Name: long-term live imaging device for improved experimental manipulation of zebrafish larvae
Uploaded: 2017-10-27T15:00:00
Description: Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

The authors would like to acknowledge primary project funding from the Morgridge Institute for Research and the Laboratory for Optical and Computational Instrumentation. We also acknowledge funding from NIH# R01GM102924 (AH and KWE). KH, JMS, RS, AH and KWE conceived and designed the study. KH and JMS performed all experiments with support from DL, KP and RS. KH, JS, RS, AH and KWE contributed to the writing of the manuscript.

Note: The base zWEDGI design was formulated for zebrafish larvae that are 2 to 4 days post-fertilization (dpf) and follow the guidelines of the University of Wisconsin-Madison Research Animals Resource Center.
1. Design and 3D Printing of Molds
<ol>
	<li>Model the PDMS component of the device with desired geometries and attributes in a 3D modeling software5. Create an assembly of a blank mold and the PDMS part and generate a negative mold for the PDMS part by creating...

<ol>
	<li>Yoo, S. K., Freisinger, C. M., LeBert, D. C., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+redox,+Src+family+kinase,+and+calcium+signaling+integrate+wound+responses+and+tissue+regeneration+in+zebrafish.">Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.</a> J. Cell Biology. 199 (2), 225-234 (2012).</li><li>Kawakami, A., Fukazawa, T., Takeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+fin+primordia+of+zebrafish+larvae+regenerate+by+a+similar+growth+control+mechanism+with+adult+regeneration.">Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.</a> Dev. Dynam. 231 (4), 693-699 (2004).</li><li>Konantz, J., Antos, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetic+morpholino+approach+using+cardiac+ventricular+injection+to+transfect+multiple+difficult-to-target+tissues+in+the+zebrafish+larva.">Reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva.</a> JoVE. (88), (2014).</li><li>Hall, C., Flores, M. F., Kamei, M., Crosier, K., Crosier, P., Sampath, K., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Innate+Immune+Cell+Behavior+During+Normal+Development,+Wound+Healing+and+Infection.">Live Imaging Innate Immune Cell Behavior During Normal Development, Wound Healing and Infection.</a> Live Imaging in Zebrafish: Insights into Development and Disease. , (2010).</li><li>Huemer, K., Squirrell, J. M., Swader, R., LeBert, D. C., Huttenlocher, A., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zWEDGI:+Wounding+and+Entrapment+Device+for+Imaging+Live+Zebrafish+Larvae.">zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.</a> Zebrafish. , (2016).</li><li>Lisse, T. S., Brochu, E. A., Rieger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+tissue+repair+in+zebrafish+larvae+with+time-lapse+brightfield+stereomicroscopy.">Capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy.</a> JoVE. (95), (2015).</li><li>Kamei, M., Isogai, S., Pan, W., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+blood+vessels+in+the+zebrafish.">Imaging blood vessels in the zebrafish.</a> Methods Cell Biol. 100, 27-54 (2010).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> JoVE. (26), (2009).</li><li>Petzold, A. M., Bedell, V. M., 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=SCORE+imaging:+specimen+in+a+corrected+optical+rotational+enclosure.">SCORE imaging: specimen in a corrected optical rotational enclosure.</a> Zebrafish. 7 (2), 149-154 (2010).</li><li>Macdonald, N. 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M., Sessa, A., 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=Transparent+Adult+Zebrafish+as+a+Tool+for+In+Vivo+Transplantation+Analysis.">Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>LeBert, D. C., Squirrell, J. M., 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=Matrix+metalloproteinase+9+modulates+collagen+matrices+and+wound+repair.">Matrix metalloproteinase 9 modulates collagen matrices and wound repair.</a> Development. 142 (12), 2136-2146 (2015).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. 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The purpose of the zWEDGI device is to capture 3D time lapse imaging by stabilizing and orienting the fish within the small working distance of a high-resolution microscope objective. While meeting these design specifications, it is also an improvement over traditional agar-based preparation for live imaging. There are three critical steps (below) in the fabrication of the zWEDGI, which, if not done correctly, can result in defective devices:
PDMS preparation (Figure 5A</s...

The regenerative capacity of zebrafish larvae Danio rerio make them an ideal model organism for examining wound response as well as healing and regrowth1,2,3,4. Access to an array of transgenic zebrafish lines and zebrafish&#39;s anatomical transparency further enhance their utility for in vivo studies of wound response events as well as longer-term regenerative processes4. Study of these biological processes using high-resolution time-lapse fluorescence microscopy therefore demands a live imaging zebrafish device that allows for high stability and minimal movement of the zebrafish larva while maintaining viability. It is key that the device allows for effective wounding while healing and regeneration occur unaffected by the device.
The standard live imaging stabilization method of embedding the larva in agarose during live imaging restricts growth and wound regeneration5 and may increase death rates since larvae begin to show sign of cardiac stress and tissue necrosis after four hours4. Therefore, removal of agarose from regions of interest is often necessary to allow normal development and regeneration6, exposing the larvae to potential damage as the agarose is cut away. Furthermore, with the agarose embedding technique, the user must orient the larvae in the short time before the agarose solidifies5,6,7. Rapidly manipulating the larva not only requires skill of the user, it also risks damage to the larva. Although methods to stabilize the larva for live imaging have been described to circumvent these drawbacks, such as ridged agar wells3 or divets8, the use of silicone vacuum grease to create an imaging chamber with PVC piping or other materials6, and rotational tubing9, many of these methods are labor intensive, messy, often non-reusable and don&#39;t allow for environmental manipulation (drug treatments, wounding etc.) after the fish has been mounted.
Therefore, the zWEDGI device (Figure 1) was designed to overcome some of the drawbacks of agar mounting for long-term live imaging of zebrafish larvae while permitting manipulation of the specimen. The zWEDGI consists of three semi-open compartmentalized chambers (Figure 1A) to allow for loading, restraint, wounding and imaging of 2 to 4 days post-fertilization zebrafish larvae. The device is fabricated from Polydimethylsiloxane (PDMS) and placed onto the cover slip of a 60 mm glass bottom imaging dish. The design presented here was intended for wound healing studies, however the use of a modular design and standard fabrication technologies make the zWEDGI design modifiable and amenable to a variety of experimental procedures, especially for procedures that require minimal restraint with experimental manipulation and long-term imaging.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fabricate molds</td>
			<td style="width:127px;"></td>
			<td style="width:145px;"></td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solidworks Professional Accedemic Research 3D modeling software</td>
			<td>Dassault Systemes</td>
			<td>SPX0117-01</td>
			<td>Fisher Unitech</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viper Si2 SLA 3D printer</td>
			<td>3D Systems Inc.</td>
			<td>23200-902</td>
			<td>3D Systems Inc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accura 60 photopolymer resin</td>
			<td>3D Systems Inc.</td>
			<td>24075-902</td>
			<td>3D Systems Inc.</td>
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			<td height="20" style="height:20px;">denatured alcohol</td>
			<td>Sunnyside</td>
			<td>5613735</td>
			<td>Menards</td>
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			<td height="20" style="height:20px;">UV post cure apparatus</td>
			<td>3D Systems Inc.</td>
			<td>23363-101-00</td>
			<td>3D Systems Inc.</td>
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			<td height="20" style="height:20px;">TouchNTuff nitrile gloves</td>
			<td>Ansell</td>
			<td>92-600</td>
			<td>McMaster Carr</td>
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			<td height="20" style="height:20px;">220B, 400B, 600 grit T414 blue-bak sandpaper&#160;</td>
			<td>Norton</td>
			<td>66261139359, 54, 52</td>
			<td>MSC</td>
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			<td height="20" style="height:20px;">borosilicate glass disc, 2&#34; diameter</td>
			<td>McMaster-Carr</td>
			<td>MIL-G-47033</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ultrasonicator cleaner</td>
			<td>Branson</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropyl rubbing alcohol 70%</td>
			<td>Hydrox</td>
			<td>54845T43</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10oz clear plastic cup</td>
			<td>WNA Masterpiece</td>
			<td>557405</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6&#34;craft stick</td>
			<td>Perfect Stix</td>
			<td>Craft WTD-500</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fabricate zWEDGI PDMS device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicon elastomeric kit&#160;</td>
			<td>Dow-Corning</td>
			<td>4019862</td>
			<td>Ellworth Adhesives&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10mL syringe</td>
			<td>Becton Dickinson</td>
			<td>305219</td>
			<td>Vitality Medical Inc</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">desiccator</td>
			<td>Bel-Art Scienceware</td>
			<td>F42027-0000</td>
			<td>Amazon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 in ratcheting bar clamp</td>
			<td>Pittsburgh</td>
			<td>68974</td>
			<td>Harbor Freight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lab oven</td>
			<td>Quincy Lab Inc.</td>
			<td>20GC</td>
			<td>Global Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tweezer set</td>
			<td>Aven</td>
			<td>549825</td>
			<td>McMaster-Carr</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">compressed air filtered nozzle</td>
			<td>Innotech</td>
			<td>TA-N2-2000FT</td>
			<td>Cleanroom Supply</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum bench vise</td>
			<td>Wilton Tool Group</td>
			<td>63500</td>
			<td>MSC Industrial</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">55mm glass bottom dish; 30mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>D60-30-1.5-N</td>
			<td>Cellvis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>Harrick Plasma</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loading Larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipetteman, P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% ethanol (diluted to 70% with water prior to use)</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-5A</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">powdered skim milk</td>
			<td></td>
			<td>2902887</td>
			<td>MP Biomedicals</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">double distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-phenylthiorurea</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tricaine (ethyl 3-aminobenzoate)</td>
			<td></td>
			<td>C-FINQ-UE</td>
			<td>Western Chemical</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">low melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat block (dry bath incubator)</td>
			<td>Fisher Scientific</td>
			<td>11-718-2</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E3 buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">large orifice pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>02-707-134</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">General purpose pipette tip, 200 uL</td>
			<td>Fisherbrand</td>
			<td>21-197-8E</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#15 scalpel blade&#160;</td>
			<td>Feather</td>
			<td>2976</td>
			<td>Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G syringe needle</td>
			<td>BD&#160;</td>
			<td>BD305122</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris imaging software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term live imaging device for improved experimental manipulation of zebrafish larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

The zWEDGI PDMS microfluidic device is a functionally compartmentalized device designed to accommodate four main functions (listed below) associated with live imaging of caudal fin wounding healing and regrowth in the zebrafish larvae. PDMS was chosen for zWEDGI fabrication because it is not only readily available and an industry standard for biocompatibility, but also works well in molds. Additionally, PDMS makes the device reusable and void of hard or sharp edges once the device is form...

Watch this Scientific Journal Video about Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae at JoVE.com

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

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