Name: a layered mounting method for extended time-lapse confocal microscopy of whole zebrafish embryos
Uploaded: 2020-01-14T12:00:00
Description: Watch this Scientific Journal Video about A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos at JoVE.com

We thank Albert Pan and Arndt Sieakmann for gifts of transgenic fish. We thank Koichi Kawakami at National Institute of Genetics, the National BioResource Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan for the gift of the transgenic zebrafish HGn39b. We also thank Fatima Merchant and Kathleen Gajewski for assistance on confocal microscopy, and Tracey Theriault for photographs.
This work was supported by grants from the National Institute of Environmental Health Sciences of the National Institutes of Health (grant number P30ES023512 and contract number HHSN273201500010C). SU was supported by a fellowship from the Keck Computational Cancer Biology program (Gulf Coast Consortia CPRIT grant RP140113) and by Hugh Roy and Lillie Endowment Fund. J-&#197; G was supported by the Robert A. Welch Foundation (E-0004).

The animal work presented here was approved by the Institutional Animal Care and Use Committees (IACUCs) of the University of Houston and Indiana University.
1. Fish husbandry
NOTE: Work with vertebrate models requires an IACUC approved protocol. It should be conducted according to relevant national and international guidelines.
<ol>
	<li>Maintain adult zebrafish as described in previously published literature9.</li>
	<li>In the afternoon, ...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Schmid, B., 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-speed+panoramic+light-sheet+microscopy+reveals+global+endodermal+cell+dynamics.">High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics.</a> Nature Communications. 4, 2207 (2013).</li><li>Megason, S. 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N., Liebel, U., Gehrig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orientation+tools+for+automated+zebrafish+screening+assays+using+desktop+3D+printing.">Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing.</a> BMC Biotechnology. 14, 36 (2014).</li><li>Weijts, B., Tkachenko, E., Traver, D., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Well+Dish+for+High-Resolution+Longitudinal+Imaging+of+the+Tail+and+Posterior+Trunk+of+Larval+Zebrafish.">A Four-Well Dish for High-Resolution Longitudinal Imaging of the Tail and Posterior Trunk of Larval Zebrafish.</a> Zebrafish. 14 (5), 489-491 (2017).</li><li>Hirsinger, E., Steventon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Versatile+Mounting+Method+for+Long+Term+Imaging+of+Zebrafish+Development.">A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.</a> Journal of Visualized Experiment. (119), (2017).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> Journal of Visualized Experiment. (69), e4196 (2012).</li><li>McCollum, C. W., 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=Embryonic+exposure+to+sodium+arsenite+perturbs+vascular+development+in+zebrafish.">Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish.</a> Aquatic Toxicology. 152, 152-163 (2014).</li><li>Pan, Y. 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=Zebrabow:+multispectral+cell+labeling+for+cell+tracing+and+lineage+analysis+in+zebrafish.">Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish.</a> Development. 140 (13), 2835-2846 (2013).</li><li>Asakawa, K., 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=Genetic+dissection+of+neural+circuits+by+Tol2+transposon-mediated+Gal4+gene+and+enhancer+trapping+in+zebrafish.">Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.</a> Proceedings of the National Academy of Science U. S. A. 105 (4), 1255-1260 (2008).</li><li>Nagayoshi, S., 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=Insertional+mutagenesis+by+the+Tol2+transposon-mediated+enhancer+trap+approach+generated+mutations+in+two+developmental+genes:+tcf7+and+synembryn-like.">Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like.</a> Development. 135 (1), 159-169 (2008).</li><li>Huang, W. 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=Combined+use+of+MS-222+(tricaine)+and+isoflurane+extends+anesthesia+time+and+minimizes+cardiac+rhythm+side+effects+in+adult+zebrafish.">Combined use of MS-222 (tricaine) and isoflurane extends anesthesia time and minimizes cardiac rhythm side effects in adult zebrafish.</a> Zebrafish. 7 (3), 297-304 (2010).</li><li>Craig, M. P., Gilday, S. D., Hove, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+effects+of+chemical+immobilization+on+the+heart+rate+of+embryonic+zebrafish.">Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish.</a> Laboratory Animal (NY). 35 (9), 41-47 (2006).</li><li>Swinburne, I. A., Mosaliganti, K. R., Green, A. A., Megason, S. 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A mounting method for extended time-lapse confocal microscopy of whole zebrafish embryos is described here. The most critical step for the mounting method is to identify the optimal concentration of agarose that will allow for unrestricted zebrafish embryo growth, and at the same time keep the embryos in a completely fixed position for confocal imaging. Because the optimal concentration of agarose is very narrow, this value is very sensitive to the errors in measurement of the weight of agarose and the volume of E3 durin...

The zebrafish has long been a model organism for developmental biology, and microscopy is the major method to visualize embryonic development. The advantages of using zebrafish embryos for developmental studies include small size, optical clarity, rapid development, and high fecundity of the adult fish. The generation of transgenic zebrafish lines expressing fluorescence in certain tissues or cells have allowed for a direct visualization of tissue development in ways not possible with larger vertebrate animals. In combination with time-lapse microscopy, details and dynamics of the tissue development can be readily studied.
A difficulty with imaging zebrafish development is that the embryos grow substantially in length; the embryo extends its length four times within the first 3 days of life1. Also, the body of the early embryo is soft, and easily becomes distorted if growth is restricted. Yet, to perform confocal microscopy, the embryo must be immobilized. To keep embryos in a fixed position for confocal time-lapse imaging, they are regularly anesthetized and mounted in 0.3-1% low-melt agarose. This has the advantage of allowing for some growth during imaging for a certain period of time, while restricting movements of the embryo. Parts of the embryo can efficiently be imaged like this. However, when using this method for imaging of the whole embryo for extended time periods, distortions are observed because of restricted growth caused by the agarose. Thus, other mounting methods are required. Kaufmann and colleagues have described an alternative mounting of zebrafish embryos for light sheet microscopy, such as selective plane illumination microscopy (SPIM), by mounting the embryos in fluorinated ethylene propylene (FEP) tubes containing low concentrations of agarose or methylcellulose2. This technique produces a superb visualization of embryogenesis over time. Schmid et al. describe mounting of up to six embryos in agarose in FEB tubes for light-sheet microscopy3 providing visualization of several embryos in one imaging session. Molds have been used to create embryo arrays for efficient mounting of larger numbers of embryos4. Masselkink et al. have constructed 3D printed plastic molds that can be used to make silicon casts that zebrafish embryos at different stages can be placed in, enabling mounting in a constant position for imaging, including confocal imaging5. 3D printing has also been used to make molds for consistent positioning of zebrafish embryos in 96-well format6. Some molds are customized for certain stages and may not permit unrestricted growth for long time periods, whereas other molds are more flexible. Recently, Weijts et al. published the design and fabrication of a four-well dish for live imaging of zebrafish embryos7. In this dish, the tail and trunk of anesthetized fish embryos are placed manually under a clear silicone roof attached just above a cover glass to form a pocket. The embryo is then fixed in this position by the addition of 0.4% agarose. This mounting allows for the imaging of the about 2 mm long posterior part (trunk and tail) of the embryo, and as up to 12 embryos can be mounted per well, the method allows for the imaging of multiple samples. Similarly, Hirsinger and Steventon recently presented a method where the head of the fish is mounted in agarose, while the tail can freely grow, and this method also efficiently facilitates imaging of the trunk and tail region of the embryo8.
This article describes a layered mounting method for zebrafish embryos that restrict the movements of the embryos while allowing for unrestricted growth. The advantages of this mounting method are that it is a low-cost, fast and easy method to mount embryos of various stages for imaging using any inverted microscope. The mounting permits long-term imaging of the whole body (head, trunk and tail) during embryo development. To showcase the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish. Two embryos per session, at two wavelengths in 3D were imaged by time-lapse microscopy for 55 consecutive hours to render movies of tissue development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Low melting agarose</td>
			<td>Sigma-Aldrich, MO</td>
			<td>A9414</td>
			<td>Store dissolved solution at 4 &#176;C</td>
		</tr>
		<tr>
			<td>35 mm glass bottom dishes with No. 0 coverslip and 10 mm diameter of glass bottom</td>
			<td>MatTek Corporation, MA</td>
			<td>P35GCOL-0-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>E10521</td>
			<td>Store dissolved solution at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>P7629</td>
			<td>Store dissolved solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Micro cover glass 22x22 mm</td>
			<td>VWR</td>
			<td>48366 067</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMi8 fluorescence microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X software</td>
			<td>Leica</td>
			<td>NA</td>
			<td>Microscope software</td>
		</tr>
		<tr>
			<td>DMC4500 digital microscope camera</td>
			<td>Leica</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1S confocal microscope</td>
			<td>Nikon Instruments Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon NIS AR Version 4.40</td>
			<td>Nikon Instruments Inc.</td>
			<td>NA</td>
			<td>Microscope software</td>
		</tr>
	</tbody>
</table>

a layered mounting method for extended time-lapse confocal microscopy of whole zebrafish embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

Development of the mounting method 
The main aim of this work was to develop a low-cost mounting technique for time-lapse imaging of zebrafish development for extended periods of time. The layered mounting method was developed to allow for full growth of the fragile zebrafish embryo body, while restricting its movements. If the agarose concentration of layer 1 is too high, the embryos will become distorted and curved (Figure 2). Embryos grown at 0.1% and 0.5% agarose ha...

Watch this Scientific Journal Video about A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos at JoVE.com

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

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