane.</li>
<li>Select the target z- plane for photoactivation and bring this region into focus. Depending on the purpose of the experiment, the region of interest can be either within or outside the GFP visible landmark. Take a single image in this z-plane. Save the image and keep it open in a separate window.</li>
<li>Set the two- photon laser wavelength to 720nm and when the laser is in mode-locked open the 'Edit bleach' menu. Define the region of interest (ROI) for photoactivation according to the image taken at 860nm (Step 4) using the 'Define Region' menu. It is crucial to select the ROI on this image after shifting the laser wavelength to 720nm as the latter action changes the x,y positions of the observed field.</li>
<li>Adjust the following parameters for photoactivation in the 'Edit bleach' menu (see step 9): a) relative laser power; b) number of iterations; c) scan speed.</li>
<li>Press 'Bleach' in the 'Edit bleach' window and wait until the laser stops.</li>
<li>Switch back to 860nm, acquire a single plane image to evaluate the intensity of the resulting photoactivated material and a second series of image acquisition to evaluate the Z-span (thickness) of the activated domain.</li> 
<li>Consider the following parameters for photoactivation:
<ol class="lalpha">
<li>One should empirically determine the optimal conditions for photoactivation by varying two parameters: laser intensity and duration of photoactivation. The relative laser power can be controlled by the Acousto-Optic Modulator (AOM) transmission. The duration of photoactivation is a function of the number of iterations and scan speed.</li>
<li>Determine the linear range of fluorescence intensity in order to avoid saturation of the uncaged fluorescence.</li>
<li>There is a linear correlation between the obtained fluorescence intensity of the uncaged molecules and the z- axis span of the photoactivated clone2.</li></ol></li>
</ol>
4. Detection of the Uncaged Fluorescein
<ol>
<li>After photoactivation, raise the embryos in the dark at 28.5&deg;C in E2 medium containing 0.1% PTU to prevent the appearance of skin pigmentation.</li>
<li>At the desired developmental stage, remove the embryo from the agarose under a dissecting microscope. Using a pointed scalpel, create a "V" shaped incision in the agarose near the embryo so that the "V" is pointing towards the embryo's head. Place a closed forceps at the point of the "V" near the head of the embryo and gently push open the forceps separating the agarose along the length of the embryo and releasing the embryo into the medium.</li>
<li>Collect the embryos with a fire-polished Pasteur pipette and fix with paraformaldehyde (PFA; 4% in 1 x PBS, PH 7.2) for either 3 hours at room temperature or overnight at 4&deg;C.</li>
<li>Remove the fixative solution and rinse 2-3 times in PBST for at least 5 minutes at room temperature.</li>
<li>Wash embryos with 100% methanol for 10 min, replenish with fresh methanol and incubate at -20&deg;C for at least 2 hr.</li>
<li>Rehydrate embryos by successive incubations (5 min each) in a series of diluted methanol in PBSTr: 80%, 60%, 40% and 20%. Wash 2 x 10 minutes in PBSTr.</li>
<li>Incubate for 30 minutes at room temperature in 500 &mu;L of Blocking solution.</li>
<li>Add an anti-Fluorescein- alkaline phosphatase (AP) Fab fragments (Roche, Palo Alto, CA, cat. no. 11426338910), diluted 1:500 in Blocking solution and incubate overnight at 4&deg;C (place microcentrifuge tubes on their sides).</li>
<li>Wash 5 x 15 minutes in PBST.</li>
<li>Wash for 5 minutes in 0.2M NaCl in TNT.</li>
<li>Wash for 5 minutes in 0.4M NaCl in TNT.</li>
<li>Equilibrate 3 x 5 minutes in freshly prepared Prestain solution.</li>
<li>Dissolve one Fast Red tablet (Roche, Palo Alto, CA, cat. no. 11496549001) in 2 mL Prestain solution. It is recommended to filter the solution using Whatman No. 2 paper to omit un-dissolved particles. Alternatively, spin down solution and use the clear supernatant liquid.</li>
<li>Add 500 &mu;L Fast Red solution to each tube, incubate in the dark at room temperature, and monitor color development until the desired signal-to-background ratio is reached (20 minutes to several hours). If background develops, transfer to 4&deg;C. After one hour of incubation, replenish with a new Fast Red staining solution.</li>
<li>Stop the staining reaction by 3 x 5 minutes washes in PBST.</li>
<li>Fix for 20 minutes in 4% PFA at room temperature.</li>
<li>Wash 2 x 5 minutes in PBST.</li>
<li>Clear embryos through a series of 25%, 50% and 75% glycerol in PBS until embryos are settled at the bottom of the tube. Embryos can be stored at 4&deg;C for a few days.</li>
</ol>
5. Fluorescent Visualization of the Uncaged Area
<ol>
<li>Prepare a microscope slide for mounting: using a 1 mL syringe apply four spots of clear silicone grease on the slide, placed at a distance of ~15mm from each other. Mount the embryo at the center in ~200 &mu;L of 75% glycerol and place a coverslip (no. 0; 18x18mm) on top and gently push it against the silicone until the glycerol solution occupies the surface between the slides.</li>
<li>Place the mounted embryo onto the stage and bring it into focus using a 40x objective. Acquire a confocal Z-series of images using 488nm Argon and 543nm HeNe lasers as the excitation source.</li>
<li>The z-axis span of the photoactivatated domain can be analyzed using 3D reconstruction of the uncaged embryo using an image analysis software.</li>
</ol>
6. Solutions
<table border="1">
<tbody>
 <tr>
 <td>100 x E3</td>
 <td>174mM NaCl, 21mM KCl, 12mM MgSO4, 18mM Ca(NO3), 15mM HEPES, pH=7.4. Sterilize by filtration and store at 4&deg;C</td>
 </tr>
 <tr>
 <td>20 x E2</td>
 <td>0.3M NaCl, 10mM KCl, 20mM CaCl2*2HOH, 20mM MgSO4*7HOH, 3mM KH2PO4, 0.8mM Na2HPO4*2HOH, filtrate and store in room temperature</td>
 </tr>
 <tr>
 <td>1 x E2</td>
 <td>Dilute 20 x E2 in double distilled water and add freshly made NaHCO3 (to final concentration of 0.7mM), Penicillin and Streptomycin (dilute pen strep solution to a final concentration of 1 x which contains 100U of penicillin and 100mg streptomycin per mL (Invitrogen, Carlsbad, CA, cat. no. 15140-122))</td>
 </tr>
 <tr>
 <td>PBST</td>
 <td>0.1% TWEEN-20 in PBS</td>
 </tr>
 <tr>
 <td>PBSTr</td>
 <td>0.3% Triton in PBS</td>
 </tr>
 <tr>
 <td>Blocking solution</td>
 <td>10% BSA, 0.3% Triton, 1% DMSO in PBS</td>
 </tr>
 <tr>
 <td>TNT</td>
 <td>100mM Tris-HCl pH-7.5, 150mM NaCl, 0.5% TWEEN-20 in double distilled water </td>
 </tr>
 <tr>
 <td>Prestain solution</td>
 <td>100mM Tris-HCl pH-8.2, 0.4M NaCl, 0.1% TWEEN-20 in double distilled water</td>
 </tr>
 </tbody>
</table>

7. Representative Results 
An example of the use of two-photon microscopy to photoactivate agents in a living zebrafish embryo is presented. Using a similar approach we previously traced the lineage of the photoactivated labeled neural progenitors in the zebrafish brain2,3. Figure 1 shows photoactivation of a caged fluorescein conjugated tracer dye in anterior neural plate of zebrafish embryos carrying a neurog1::gfp reporter transgene, which served as a live intrinsic landmark.
Embryos were injected with caged fluorescein at the one cell stage and embedded in agarose at bud stage. At 3- 5-somite stage the spatial coordinates were measured in the neurog1::gfp transgenic embryo to determine the region of interest (ROI) for uncaging (Figure 1A, A'). We uncaged the fluorescein lineage tracer in a specific domain of the neural plate (Figure 1B, B'). Subsequently, the fate of this given labeled neural progenitor subdomain was determined at prim5 (24 hours post fertilization) stage by immunostaining of the uncaged fluorescein (Figure 1C, C').
The extent of two-photon laser uncaging, i.e. the intensity of the uncaged fluorescence and the thickness of the photoactivated domain (z-span), can be controlled by adjusting two parameters: laser intensity and duration. The latter can be controlled by adjustment of the iteration number and scan speed (see part 3, step 9; Russek-Blum, 2009). In the example shown in Figure 1, we used relative laser power of 12% AOM transmission, 20 iterations and scan speed of 25.6 &mu;sec/ pixel. Using these settings we labeled a ROI containing 9-10 cells and a photoactivated z- span of 30&mu;m (~1-2 cell rows).
<img src="/files/ftp_upload/1902/1902fig1.jpg" alt="Figure 1" /> 
Figure 1. A Representative result of photoactivation of caged fluorescein in live zebrafish embryo using two-photon microscopy.
Schematic illustration (A-C) and representative images (A'-C') of the photoactivation process. Live zebrafish embryo (A, A') expressing GFP under the control of neurogenin-1 promoter (neurog1::gfp) that was injected with caged fluorescein tracer dye at the 1 cell stage. At the 3-5- somite stage, a discrete area of the forebrain primordium (diencephalon) was photoactivated and the uncaged fluorescein tracer dye could be detected (B, B'). Subsequent to the photoactivation procedure, the embryo was incubated at 28.5&deg;C and brain cells containing the uncaged fluorescein were traced by anti-Fluorescein immunostaining at 24 hours post fertilization (hpf; C, C'). Dien., Diencephalon; Tel., Telencephalon.

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book:+Guide+for+the+Laboratory+Use+of+Zebrafish+(Brachydanio+rerio.">The Zebrafish Book: Guide for the Laboratory Use of Zebrafish (Brachydanio rerio.</a> , (1995).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Russek-Blum, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neuronal+cluster+size+is+determined+during+early+forebrain+patterning.">Dopaminergic neuronal cluster size is determined during early forebrain patterning.</a> Development. 135, 3401-3413 (2008).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Staudt, N., Houart, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prethalamus+Is+Established+during+Gastrulation+and+Influences+Diencephalic+Regionalization.">The Prethalamus Is Established during Gastrulation and Influences Diencephalic Regionalization.</a> PLoS Biol. 5, e69-e69 (2007).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Hatta, K., Tsujii, H., Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+a+photoconvertible+fluorescent+protein.">Cell tracking using a photoconvertible fluorescent protein.</a> Nat Protoc. 1, 960-967 (2006).</li><li>Boyden, E. 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Photo-activatable compounds are molecules whose function is masked until they are illuminated with a specific wavelength (usually UV), inducing a photochemical reaction that converts the molecules into a biologically or chemically active state. These probes provide very powerful tools in cell biology research, since the activation can be precisely controlled temporally and spatially by limiting their exposure to light. 
The significant advantage of multi-photon microscopy is its relatively d...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3310</td>
<td>molecular probes</td>
</tr>
<tr>
<td>Agarose for injection trough and coated plates</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Thin Wall Glass Capillaries with filament</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-6</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Microloader tip</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic picopump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PV820</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>22290-9</td>
<td>&nbsp;</td>
<tr>
<td>Low melting point agarose for embryo mounting</td>
<td>&nbsp;</td>
<td>Ultra Pure LMP agarose</td>
<td>16520100</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>Fast Red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11496549001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

two-photon-based photoactivation in live zebrafish embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

two-photon-based-photoactivation-in-live-zebrafish-embryos

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11.5K Views.  Weizmann Institute of Science. Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Video: Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...