Name: microstructured devices for optimized microinjection and imaging of zebrafish larvae
Uploaded: 2017-12-08T13:00:00
Description: Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

The authors would like to thank David Langenau for generously providing aquarium space; Eric Stone, John C. Moore and Qin Tang for help with zebrafish maintenance and reagents, and Anne Robertson and Elliott Hagedorn from Leonard Zon&#39;s lab for procuring the zebrafish strain used here. They would also like to thank Octavio Hurtado for advice on photolithographic techniques. FE was funded by Fellowships from Shriner&#39;s Hospital for Children and the American Australian Association. This work was funded by NIH grant GM92804.

Microinjection of larvae was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care under Protocol 2011N000127.
1. Device Fabrication
NOTE: All computer assisted drawing (CAD) files used to design photolithography masks described here (Figure 1) are available for download. See Table of Materials for links.
<ol>
	<li>Fabricate the master mold wafer in a Class 1,000 clean room using standard photolithographi...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+student+team+in+a+University+of+Michigan+biomedical+engineering+design+course+constructs+a+microfluidic+bioreactor+for+studies+of+zebrafish+development.">A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.</a> Zebrafish. 6 (2), 201-213 (2009).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+on+a+chip:+a+novel+platform+for+real-time+monitoring+of+drug-induced+developmental+toxicity.">Zebrafish on a chip: a novel platform for real-time monitoring of drug-induced developmental toxicity.</a> PLoS One. 9 (4), e94792 (2014).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+embryo+array+for+automated+trapping,+immobilization+and+microperfusion+of+zebrafish+embryos.">Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.</a> PLoS One. 7 (5), e36630 (2012).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+on+chips:+Microfluidic+living+embryo+array+for+accelerated+in+vivo+angiogenesis+assays.">Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays.</a> Sens Actuators B Chem. 189, 11-20 (2013).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mapping+of+brain-wide+activity+in+awake+and+drug-responsive+vertebrates.">High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.</a> Lab Chip. 15 (3), 680-689 (2015).</li><li>Noori, A., Selvaganapathy, P. 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Here, we describe the use of devices we recently developed to facilitate 2 dpf zebrafish microinjection5, and introduce a simple agarose-free mounting device for convenient imaging of embryos. These tools highlight the utility of photolithographic techniques for fabrication of devices useful for zebrafish techniques.
We have found MSA devices particularly useful for injection of cells or particles prone to aggregation within the microinjection needle, such as fungal con...

Zebrafish have emerged as a powerful model for many fields, from studies of fundamental developmental biology to large-scale genetic and chemical screens1,2. Routine genetic manipulations, such as gene overexpression, knockdown, CRISPR/Cas9 mutagenesis, and transgenesis rely on microinjection of genetic material into the single-cell zygote, which has led to the development of simple, easy-to-use, commercially available tools for orienting and stabilizing eggs for injection3. Other approaches, such as transplantation and infection, often require microinjection into later stage embryos and larvae using larger gauge capillary needles4. However, use of larger gauge needles presents significant technical challenges, as it is more difficult to penetrate the target tissue without pushing or rolling the embryo. Under these conditions, obtaining the appropriate water tension required to stabilize the embryo while avoiding drying during the procedure is difficult, and embryos may not be ideally oriented for injection into the target tissue.
Following microinjection, it is often useful to screen injected embryos to select those that have been successfully injected, and to capture images of the initial time point. To address these challenges, we have developed a range of microstructured devices that help to stabilize 2 dpf embryos in various orientations both for microinjection5, and for rapid image-based screening post-injection.
To obtain sufficient structural resolution in these devices, we utilized photolithographic techniques. Commonly used in microelectronic industries and more recently extrapolated to microfluidic fabrication, these approaches can achieve vertical structures ranging from 1-1,000 &#181;m, a scale well suited to manipulation of zebrafish embryos and larvae. All devices were fabricated using polydimethylsiloxane (PDMS), which is cheap, physically robust, biologically inert, and transparent.
Microstructured surface arrays (MSAs) were formatted as blocks of PDMS with a patterned top surface, analogous to the simple channels in agarose blocks commonly used for egg microinjection. For post-injection screening, 6 imaging devices can be arrayed in a standard glass-bottomed 6-well plate. These devices are designed for easy loading of embryos, while the unloading procedure conveniently allows rescue of specific embryos, facilitating image-based screening approaches in a more user-friendly manner than those devices previously developed by the Beebe laboratory6.

<table>
	<tbody>
		<tr>
			<td>Dow Corning Sylgard 184&#160; Polydimethylsiloxane (PDMS)&#160;</td>
			<td>Ellsworth Adhsives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>For casting the devices. Kit includes PDMS monomer and Initiator</td>
		</tr>
		<tr>
			<td>Low gelling temperature agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-10G</td>
			<td>For casting agarose devices</td>
		</tr>
		<tr>
			<td>PFDTS silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>For casting of negative PDMS molds</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma Aldrich</td>
			<td>E10521-10G</td>
			<td>To anesthetize&#160; zebrafish&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine Dextran 70,000 Da</td>
			<td>ThermoFisher</td>
			<td>D1818</td>
			<td>To trace microinjections</td>
		</tr>
		<tr>
			<td>Leukotriene B4 (LTB4)</td>
			<td>Cayman Chemicals</td>
			<td>20110</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td>
			<td>Sigma Aldrich</td>
			<td>F3506-50MG</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>15 cm Petri dish</td>
			<td>Fisher scientific</td>
			<td>08-757-148</td>
			<td>For Casting from the master wafer</td>
		</tr>
		<tr>
			<td>Glass-bottom 6-well plates</td>
			<td>MatTek</td>
			<td>P06G-0-20-F</td>
			<td>For imaging devices</td>
		</tr>
		<tr>
			<td>Borosilicate glass microcapillaries</td>
			<td>World Scientific Instruments</td>
			<td>TW-100-4</td>
			<td>For microinjection needles</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma Aldrich</td>
			<td>Z350796</td>
			<td>For transferring zebrafish embryos</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Fisher scientific</td>
			<td>E5242956003</td>
			<td>For loading the microinjection needles</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 1.5 mm punch</td>
			<td>Ted Pella Inc.</td>
			<td>15111-15</td>
			<td>To punch ports in PDMS imaging devices</td>
		</tr>
		<tr>
			<td>No.&#160;11 Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For cutting PDMS&#160;</td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-10</td>
			<td>For dechorionating embryos and breaking microinjection needle tips</td>
		</tr>
		<tr>
			<td>Marzhauser Micromanipulator</td>
			<td>ASI&#160;</td>
			<td>MM33-R</td>
			<td>For manipulating microinjection needle</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>MSC</td>
			<td>SPI - 87242624</td>
			<td>For mounting micromanipulator</td>
		</tr>
		<tr>
			<td>MPPI-3 Picopump controller</td>
			<td>ASI</td>
			<td>MPPI-3</td>
			<td>To control microinjection volume and timing</td>
		</tr>
		<tr>
			<td>EVOS inverted fluorescent microscope</td>
			<td>ThermoFisher</td>
			<td>EVOS FL</td>
			<td>To image injected embryos</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>For visualizing microinjecion</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td></td>
			<td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &#160;</td>
		</tr>
	</tbody>
</table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

The approach described here demonstrates the design (Figure 1) and fabrication of devices for use with 2 dpf zebrafish, using photolithographic (Figure 2) and soft-lithographic (Figure 3) techniques. This method allows rapid testing of many design iterations and modifications, and alterations and optimization of microstructure dimensions for use with zebrafish at other stages of development may exten...

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

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