The authors thank Thomas Schilling for kindly gifting the Tg(sox10:eGFP) fish and Mary Halloran for kindly gifting the Tg(foxd3:GFP) fish.

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cranial+neural+crest+cell+migration+and+patterning+in+craniofacial+development.">Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development.</a> Development. 137, 2605-2621 (2010).</li><li>Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neural+crest+cell+formation+and+migration+in+mouse+embryos.">Specification of neural crest cell formation and migration in mouse embryos.</a> Sem Cell Dev Biol. 16, 683-693 (2005).</li><li>Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L., Coulombre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+avian+ocular+and+periocular+tissues.">Origins of avian ocular and periocular tissues.</a> Exp Eye Res. 29, 27-43 (1979).</li><li>Bohnsack, B. L., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+and+retinoic+acid+interact+to+regulate+zebrafish+craniofacial+neural+crest+development.">Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development.</a> Dev Biol. 373, 300-309 (2013).</li><li>Chawla, B., Schley, E., Williams, A. L., Bohnsack, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+and+pitx2+regulate+early+neural+crest+survival+and+migration+in+craniofacial+and+ocular+development.">Retinoic acid and pitx2 regulate early neural crest survival and migration in craniofacial and ocular development.</a> Birth Defects Res B Dev Reprod Toxicol. , (2016).</li><li>Trainor, P. A., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+cells+of+the+mouse+embryo-codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+branchial+arches.">Cranial paraxial mesoderm and neural crest cells of the mouse embryo-codistribution in the craniofacial mesenchyme but distinct segregation in branchial arches.</a> Development. 121 (8), 2569-2582 (1995).</li><li>Hong, C. S., Saint-Jeannet, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+proteins+and+neural+crest+development.">Sox proteins and neural crest development.</a> Sem Cell Dev Biol. 16, 694-703 (2005).</li><li>Steventon, B., Carona-Fontaine, C., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+network+during+neural+crest+induction:+from+cell+specification+to+cell+survival.">Genetic network during neural crest induction: from cell specification to cell survival.</a> Sem Cell Dev Biol. 16, 647-654 (2005).</li><li>Dressler, 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=Dental+and+craniofacial+anomalies+associated+wth+Axenfeld-Rieger+syndrome+with+PITX2+mutation.">Dental and craniofacial anomalies associated wth Axenfeld-Rieger syndrome with PITX2 mutation.</a> Case Report Med. , 621984 (2010).</li><li>Ozeki, H., Shirai, S., Ikeda, K., Ogura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalies+associated+with+Axenfeld-Rieger+syndrome.">Anomalies associated with Axenfeld-Rieger syndrome.</a> Graefes Arch Clin Exp Ophthalmol. 237 (9), 730-734 (1999).</li><li>Strungaru, M. H., Dinu, I., Walter, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype-phenotype+correlations+in+Axenfeld-Rieger+malformation+and+glaucoma+patients+with+FOXC1+and+PITX2+mutations.">Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations.</a> Invest Ophthalmol Vis Sci. 48, 228-237 (2007).</li><li>Tumer, Z., Bach-Holm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenfeld-Rieger+syndrome+and+spectrum+of+Pitx2+and+Foxc1+mutations.">Axenfeld-Rieger syndrome and spectrum of Pitx2 and Foxc1 mutations.</a> Eur J Hum Genet. 17, 1527-1539 (2009).</li><li>Schoner, 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=Hydrocephalus,+agenesis+of+the+corpus+callosum,+and+cleft+lip/palate+represent+frequent+associations+in+fetuses+with+Peters+plus+syndrome+and+B3GALTL+mutations.+Fetal+PPS+phenotypes,+expanded+by+Dandy+Walker+cyst+and+encephalocele.">Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.</a> Prenat Diagn. 33 (1), 75-80 (2013).</li><li>Aliferis, 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=A+novel+nonsense+B3GALTL+mutation+confirms+Peters+plus+syndrome+in+a+patient+with+multiple+malformations+and+Peters+anomaly.">A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.</a> Ophthalmic Genet. 31 (4), 205-208 (2010).</li><li>Lesnik Oberstein, ., S, 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=Peters+Plus+syndrome+is+caused+by+mutations+in+B3GALTL,+a+putative+glycosyltransferase.">Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.</a> Am J Hum Genet. 79 (3), 562-566 (2006).</li><li>Brittijn, S. 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=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Bohnsack, B. L., Kasprick, D., Kish, P. E., Goldman, D., Kahana, 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+zebrafish+model+of+Axenfeld-Rieger+Syndrome+reveals+that+pitx2+regulation+by+retinoic+acid+is+essential+for+ocular+and+craniofacial+development.">A zebrafish model of Axenfeld-Rieger Syndrome reveals that pitx2 regulation by retinoic acid is essential for ocular and craniofacial development.</a> Invest Ophthalmol Vis Sci. 53 (1), 7-22 (2012).</li><li>Williams, A. L., Eason, J., Chawla, B., Bohnsack, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyp1b1+regulates+ocular+fissure+closure+through+a+retinoic+acid-independent+pathway.">Cyp1b1 regulates ocular fissure closure through a retinoic acid-independent pathway.</a> Invest Ophthalmol Vis Sci. 58 (2), 1084-1097 (2017).</li><li>Curran, K., Raible, D. W., Lister, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxd3+controls+melanophore+specification+in+the+zebrafish+neual+crest+by+regulation+of+Mitf.">Foxd3 controls melanophore specification in the zebrafish neual crest by regulation of Mitf.</a> Dev Biol. 332 (2), 408-417 (2009).</li><li>Dutton, K., Dutton, J. R., Pauliny, A., Kelsh, R. 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Multi-photon time-lapse imaging enables the in vivo tracking of transient and migratory cell populations. This powerful technique can be used to study embryonic processes in real time, and in the present study, the results of this method enhanced the current knowledge of neural crest cell migration and development. Previous time-lapse imaging studies typically utilize confocal laser scanning microscopy.29,30,31,</s...

Congenital eye diseases can cause childhood blindness and are often due to abnormalities of the cranial neural crest. Neural crest cells are transient stem cells that arise from the neural tube and form numerous tissues throughout the body.1,2,3,4,5 Neural crest cells, derived from the prosencephalon and mesencephalon, give rise to the bone and cartilage of the midface and frontal regions, and the iris, cornea, trabecular meshwork, and sclera in the anterior segment of the eye.4,6,7,8 Neural crest cells from the rhombencephalon form the pharyngeal arches, jaw, and cardiac outflow tract.1,3,4,9,10 Studies have highlighted the contributions of the neural crest to ocular and periocular development, emphasizing the importance of these cells in vertebrate eye development. Indeed, disruption of neural crest cell migration and differentiation lead to craniofacial and ocular anomalies as observed in Axenfeld-Rieger Syndrome and Peters Plus Syndrome.11,12,13,14,15,16,17 Thus, a comprehensive understanding of the migration, proliferation and differentiation of these neural crest cells will provide insight into the complexities underlying congenital eye diseases.
The zebrafish is a powerful model organism for studying ocular development, as the structures of the zebrafish eye are similar to their mammalian counterparts, and many genes are evolutionarily conserved between zebrafish and mammals.18,19,20 In addition, zebrafish embryos are transparent and oviparous, facilitating the visualization of eye development in real-time.
Expanding on previously published work,6,7,20 the migratory pattern of neural crest cells was described using multi-photon fluorescence time-lapse imaging on transgenic zebrafish lines labeled with green fluorescent protein (GFP) under the transcriptional control of the SRY (sex-determining region Y)-box 10 (sox10) or Forkhead Box D3 (foxd3) gene regulatory regions.21,22,23,24. Multi-photon fluorescence time-lapse imaging is a powerful technique that combines the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with fluorophores.25,26,27 The use of the multi-photon laser has distinct advantages over standard confocal microscopy, including increased tissue penetration and decreased fluorophore bleaching.
Using this method, two distinct populations of neural crest cells varying in timing of migration and migratory pathways were discriminated, namely foxd3-positive neural crest cells in the periocular mesenchyme and developing eye and sox10-positive neural crest cells in the craniofacial mesenchyme. With this method, an approach to visualize the migration of ocular and craniofacial neural crest migration in zebrafish is introduced, making it easy to observe regulated neural crest migration in real time during development.
This protocol provides information for generating time-lapse videos during early eye development in Tg(sox10:EGFP) and Tg(foxd3:GFP) transgenic zebrafish, as an example. This protocol can be further applied for the high-resolution, three-dimensional, real-time visualization of the early development of any ocular and craniofacial structure derived from neural crest cells in zebrafish. Moreover, this method can further be applied for the visualization of the development of other tissues and organs in zebrafish and other animal models.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">RC-40HP</td>
			<td style="width:391px;">High Profile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass Coverslips</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">12-545-102</td>
			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">High Vacuum Grease</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">14-635-5C</td>
			<td style="width:391px;">2.0-lb. tube. DOW CORNING CORPORATION 
			1658832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Quick Exchange Platform</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">QE-1</td>
			<td style="width:391px;">35 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Stage Adapter</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">SA-20LZ-AL</td>
			<td style="width:391px;">16.5 x 10 cm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">TC SP5 MP multi-photon microscope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mai Tai DeepSee Ti-Sapphire Laser</td>
			<td style="width:175px;">SpectraPhysics</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Laser Safety Box</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Leica Application Suite X (LAS X)&#160; Software</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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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.

Multi-photon fluorescence time-lapse imaging generated a series of videos that revealed the migration patterns of cranial neural crest cells that give rise to the craniofacial structures and anterior segment of the eye in the Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish lines. As an example, sox10-positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the craniofacial region (Video 1, F...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

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

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 ...

The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissues and allows for longer periods of image acquisition. To set up for embryo collection between 3:00 and 9:00 p.m. assemble breeding tanks and fill them with reverse osmosis, or RO water.Transfer up to three females and three males into opposing sides of the divided tank. The next morning, remove the divider shortly after the lights turn on in the vivarium. Allow undisturbed mating for 20 minutes or until sufficient numbers of embryos are produced.The embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish, and quickly place them into a clean, solid outer tank filled with RO water. Use an egg collecting screen to collect eggs from the outer tank and transfer the eggs to Petri dishes containing 30 milliliters of 1X embryo medium.Incubate the collected eggs at 28.5 degrees Celsius. For transgenic embryos, at 24 hours post-fertilization or HPF, remove the dead eggs and assess the developmental stage of the embryos. Using forceps, remove the chorions from three to four embryos that express GFP.Under a dissecting fluorescence stereo microscope with a standard 460 to 490 nanometer band pass excitation filter, screen for GFP positive embryos. Transfer these embryos to a separate Petri dish containing 30 milliliters of fresh 1X embryo medium. After 20 HPF, place the screened and dechorionated GFP positive embryos in 0.003%PTU solution to inhibit pigmentation.Avoid initiating treatment earlier as it can have adverse effects on neural crest and neuroepithelial development. Add 0.4 grams of LowMelt Agarose powder to 20mL of 1x embryo medium. Heat the mixture for one to two minutes or until the solution is clear and all particles are dissolved.Aliquot approximately 1mL of the Agarose solution into fresh 1.5mL centrifuge tubes for storage. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass cover slip onto the base and screw the top of the open bath chamber onto the base until it is tight.Pipette approximately 500-700 microliters of 2%Agarose solution into the open bath chamber until the base is approximately 3/4s filled. Wait 30 seconds to allow the Agarose to cool slightly without completely polymerizing and subsequently transfer a single embryo to the center of the base. Under a fluorescent stereo microscope, use a 10 microliter micro pipette tip to orient the embryo and position it near the bottom of the Agarose so that it doesn't float away when the embryo medium is added.A critical step is to make sure that the embryo is positioned correctly in the Agarose. In these experiments, we orient the embryo laterally but the embryo can also be positioned to focus on dorsal and ventral areas. Monitor and reposition the embryo until the Agarose has set.Then use a transfer pipette to entirely fill the assembled open chamber with time-lapse embryo medium. After placing the entire set up onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position and use the fine focus to position the embryo in the middle of the microscope range.Manually lower the stage and change the five time objective to the 25 time water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the XYTZ mode for obtaining multiple images at time or T intervals in the XY plane over a depth of Z.Use the epifluorescence or bright field view to find the depth of focus in the area of interest which will demarcate the z-stack.For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here, in the software click the begin button. After focusing down to the mid-line of the embryo, which is the end of the z-stack, click the end button.A critical step is to make sure that the Z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to the medial edges. Click on the menu for adjusting the acquisition time and the imaging frequency.For adequate recovery of the fluor4 and survival of the embryo, allow for a ratio of at least one to three between z-stack acquisition when laser power is on and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Then set the total length of time for the experiment in the appropriate window.Now, turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera.Make sure that the area of interest remains within the frame through the duration of the experiment. During time-lapse acquisition, turn off the epifluorescence light source, cover the stage with the laser safety box which is adequate for protection against background light, then press start. Access the open bath chamber through the sliding doors on the laser safety box to refill it with time lapse embryo medium every 8-12 hours.Following imaging acquisition, open the files in the image processing software, highlight the correct image series. In the software, choose the process menu, click on 3D deconvolution and apply to deconvolve each z-stack. Import individual tif files into video processing software, then select all tif files and drag them into the video editor.Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a mov or mp4 file. Seen here by multi-photon fluorescence imaging are the migration patterns of cranial neural crest cells that give rise to the cranial facial structures and anterior segment of the eye in transgenic sox10:EGFP and foxd3:GFP zebrafish lines.Sox 10 positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the cranial facial region. The cells from the prosencephalon and mesencephalon migrate dorsally and ventrally to the developing eye to populate the periocular mesencine. As indicated here, the sox10 positive cells form the frontal nasal process.Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time lapse imaging of foxd3 positive neural crest cells between 30 and 60 hpf show that these cells migrated between the optic cup and surface ectoderm and though the ocular fissure marked by the arrow and coalesced around the lens marked by the asterisk forming the iris stroma. Once mastered, this technique from start to finish can be done in four days if it is performed properly.When attempting this procedure, it is important to remember to choose embryos with a high amount of fluorescence. Setting up the software initially can be challenging but once the parameters are determined, additional changes are typically minimal. After watching this video, you should have a good understanding of how to use multi-photon time-lapse microscopy to image migrating cells in the embryo.

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Research

JoVE Journal

Developmental Biology

7.4K Views.  University of Michigan. The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissu...