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>
	<li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+toto+imaging+of+embryogenesis+with+confocal+time-lapse+microscopy.">In toto imaging of embryogenesis with confocal time-lapse microscopy.</a> Methods in Molecular Biology. 546, 317-332 (2009).</li><li>Masselink, W., Wong, J. C., Liu, B., Fu, J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+silicone+imaging+casts+for+zebrafish+embryos+and+larvae.">Low-cost silicone imaging casts for zebrafish embryos and larvae.</a> Zebrafish. 11 (1), 26-31 (2014).</li><li>Wittbrodt, J. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Long-Term+Imaging+of+Embryos+with+Genetically+Encoded+alpha-Bungarotoxin.">Improved Long-Term Imaging of Embryos with Genetically Encoded alpha-Bungarotoxin.</a> PLoS One. 10 (8), 0134005 (2015).</li></ol>

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 ...

We have developed a mounting method for time-lapse imaging that allows for unrestricted growth of fragile zebrafish embryos and at the same time keeps them in a completely fixed position. This mounting method is fast, easy, and cheap and it works for lab imaging on any inverted microscope. We developed this method for imaging of live zebrafish embryos, but it could be modified for imaging of other small aquatic animal models.Start by breeding the adult zebrafish. In the afternoon, place the fish into breeding tanks at a male-to-female ratio of one-to-two. Then prepare a stock solution of 1%low melt agarose in embryo media and dissolve it by heating it in a microwave oven.Aliquot the agarose solution into 1.5 milliliter tubes. Make a stock solution of 4%tricaine in distilled water and store the agarose and tricaine at four degrees Celsius. Tricaine is toxic and has to be weighed and prepared in a fume hood.After mating, harvest the zebrafish embryos in E3 in a Petri dish and incubate them at 26.5 degrees Celsius for about 28 hours before mounting. Anesthetize the embryos with 0.02%tricaine and dechorionate them under a microscope by gently gripping and pulling the chorion apart to release the embryo. Just before mounting, heat the agarose solution to 65 degrees Celsius and then let it cool to approximately 30 degrees Celsius so the embryo is not harmed by the heat and add tricaine to the agarose solution.Use 35 millimeter glass bottom dishes with a number zero coverglass bottom. Gently place a dechorionated embryo with one of its lateral sides toward the bottom of the dish with a glass pipette or a micropipette, then carefully remove any remaining E3 with a micropipette. Add the first agarose solution to the small well created by the coverglass attached to the bottom of the dish to cover the embryo making sure that the agarose covers the small well but does not overflow it.Cover the small well with a coverglass to create a narrow agarose-filled space with the embryo between the two coverglasses. Place a layer of 1%agarose solution on top of the coverglass all over the bottom of the dish which will keep the coverglass in place as it solidifies. Then fill the remaining portion of the dish with E3 containing 0.02%tricaine to keep the system hydrated.To identify the optimal agarose concentration for layer one, mount the embryos in increasing concentrations of agarose and perform time-lapse imaging to identify the concentration that minimizes embryo distortion and motility, then test a finer range of concentrations based on the results. The most critical step of this protocol is to identify the optimal concentration of agarose for layer one. Test different nano concentrations of agarose such as 0.030%0.032%etc.to find the optimal concentration. To perform time-lapse imaging of the embryos, use a microscope stage with an incubator set to 28.5 degrees Celsius and image for up to 55 hours. To simultaneously image several embryos, use a stage adapter that holds several dishes and rotate the dishes one by one so that the embryos are horizontally positioned.Select a low magnification objective, then locate the embryos using the eyepiece and finally align them horizontally by rotating the dishes. Record the embryos'location in the software and create a filename for autosaving the data. Set the pinhole size, scan speed, image size, and zoom.Then select a higher magnification for capturing images and the fluorescence channels to be imaged. For each channel, adjust the laser power and the detector high voltage making sure to collect the best dynamic range possible while avoiding saturation and limiting photo bleaching. Next, to image the whole embryo with a 10X objective, set the large image capture settings in the software to image several fields of view and stitch them together adjusting the position of the embryo to make sure it fits within this large image area.Configure the settings for capturing Z-stacks according to the manuscript directions. In order to maintain focus of multiple embryos during long-term time-lapse imaging, use an automated focus and adjust the individual offset levels for each embryo to focus at the reference plane. Then set up time-lapse parameters in the microscope software to image for a duration of 55 hours at one-hour intervals.At each cycle, capture two embryo datasets sequentially and save data automatically. Use the microscope software to process the images. If more than one embryo was imaged, create independent files for each embryo by splitting the datasets based on the location of the imaging.Convert the 3D time datasets into 2D time datasets using maximum intensity projections or a similar tool. Finally, create movie files by saving as AVI and selecting the no compression option with 200 millisecond intervals for a playback speed of five frames per second. This layered mounting method makes it possible to image zebrafish embryos while allowing them to grow but at the same restricting their movement.If the agarose concentration is too high, the embryos become distorted. But if it is too low, they will move out of the field of view during time-lapse microscopy. The optimal agarose concentration was found to be between 0.028 and 0.034%Live transgenic zebrafish expressing RFP in the entire embryo and GFP in the endothelial cells of the vasculature were imaged for 55 hours during which the intersegemental vessel sprouted, subintestinal and head vasculature developed, and caudal vein plexus condensation with trunk extension occurred.Embryos expressing GFP and motor neurons were imaged to visualize motor neuron development. The motor neuron axons sprout from the ventral neural tube over the somites towards the ventral side of the embryo. The co-development of the dorsal sprouting of motor neuron axons in relation to the position of intersegmental vessels was imaged using a higher magnification.This made it possible to visualize the finer details of ventral axon sprouting as well as dorsal axon sprouting from the neural tube. As somite numbers increased, the somites also extend in length and width. This process was imaged using transgenic embryos expressing GFP in muscles.Because the optimal concentration of agarose is very narrow, it is very sensitive to small variations in measurements when preparing the stock solution of agarose and it needs to be redefined for each new batch of agarose solution. It is important to carefully set the imaging parameters including image size and resolution, scan speed, zoom, detector high voltage, and laser power to ensure consistency between experiments and also to reduce the possibility of photo toxicity and photo bleaching during long-term time-lapse imaging. Using this mounting method for transgenic zebrafish embryos followed by extended time-lapse imaging of the whole organism, we can visualize how different tissues develop together.

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Developmental Biology

8.7K visualizações.  University of Houston. Desenvolvemos um método de montagem para imagens de lapso de tempo que permite o crescimento irrestrito de embriões frágeis de zebrafish e, ao mesmo tempo, os mantém em uma posição completamente fixa. Este método de montagem é rápido, fácil e barato e funciona para imagens de laboratório em qualquer microscópio invertido. Desenvolvemos este método para a imagem de embriões de zebrafish vivos, mas ele poderia ser modificado para imagens de outros pequenos modelos de animais aquá...