The authors acknowledge Dr. Kenzo Ivanovitch for previous work on this method and the group of Dr. Shigenori Nonaka (National Institutes of Natural Sciences, Japan) for providing the initial expertise on embryo mounting. This study was supported by Grant PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovaci&#243;n and Grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to MT and Grant 1380918 from the FEDER Andaluc&#237;a 2014-2020 Operating Program to JND. MS was supported by a La Caixa Foundation PhD fellowship (LCF/BQ/DE18/11670014) and The Company of Biologists travelling fellowship (DEVTF181145). The CNIC is supported by the Spanish Ministry of Science and the ProCNIC Foundation.

All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Reference PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
This protocol includes the use of two males from the fluorescent transgenic mouse line reporting NOTCH activity Tg(CBF:H2BVenus,+)<sup class="x...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allocation+and+early+differentiation+of+cardiovascular+progenitors+in+the+mouse+embryo.">Allocation and early differentiation of cardiovascular progenitors in the mouse embryo.</a> Trends in Cardiovascular Medicine. 11 (5), 177-184 (2001).</li><li>Lawson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+the+mouse+embryo.">Fate mapping the mouse embryo.</a> International Journal of Developmental Biology. 43 (7), 773-775 (1999).</li><li>Ivanovitch, K., Temi&#241;o, S., Torres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+heart+tube+development+in+mouse+reveals+alternating+phases+of+cardiac+differentiation+and+morphogenesis.">Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis.</a> eLife. 6, 30668 (2017).</li><li>Meilhac, S. M., Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deployment+of+cell+lineages+that+form+the+mammalian+heart.">The deployment of cell lineages that form the mammalian heart.</a> Nature Reviews Cardiology. 15 (11), 705-724 (2018).</li><li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nature Reviews Genetics. 6 (11), 826-835 (2005).</li><li>Meilhac, S. M., Lescroart, F., Blanpain, C. D., Buckingham, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+complexity+of+early+heart+progenitor+cells.">Dissecting the complexity of early heart progenitor cells.</a> Journal of Cardiovascular Development and Disease. 9 (1), 5 (2022).</li><li>McDole, 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=In+toto+imaging+and+reconstruction+of+post-implantation+mouse+development+at+the+single-cell+level.">In toto imaging and reconstruction of post-implantation mouse development at the single-cell level.</a> Cell. 175 (3), 859-876 (2018).</li><li>Saykali, 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=Distinct+mesoderm+migration+phenotypes+in+extra-embryonic+and+embryonic+regions+of+the+early+mouse+embryo.">Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo.</a> eLife. 8, 42434 (2019).</li><li>Ichikawa, T., 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=Live+imaging+of+whole+mouse+embryos+during+gastrulation:+Migration+analyses+of+epiblast+and+mesodermal+cells.">Live imaging of whole mouse embryos during gastrulation: Migration analyses of epiblast and mesodermal cells.</a> PLoS ONE. 8 (7), 64506 (2013).</li><li>Tyser, R. C. V., 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=Single-cell+transcriptomic+characterization+of+a+gastrulating+human+embryo.">Single-cell transcriptomic characterization of a gastrulating human embryo.</a> Nature. 600 (7888), 285-289 (2021).</li><li>Aguilera-Castrejon, 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=Ex+utero+mouse+embryogenesis+from+pre-gastrulation+to+late+organogenesis.">Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.</a> Nature. 593 (7857), 119-124 (2021).</li><li>Yue, Y., 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=in+toto+live+imaging+of+cardiomyocyte+behaviour+during+mouse+ventricle+chamber+formation+at+single-cell+resolution.">in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution.</a> Nature Cell Biology. 22 (3), 332-340 (2020).</li><li>Nowotschin, S., Xenopoulos, P., Schrode, N., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+single-cell+resolution+live+imaging+reporter+of+Notch+signaling+in+the+mouse.">A bright single-cell resolution live imaging reporter of Notch signaling in the mouse.</a> BMC Developmental Biology. 13 (1), 15 (2013).</li><li>Cold Spring Harbor Protocols. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharpened+tungsten+needles.">Sharpened tungsten needles.</a> Cold Spring Harbor Protocols. , (2012).</li><li>Tam, P. P., Snow, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+culture+of+primitive-streak-stage+mouse+embryos.">The in vitro culture of primitive-streak-stage mouse embryos.</a> Journal of Embryology and Experimental Morphology. 59, 131-143 (1980).</li><li>Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+for+culturing+mouse+embryos.">Preparation of rat serum for culturing mouse embryos.</a> Cold Spring Harbor Protocols. 2011 (4), 5593 (2011).</li><li>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=Postimplantation+mouse+development:+Whole+embryo+culture+and+micro-+manipulation.">Postimplantation mouse development: Whole embryo culture and micro- manipulation.</a> International Journal of Developmental Biology. 42 (7), 895-902 (1998).</li><li>Optimizaci&#243;n de propiedades fisicoqu&#237;micas y medios de cultivo para el cultivo del embri&#243;n de rat&#243;n ex vivo. Universidad de Ja&#233;n. Biolog&#237;a Experimental Available from: <a target="_blank" href="https://hdl.handle.net/10953.1/1400">https://hdl.handle.net/10953.1/1400</a> (2021)</li><li>Behringer, R., Gertsenstein, M., Vintersen Nagy, K., Nagy, 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> Manipulating the mouse embryo: A laboratory manual, Fourth Edition. , 814 (2014).</li><li>Shea, K., Geijsen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+6.5+dpc+mouse+embryos.">Dissection of 6.5 dpc mouse embryos.</a> Journal of Visualized Experiments. 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Early heart progenitors organize in a primitive heart tube that starts beating while it is still forming. Understanding how this process takes place is key to pinpoint the wide spectrum of congenital heart defects to specific morphogenetic events. For that, live imaging offers an opportunity to study normal and defective embryonic development with increased temporal resolution. This is especially useful to study early cardiac progenitor cells as they transition quickly through multiple differentiation and migration behav...

The heart forms early during embryogenesis to start pumping nutrients to the whole embryo, while it continues developing1. In mouse embryos, one and a half days after the initiation of gastrulation, a rudimentary heart organ assembles at the anterior pole2,3. By Early Streak (ES) stage, cardiac progenitors in the epiblast ingress through the primitive streak to the nascent mesodermal layer4,5,6 and start migrating to the anterior pole, where they differentiate to form the primitive heart tube. Throughout this process, early heart progenitors undergo cell rearrangements, shape transformations, and differentiation, in addition to migration7 (Figure 1).
Early cardiac progenitors have attracted researchers for nearly a century due to their remarkable ability to differentiate and build a functional organ simultaneously. Over the last two decades, clonal analysis and conditional knockout models have shown that early heart development implicates distinct cell sources in a highly dynamic process8,9,10. However, the 3D structure of the primitive heart tube and the dynamic nature of its morphogenesis make it challenging to study (Figure 1), and we are far from understanding its full complexity11.
To study these dynamic cellular processes, live imaging methods now offer an unprecedented detail7,12,13,14. In the mouse model, live approaches have been key to interrogating developmental topics that are difficult to address by static analysis7,13,15. While long-term ex vivo culture and robust microscope setups are advancing fast16,17, few researchers have the expertise to successfully image live embryos. Although paper-based publications provide enough technical details to reproduce live imaging experiments, some skills and tricks are hard to grasp without visual examples or peer-to-peer assistance. To accelerate this learning process and spread the use of live imaging among laboratories, we assembled a video protocol (Figure 2) that gathers the necessary skills to perform live imaging on gastrulating mouse embryos.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64273/64273fig01.jpg" /> 
Figure 1: Early differentiation of cardiac progenitor cells in the mouse embryo from the onset of gastrulation to the stage preceding primitive heart tube formation. Cardiac progenitor cells ingress the mesoderm soon after the start of gastrulation, migrating to the opposite side of the embryo. Morphological and embryonic day (E) stage are written on top of the diagrams. Dashed arrows depict the migration trajectory of primitive heart tube progenitors during gastrulation. This figure was adapted from11. Abbreviations: ES = Early Streak; MS = Middle Streak; EHF = Early Head Fold. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64273/64273fig02.jpg" /> 
Figure 2: Workflow diagram for live imaging of early heart progenitors. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#55 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish with glass coverslip bottom 14 mm Diameter</td>
			<td>Mattek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm vise table&#160;</td>
			<td>Grandado</td>
			<td>SKU 8798771617573</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td>&#160;with L-Glutamine, without Glucose, without Na Pyruvate</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>10438-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+)&#160;</td>
			<td>JAX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorobrite DMEM</td>
			<td>ThermoFisher</td>
			<td>A1896701&#160;</td>
			<td>DMEM for live-cell imaging</td>
		</tr>
		<tr>
			<td>High-vacuum silicone grease</td>
			<td>Dow Corning</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder for wires</td>
			<td>Perlen Pressen</td>
			<td>pwb1</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 780 Upright microscope</td>
			<td>Zeiss&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai Deepsee far red pulsed-laser tuned at 980 nm</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non Descanned Detectors equipped with the filter sets 
			cyan-yellow (BP450-500/BP520-560), green-red (BP500-520/BP570-610) and yellow-red (BP520-560/BP645-710)</td>
			<td>Zeiss&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Obj: 20x water dipping 1.0 NA, long working distance</td>
			<td>Zeiss&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 and P200 pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Oil</td>
			<td>Nidacon</td>
			<td>VNI0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>(the final concentration should be 50 &#956;g/mL penicillin and 50 &#956;g/mL streptomycin)</td>
		</tr>
		<tr>
			<td>Petri dishes 35 mm x 10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polymethyl methacrylate&#160;</td>
			<td></td>
			<td></td>
			<td>Reused from old laboratory equipment</td>
		</tr>
		<tr>
			<td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td>
			<td>Janvier Labs</td>
			<td>9979</td>
			<td></td>
		</tr>
		<tr>
			<td>Set of 160 mm fines</td>
			<td>RS PRO</td>
			<td>541-6933</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard 1.0 mm glass capillaries</td>
			<td>Anima Lab</td>
			<td>&#160;1B100F-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.22 &#956;m syringe filter</td>
			<td>Corning</td>
			<td>431218</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 5 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>15809152</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic homogeniser (sonicator)</td>
			<td>Bandelin</td>
			<td>BASO_17021</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of early cardiac progenitors in the mouse embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We used the protocol to visualize NOTCH signaling activation in early cardiac progenitors about to differentiate to endothelial cells during primitive heart tube morphogenesis. For that, we crossed wild-type C57BL/6-N mice with Tg(CBF:H2BVenus,+) mice18 to obtain embryos reporting NOTCH activity through yellow fluorescent protein Venus. At E7.5, Venus fluorescence is present throughout the neural ectoderm, with a few positive nuclei at the splanchnic and extraembryonic mesoderm. After 4 h, endothe...

Watch this Scientific Journal Video about Live Imaging of Early Cardiac Progenitors in the Mouse Embryo at JoVE.com

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

This protocol allows imaging mouse embryos as they develop. This enables studying early cardiogenesis in detail. Like taking still images of fake embryos, live imaging gives full access to study dynamic process in embryo development.The skills required for life imaging are hard to grasp from text only publication. Watching another person doing it is essential to learn. To begin, cut one centimeter long tungsten wire pieces and sharpen them to 0.02 to 0.05 millimeters in diameter by submerging the tip of the wire in a saturated sodium nitrate solution.To prepare elbowed glass capillaries, place the midpoint of a standard one millimeter glass capillary over a bunsen lighter for three to six seconds, pulling slowly from both ends until the capillary turns thin and flexible. Then, break the capillary in half and heat it brief briefly to produce 90 degree bend two centimeters from the tip. Saw a piece of polymethyl methacrylate measuring approximately seven millimeters long, four millimeters wide, and two millimeters thick.Hold the methacrylate piece using a bench vice, then drill customized holes transversely through the whole piece. Rotate the drill slowly while applying low but constant pressure. Use a fine file to smooth the holder's edge and adjust its size.Additionally, create a mild slope on the edges of the holder to facilitate embryo positioning. Place the finished holder in a dish with distilled water to rinse it. Under the stereo microscope, check the holder to see if the holes contain drilling dust.If dust is present, use tungsten needles to remove it. To sterilize the holder, place it in a conical tube full of distilled water and sonicate for 20 minutes with a minimum of 20 watt power output. Extract the uterus from a euthanized embryonic day 6.75 to 7.5 pregnant mouse and place it on a dry and clean paper wipe.Then, cut the uterus, inserting the tip of fine scissors from the mesometrial side, and sliding the blade along to expose the decidui, and transfer them to the dissection medium. Dissect the embryos, keeping the ectoplacental cone intact. Then, peel off the Reichter's membrane, leaving part of it on the extra embryonic region.To keep the dissection medium warm, replace it every 10 minutes. Moreover, dissect decidui in groups of three to four while keeping the rest in a dissection medium placed in 37 degrees Celsius bath. Immediately place the dissected embryos in a culture medium.Select the embryos with the desired fluorescence features using a fluorescence stereo microscope and place them in the tube containing the culture medium in the cell culture incubator to recover for two hours before imaging. After turning on all microscope components, including computer, software and lasers, turn on the carbon dioxide controller and set it to 7%Place a drop of high vacuum silicon grease on a 35 millimeter dish with a glass cover slip bottom. Place the holder on top of the drop and press gently to immobilize it.For embryo mounting, transfer two to three embryos from the incubator to the dish with a holder. Using a pair of forceps and a tapered tungsten needle, set the embryos in the holes. Use the needle to hook the embryo by the ectoplacental cone and insert it into a size matching hole.Carefully move the dish containing the embryos and the holder to the microscope plate. Lower the objective until a liquid meniscus is formed at the medium objective interface. After placing the embryo in focus, mount the incubation chamber and seal it around the objective using tape.Using a live capture at the microscope control software, adjust the laser output and gain levels. Then, cover the medium with paraffin oil by dripping it slowly on the objective. Set up a time lapse recording.Set a five to 10 minutes interval with three to five micron Z space between stacks. Leave a blank space on top of the Z-Stack to anticipate embryo growth. A minimum of 50 microns, adding 30 microns for every hour the acquisition will not be supervised.Enable the auto save option to allow supervision of the acquisition from a computer to allow the adjustment of the Z-Stack size if needed, to fit the area of interest within focus. Live whole body embryo heart development by multiphoton imaging is shown. At embryonic day 7.5, venous fluorescence is present throughout the neural ectoderm, with a few positive nuclei at the splanchnic and extra embryonic mesoderm.After four hours, endothelial progenitors activate venous and assemble to form endocardial tube and underlying aortas, which become enclosed structures after seven hours. Be careful while removing the Reichter's membrane. It can be really stuck to the embryo, especially on prevacillation stages.During removal, try to pinch the membrane by the extra embryonic region.

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Research

JoVE Journal

Developmental Biology

1.2K Aufrufe.  Centro Nacional de Investigaciones Cardiovasculares (CNIC). Dieses Protokoll ermöglicht die Bildgebung von Mausembryonen während ihrer Entwicklung. Dies ermöglicht es, die frühe Kardiogenese im Detail zu untersuchen. Wie bei der Aufnahme von Standbildern von gefälschten Embryonen bietet die Live-Bildgebung vollen Zugriff auf die Untersuchung des dynamischen Prozesses in der Embryonalentwicklung.Die Fähigkeiten, die für die Bildgebung des Lebens erforderlich sind, sind durch reine Textveröffentlichungen schwer zu erfassen. Einer anderen ...