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, place adult zebrafish in breeding tanks. Breed males to females at a ratio of 1:2.</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Make a stock solution of 1% low melt agarose in embryo media (E3: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, adjusted to pH 7.0). Aliquot the stock solution into 1.5 mL tubes and store them at 4 &#176;C.</li>
	<li>Make a stock solution of 4% (w/v) Tricaine (MS-222) in distilled water. Store at 4 &#176;C in a dark bottle. 
	CAUTION: Tricaine is toxic and should be weighed and dissolved in a fume hood.</li>
	<li>Make a stock solution of 20 mM N-phenylthiourea (PTU) in distilled water. Store at -20 &#176;C.</li>
</ol>
3. Preparation of embryos
<ol>
	<li>After mating, harvest embryos in E3 in a Petri dish and incubate them at 26.5 &#176;C for about 28 h before mounting. 
	NOTE: This slows down the development of the embryos so that the embryos are approximately at 30 somite stage at the beginning of imaging.</li>
	<li>Anesthetize embryos in 0.016-0.020% Tricaine in E3. To inhibit pigmentation, add PTU to a concentration of 200 &#181;M.</li>
	<li>Dechorionate the embryos using forceps under a dissecting microscope. Using two forceps, grip and gently pull the chorion apart to release the embryo.</li>
</ol>
4. Mounting in agarose
NOTE: The developed mounting method requires two different concentrations of low-melt agarose in E3 with 0.02% Tricaine and PTU as needed. The first agarose solution contains an optimal concentration of agarose at which the distortion and motility are at a minimum. The optimization is described in step 5 below.
<ol>
	<li>Heat the agarose solutions for the two layers (concentration defined in step 5 below and 1%) to 65 &#176;C. Let the agarose cool down to approximately 30 &#176;C just before mounting so that the embryo is not harmed by the heat. For mounting, use 35 mm glass bottom dishes with a No. 0 cover glass bottom. The cover glass attached to the bottom of the dish creates a 10 mm shallow (approx. 1.2 mm deep) well, in which the embryo is to be placed. 
	NOTE: In this case, the concentration with the least motility and distortions were between 0.025 to 0.040% agarose.</li>
	<li>Gently place a dechorionated embryo with one of its lateral sides toward the bottom of the dish using a glass pipette or micropipette. If using a micropipette, cut the outer part of the tip to increase the size of the opening to fit the embryo (Figure 1A). Carefully remove any remaining E3 with a micropipette.</li>
	<li>Add the first agarose solution to the small well created by the cover glass attached to the bottom of the dish to cover the embryo (Layer 1) (Figure 1B). Ensure that the agarose covers the small well but will not overflow it.</li>
	<li>Cover the small well with a cover glass (22 mm x 22 mm) (Figure 1C) to create a narrow agarose filled space with the embryo between the two cover glasses.</li>
	<li>Place a layer of 1% agarose solution on top of the cover glass all over the bottom of the dish (Layer 2) (Figure 1D). As this layer solidifies, it holds the cover glass in place.</li>
	<li>Fill the remaining portion of the dish with E3 containing 0.02% Tricaine to keep the system hydrated (Layer 3) (Figure 1E). 
	NOTE: In this setup, the cover glass and 1% agarose protect the bottom layer from getting diluted.</li>
</ol>
5. Optimization of agarose solution for layer 1
<ol>
	<li>To identify the optimal concentration of agarose for Layer 1, use a multiscale grid search approach. Mount embryos in increasing concentrations of agarose ranging from 0.01% to 1% followed by time-lapse imaging of embryo growth restriction and motility in the field of view. Identify the concentrations where both the distortion and motility are at a minimum.</li>
	<li>To optimize the concentration of agarose further, mount the embryos using a finer range of concentrations of agarose (e.g., between 0.025 and 0.040% agarose) depending on the concentration found to be best in step 5.1 (e.g., 0.025%, 0.028%, 0.031%, etc.). 
	NOTE: In our laboratory, the optimal agarose concentration was around 0.03%.</li>
</ol>
6. Time-lapse imaging
NOTE: This mounting method works for any inverted microscope with time-lapse functionality for fluorescence and bright field imaging.
<ol>
	<li>Perform time-lapse imaging for the whole embryo or parts of it for up to 55 h. For imaging of several embryos, use a stage adaptor that holds several dishes with one embryo mounted per dish. Rotate the dishes one by one so that the embryos are approximately horizontally positioned as determined by eye. For optimal embryo growth and development, use a microscope stage with an incubator set at 28.5 &#176;C.</li>
	<li>Select a low magnification objective in the microscope software. Under the eye piece, locate and finely align the embryos horizontally by rotating the dishes using bright field illumination and record their locations in the software. Create a filename for automatic saving of the data. 
	NOTE: In this experiment, a plan apo &#955; 4x 0.2 NA objective and the XY tab within the ND acquisition window were used to record the locations of the embryos. Data were saved in .nd2 format. The objective was selected by clicking on its icons in the Ti Pad tab.</li>
	<li>Set the pinhole size, scan speed, image size, and zoom. Next, select a higher magnification objective for capturing the images. 
	NOTE: In this experiment, a plan-apo 10x 0.45 NA objective was used for imaging of whole embryos, and a super-fluor 20x 0.75 NA objective was used for the higher magnification imaging of parts of the embryo. The pinhole was set to 1.2 AU (19.2 &#181;m for the 10x lens), the scan speed was set for a pixel dwell time of 2.4 &#181;s and the image size was set to 1024 x 1024 pixels with a scan zoom of one, giving a pixel size of 1.24 &#181;m for the 10x lens and 0.62 &#181;m for the 20x lens.</li>
	<li>Select the channels for the fluorescence to be imaged. Adjust the image capture settings one channel at a time. For each fluorescence channel adjust the laser power and the detector high voltage, making sure to collect the best dynamic range possible while avoiding the saturation and limiting photobleaching. 
	NOTE: In this experiment, GFP was imaged with the 488 green channel, (488 nm laser and emission between 500 and 550 nm), and RFP with the 561 red channel (561 nm laser and emission between 570 and 600 nm), and the transmission image was collected using the 561 nm laser and the transmitted light detector (TD channel).</li>
	<li>To image the whole embryo with the 10x objective, image several fields of view with overlap and stitch them together using the microscope software. 
	NOTE: As the embryos will grow substantially in size during the imaging, make sure that there is space in the field of view anterior and posterior to the embryo. Use the Scan Large Image tab of the ND acquisition window and select a 4 x 1 pattern with 10% overlap to capture four adjacent fields of view.</li>
	<li>Configure the settings for capturing the Z-stacks using the microscope software. 
	NOTE: Because the embryo is mounted close to the bottom of the glass dish, its center will move away from the bottom as it grows. Use the Z tab of the ND acquisition window and select the Asymmetrical relative range. With this method, the current focus plane is used as the Reference plane and the rest of the planes are asymmetrically distributed above and below to include the whole embryo within the Z-stack volume, with enough space to account for the specimen&#39;s growth. In all experiments, the interval was set to 11 &#181;m and the total range to about 45 planes.</li>
	<li>In order to maintain the focus of multiple embryos during long-term time-lapse imaging, use an automated focus. If imaging several embryos, adjust the individual offset levels for each embryo to focus at the Reference plane. 
	NOTE: In this experiment, a laser-based focus stabilization system was used.</li>
	<li>Set up time-lapse parameters in the microscope software and image for a duration of 55 h at about 1 h intervals. At each cycle, capture two embryo datasets sequentially and save data automatically after each cycle.</li>
	<li>Process images using an on-line or off-line version of the microscope software. If more than one embryo was imaged, create independent files for each embryo by splitting the dataset based on the location of imaging. Use maximum intensity projections or a similar tool to convert 3D time data sets into 2D time data sets. Create movie files by using the Save as menu option, selecting .avi as the file format. Select the No compression option with 200 ms intervals for a playback speed of 5 frames /s. 
	NOTE: The original dataset of two embryos in this experiment was split using the Split locations function in the software (File | Import/Export | Split Multipoints). 3D time datasets were converted to 2D time data sets using the Maximum intensity projection function (Image | ND Processing| Create Maximum Intensity Projection Image in Z).</li>
</ol>

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	<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. 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The authors have nothing to disclose.

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><tbody><tr><td>Low melting agarose</td><td>Sigma-Aldrich, MO</td><td>A9414</td><td>Store dissolved solution at 4 &amp;deg;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 &amp;deg;C</td></tr><tr><td>N-phenylthiourea (PTU)</td><td>Sigma-Aldrich, MO</td><td>P7629</td><td>Store dissolved solution at -20 &amp;deg;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 ...

a-layered-mounting-method-for-extended-time-lapse-confocal-microscopy

Research

JoVE Journal

Developmental Biology

8.9K Views.  University of Houston. We hebben een montagemethode ontwikkeld voor time-lapse beeldvorming die onbeperkte groei van fragiele zebravisembryo's mogelijk maakt en tegelijkertijd in een volledig vaste positie houdt. Deze montagemethode is snel, eenvoudig en goedkoop en werkt voor lab imaging op elke omgekeerde microscoop. We ontwikkelden deze methode voor beeldvorming van levende zebravisembryo's, maar het kan worden aangepast voor beeldvorming van andere kleine aquatische diermodellen.Begin met het fokken van ...

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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscience

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

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.  ...

Biology

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Layered Agar Mounting: Preparing Live Zebrafish Embryos for Long-Term Imaging with an Inverted Microscope

Source: Upadhyay S., et.al. <a href="https://www.jove.com/v/60321/" target="_blank">A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos</a>. J. Vis. Exp. (2020).
This article describes a method to mount live zebrafish embryos for long term imaging. This method is cost effective and easy to perform using regular glass-bottom microscopy dishes for imaging on Inverted Microscope. The mounting is performed in layers of agarose at different concentrations.

Source: Upadhyay S., et.al. A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos. J. Vis. Exp. (2020).
This ...

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

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