The authors have nothing to disclose.

<ol>
	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Dev. Biol. 100 (1), 64-119 (1983).</li><li>Kimmel, C. B., Warga, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+cell+lineages+originate+in+the+gastrula+of+the+zebrafish.">Tissue-specific cell lineages originate in the gastrula of the zebrafish.</a> Science. 231 (4736), 365-368 (1986).</li><li>Nishida, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+in+ascidian+embryos+by+intracellular+injection+of+a+tracer+enzyme.+III.+Up+to+the+tissue+restricted+stage.">Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage.</a> Dev. Biol. 121 (2), 526-541 (1987).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22 (3), 451-461 (1999).</li><li>Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A., Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-62 (2007).</li><li>Fernandez, R., Das, P., Mirabet, V., Moscardi, E., Traas, J., Verdeil, J. L., Malandain, G., Godin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+plant+growth+in+4D:+robust+tissue+reconstruction+and+lineaging+at+cell+resolution.">Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution.</a> Nat. Methods. 7 (7), 547-553 (2010).</li><li>Olivier, N., 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=Cell+lineage+reconstruction+of+early+zebrafish+embryos+using+label-free+nonlinear+microscopy.">Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy.</a> Science. 329 (5994), 967-971 (2010).</li><li>Kitajima, T. S., Ohsugi, M., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+kinetochore+tracking+reveals+error-prone+homologous+chromosome+biorientation+in+mammalian+oocytes.">Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.</a> Cell. 146 (4), 568-581 (2011).</li><li>Schr&#246;del, T., Prevedel, R., Aumayr, K., Zimmer, M., Vaziri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-wide+3D+imaging+of+neuronal+activity+in+Caenorhabditis+elegans+with+sculpted+light.">Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light.</a> Nat. Methods. 10 (10), 1013-1020 (2013).</li><li>Du, Z., Santella, A., He, F., Tiongson, M., Bao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+inference+of+systems-level+mechanistic+models+of+development+from+live-imaging-based+phenotype+analysis.">De novo inference of systems-level mechanistic models of development from live-imaging-based phenotype analysis.</a> Cell. 156 (1-2), 359-372 (2014).</li><li>Amat, F., 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=Fast,+accurate+reconstruction+of+cell+lineages+from+large-scale+fluorescence+microscopy+data.">Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data.</a> Nat. Methods. 11 (9), 951-958 (2014).</li><li>He, B., Doubrovinski, K., Polyakov, O., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+constriction+drives+tissue-scale+hydrodynamic+flow+to+mediate+cell+elongation.">Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.</a> Nature. 508 (7496), 392-396 (2014).</li><li>Singh, D., Pohl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+rotational+cortical+flow,+asymmetric+midbody+positioning,+and+spindle+rotation+mediates+dorsoventral+axis+formation+in+C.+elegans.">Coupling of rotational cortical flow, asymmetric midbody positioning, and spindle rotation mediates dorsoventral axis formation in C. elegans.</a> Dev. Cell. 28 (3), 253-267 (2014).</li><li>Singh, D., Pohl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+function+for+the+midbody+remnant+in+embryonic+patterning.">A function for the midbody remnant in embryonic patterning.</a> Commun. Integr. Biol. 7, e28533 (2014).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, 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=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J. Struct. Biol. 116 (1), 71-76 (1996).</li><li>Henriksson, J., Hench, J., Tong, Y. G., Johansson, A., Johansson, D., B&#252;rglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endrov:+an+integrated+platform+for+image+analysis.">Endrov: an integrated platform for image analysis.</a> Nat. Methods. 10 (6), 454-456 (2013).</li><li>Thielicke, W., Stamhuis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIVlab+-+Towards+User-friendly,+Affordable+and+Accurate+Digital+Particle+Image+Velocimetry+in+MATLAB.">PIVlab - Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB.</a> Journal of Open Research Software. 2 (1), e30 (2014).</li><li>Kim, E., Sun, L., Gabel, C. V., Fang-Yen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+imaging+of+Caenorhabditis+elegans+using+nanoparticle-mediated+immobilization.">Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization.</a> PLoS One. 8 (1), e53419 (2013).</li><li>Dickinson, D. J., Ward, J. D., Reiner, D. J., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+Caenorhabditis+elegans+genome+using+Cas9-triggered+homologous+recombination.">Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.</a> Nat. Methods. 10 (10), 1028-1034 (2013).</li><li>Schonegg, S., Constantinescu, A. T., Hoege, C., Hyman, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rho+GTPase-activating+proteins+RGA-3+and+RGA-4+are+required+to+set+the+initial+size+of+PAR+domains+in+Caenorhabditis+elegans+one-cell+embryos.">The Rho GTPase-activating proteins RGA-3 and RGA-4 are required to set the initial size of PAR domains in Caenorhabditis elegans one-cell embryos.</a> Proc. Natl. Acad. Sci. U. S. A. 104 (38), 14976-14981 (2007).</li><li>Schmutz, C., Stevens, J., Spang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+the+novel+RhoGAP+proteins+RGA-3+and+RGA-4+in+the+germ+line+and+in+the+early+embryo+of+C.+elegans.">Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans.</a> Development. 134 (19), 3495-3505 (2007).</li><li>Naganathan, S. R., F&#252;rthauer, S., Nishikawa, M., J&#252;licher, F., Grill, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+torque+generation+by+the+actomyosin+cell+cortex+drives+left-right+symmetry+breaking.">Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking.</a> Elife. 3, e04165 (2014).</li><li>Kaletta, T., Schnabel, H., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binary+specification+of+the+embryonic+lineage+in+Caenorhabditis+elegans.">Binary specification of the embryonic lineage in Caenorhabditis elegans.</a> Nature. 390 (6657), 294-298 (1997).</li><li>Schnabel, R., 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=Global+cell+sorting+in+the+C.+elegans+embryo+defines+a+new+mechanism+for+pattern+formation.">Global cell sorting in the C. elegans embryo defines a new mechanism for pattern formation.</a> Dev. Biol. 294 (2), 418-431 (2006).</li><li>Murray, J. I., Bao, Z., Boyle, T. J., Waterston, R. 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+lineaging+of+fluorescently-labeled+Caenorhabditis+elegans+embryos+with+StarryNite+and+AceTree.">The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree.</a> Nat. Protoc. 1 (3), 1468-1476 (2006).</li><li>Giurumescu, C. 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=Quantitative+semi-automated+analysis+of+morphogenesis+with+single-cell+resolution+in+complex+embryos.">Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.</a> Development. 139 (22), 4271-4279 (2012).</li><li>Dejima, K., Kang, S., Mitani, S., Cosman, P. C., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan+defines+precise+spindle+orientation+by+modulating+Wnt+signaling+in+C.+elegans.">Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans.</a> Development. 141 (22), 4354-4365 (2014).</li><li>Schnabel, R., Hutter, H., Moerman, D., Schnabel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+normal+embryogenesis+in+Caenorhabditis+elegans+using+a+4D+microscope:+variability+of+development+and+regional+specification.">Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification.</a> Dev Biol. 184 (2), 234-265 (1997).</li></ol>

Research in the lab of CP is funded by the Deutsche Forschungsgemeinschaft (EXC 115; FOR 1756) and the LOEWE Research Cluster Ubiquitin Networks. CP is further supported by the European Union Framework Program 7 (Marie Curie Actions Project 326632).

من خلال تتبع الكائن في التنمية ولا سيما تتبع النووي، كان، فإنه من الممكن توضيح الآليات الزخرفة المركزية للC. ايليجانس مرحلة التطور الجنيني 1،23،24. توسيع هذه الاستراتيجية إلى ارتفاع الإنتاجية، فقد كان من الممكن مؤخرا للكشف عن قواعد الزخرفة إضافية واقتراح ال...

مع التحسن المطرد من البروتينات الفلورية، والهندسة الجينية، المجهر الضوئي، والبرنامج التشغيلى الكمبيوتر والأجهزة، أصبح من الممكن الآن لتسجيل تطوير العديد من الكائنات النموذجية في القرار المكانية والزمانية لم يسبق له مثيل. هذا يسمح للباحثين لطرح الأسئلة التي لا يمكن معالجتها سابقا أو لإعادة النظر في العمليات التنموية المعروفة من أجل البحث عن الجوانب تجاهلها. وأثار هذا التقدم في مجال البيولوجيا التطورية الكمي، الذي يهدف إلى تحويل، ونماذج رسمية النوعية في النماذج الكمية التي كتبها قياسات دقيقة والتحليلات الإحصائية. جعلت خلايا تتبع والهياكل التحت خلوية من الممكن استخلاص النماذج الكمية من التطور الجنيني، نشاط الجهاز العصبي، أو انقسام الخلايا 12/01. من خلال تتبع بقايا انقسام الخلايا خلال التنمية في وقت مبكر من C. ايليجانس الجنين، يمكن أن تكشف مؤخرا أنها تتبع جهاز ستيريوعوامل كتبته مسار ويشكل الاستقطاب المهم 13،14. هنا، يتم عرض البروتوكولات التي تجعل النهج البيولوجيا التطورية الكمي للوصول لغير الخبراء. التركيز يكمن في ثلاث على التوالي إلى الأمام، والأدوات المتاحة بحرية التي يمكن أن تنفذها في أي مختبر لديه حق الوصول إلى معيار المجهري متحد البؤر وأجهزة الكمبيوتر. وتشمل هذه بروتوكول لتوليد الأشكال خلية 3D، وبروتوكول لتعقب فلول انقسام الخلايا، وبروتوكول لوصف كميا ديناميات أكتوميوزين القشرية. الديدان الخيطية C. يستخدم ايليجانس كحالة نموذجية، ومع ذلك، فإن أساليب وأدوات مناقشتها هنا هي مناسبة لمجموعة متنوعة من الأسئلة في النماذج البيولوجية الأخرى، على سبيل المثال، الخلايا المستزرعة، إإكسبلنتس الأنسجة، organoids أو الكروية والأجنة الأخرى، الخ عموما، بعض التحليلات هو موضح هنا يمكن أيضا أن يؤديها مع شعبية مفتوحة المصدر أداة يماغيج (http://imagej.nih.gov/ij/docs/index.أتش تي أم أل. أو في فيجي، &quot;وشملت بطاريات&quot; إصدار يماغيج، http://fiji.sc/Fiji) التي هي العديد من الإضافات للالتحليلات الكمية المختلفة المتاحة. ومع ذلك، فإن برامج مناقشتها هنا مصممة لمعالجة مشاكل محددة. أولا، IMOD، ومعالجة الصور، والنمذجة وعرض جناح يمكن استخدامها لإعادة بناء 3D من مسلسل أقسام من الإلكترون المجهري أو ضوء 15. كما يحتوي IMOD أدوات للعرض البيانات 3D من أي اتجاه. ثانيا، Endrov، وهو برنامج جافا المصممة لإجراء تحليل الصورة، ومعالجة البيانات، والشرح الشبكات أو المسارات (وغيرهم) على أساس العمارة المساعد طويلة، مع يماغيج المساعد التوافق 16. أنه يحتوي على أكثر من 140 عمليات لمعالجة الصور واجهة المستخدم الموسعة التي يتم فيها عرض النماذج والبيانات الخام على حدة. ويمكن الاطلاع على شفرة المصدر في https://github.com/mahogny/Endrov. ثالثا، PIVlab، وMأداة ATLAB عن الجسيمات الصور الرقمية velocimetry التي تسمح للمستخدم لكميا ونوعيا تحليل حقول تدفق الجسيمات 17. استخدام هذه البرامج يتطلب ترخيص MATLAB الذي يتضمن أدوات معالجة الصور (http://mathworks.com). PIVlab هو برنامج مصمم لوصف تدفق كميا. وتحسب توزيع سرعة وحجم، الدردورية، الاختلاف، أو القص ضمن أزواج صورة أو سلسلة. لهذا، فإنه عبر يرتبط مناطق صغيرة من الصور (وتسمى &#39;يمر&#39; في قسم البروتوكول) من الزوج الصورة لاشتقاق النزوح الجسيمات الأكثر احتمالا. هذا الارتباط عبر ينتج مصفوفة الارتباط التي يمكن تحليلها في مجال الفضاء أو التردد باستخدام إما مباشرة عبر ارتباط أو تحول فورييه السريع (الاتحاد الفرنسي للتنس)، على التوالي. المعدات المستخدمة هنا هي مجهر مقلوب مجهزة نيبكو (&#39;الغزل&#39;) القرص، وهو EMCCD، 488 و 561 نانومتر solid- القياسيةليزر الدولة، و20x والهواء أو 40X 60X أو خطة / امزيغ أهداف بالنفط أو الغمر بالمياه. ومع ذلك، فمن الممكن أيضا أن أداء الوقت الفاصل بين التصوير مع طرائق التصوير الأخرى، على سبيل المثال، راهنا، line- أو القائم على الليزر المجهر رقة المسح الضوئي، متعدد الفوتون المجهري، وكذلك برنامج التحصين الموسع ومضان المجهر جنبا إلى جنب مع deconvolution أو منظم إضاءة. وميزة استخدام نظام القرص نيبكو هي الحصول على الصور سريع للغاية، وخاصة إذا وضع تدفق (حركة مستمرة والمسح الضوئي للجسم في البعد ض) هو متاح. بالإضافة إلى ذلك، لتحسين القرار، مكبرة الموسع 1.5 أضعاف أمام EMCCD يمكن استخدامها.

<table><tbody><tr><td>Stereo microscope</td><td>Motic/VWR</td><td>OT4005S</td><td>Stereo microscope for dissection and mounting</td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>18329</td><td>Embryo mounting</td></tr><tr><td>20 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>876</td><td>Adult animal mounting</td></tr><tr><td>0.1 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>631-0902</td><td>Adult animal mounting</td></tr><tr><td>Cover glass 18x18 mm</td><td>VWR</td><td>631-1331</td><td>Embryo/adult mounting</td></tr><tr><td>Cover glass 24x60 mm</td><td>VWR</td><td>631-1339</td><td>Embryo mounting</td></tr><tr><td>Scalpel</td><td>VWR</td><td>233-5455</td><td>Embryo dissection</td></tr><tr><td>Silicone tubing</td><td>VWR</td><td>228-1501</td><td>Tubing for mouth pipette</td></tr><tr><td>30 mm PTFE membrane filter</td><td>NeoLab</td><td>Jul-01</td><td>Filter for mouth pipette</td></tr><tr><td>Capillary tubes</td><td>VWR</td><td>621-0003</td><td>Pipette tip for mouth pipette</td></tr><tr><td>Vaseline</td><td>Roth</td><td>E746.1</td><td>Embryo/adult mounting</td></tr><tr><td>Agar</td><td>Roth</td><td>5210.5</td><td>Adult animal mounting</td></tr><tr><td>Potassium-di-hydrogenphosphate</td><td>Roth</td><td>P018.2</td><td>M9 buffer</td></tr><tr><td>Di-sodium- hydrogenphosphate</td><td>Roth</td><td>P030.2</td><td>M9 buffer</td></tr><tr><td>Sodium chloride</td><td>Roth</td><td>3957.1</td><td>M9 buffer</td></tr><tr><td>VisiScope Spinning Disc Confocal System</td><td>Visitron Systems</td><td>n/a</td><td>Confocal microscopy</td></tr></tbody></table>

tracking and quantifying developmental processes in c. elegans using open-source tools

التقاط كميا العمليات التنموية أمر بالغ الأهمية لاستخلاص النماذج الميكانيكية والمفتاح لتحديد ووصف الظواهر متحولة. هنا يتم عرض بروتوكولات لإعداد الأجنة وC. الكبار ايليجانس الحيوانات لقصيرة وطويلة الأجل الوقت الفاصل بين المجهر وطرق تتبع وتقدير من العمليات التنموية. الأساليب المقدمة تستند فقط على C. سلالات ايليجانس المتاحة من مركز علم الوراثة انواع معينة وعلى البرمجيات مفتوحة المصدر التي يمكن تنفيذها بسهولة في أي مختبر مستقل عن نظام المجهر المستخدمة. A إعادة بناء نموذج 3D خلية الشكل باستخدام IMOD نماذج البرمجيات، وتتبع اليدوي الهياكل التحت خلوية fluorescently المسمى باستخدام متعددة الأغراض برنامج تحليل الصور Endrov، وتحليل تدفق مقلص القشرية باستخدام PIVlab (حل الزمن الرقمي الجسيمات صورة Velocimetry وأظهرت أداة لMATLAB). تم مناقشته كيف يمكن أيضا أن يتم نشر هذه الأساليب رس التقاط كميا العمليات التنموية الأخرى في نماذج مختلفة، على سبيل المثال، تتبع الخلايا والنسب تتبع، تتبع تدفق الحويصلة.

باستخدام بروتوكولات 2، 3، و 4، والتصوير الوقت الفاصل بين الغدد التناسلية في البرية من نوع C. ايليجانس يتم تنفيذ البالغين (سلالة OD58 (UNC-119 (ED3) الثالث؛ ltIs38 [pAA1؛ فطيرة-1 :: :: GFP PH (PLC1delta1) + UNC-119 (+)])، معربا عن علامة الغشاء من المروج سلالة الجرثومية ). التركيز على منعطف من الغدد ?...

Watch this Scientific Journal Video about Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools at JoVE.com

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

تتبع وقياس العمليات التنموية في<em&gt; C. ايليجانس</em&gt; استخدام أدوات المصدر المفتوح

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

tracking-quantifying-developmental-processes-c-elegans-using-open

Research

JoVE Journal

Biology

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

8.8K Views.  Goethe University. Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Video: Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

انجذاب كهربي المستندة ميكروفلويديك للتحليل الكمي بناء على الطلب<em&gt; انواع معينة ايليجانس</em&gt; &#39;تنقل

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Bioengineering

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. ايليجانس تتبع وقياس السلوكية

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

Neuroscience

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

المجهر الضوئي ورقه الخفيفة والمؤتمتة الخلايا النسب تحليلات لكتالوج Caenorhabditis الاجنه الجنينية مع القرار تحت الخلوية

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Developmental Biology

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

تحليل مقارن للطرق التجريبية لقياس النشاط الحيواني في نماذج Elegans Caenorhabditis من مرض الميتوكوندريا

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Behavior

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

طريقه التصوير شبه عاليه الانتاجيه ومجموعه أدوات التصور البيانات لتحليل التنمية الجنينية c. اليجس

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers الاغاروز لفترة طويلة التصوير الكالسيوم من<em&gt; انواع معينة ايليجانس</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

التصوير C. ايليجانس الأجنة باستخدام مجهر Epifluorescent وبرمجيات المصدر المفتوح

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

تحليل الآلي لل<em&gt; C. ايليجانس</em&gt; السباحة السلوك باستخدام CeleST البرمجيات

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

الآلي التحليل السلوكي لكبير C. ايليجانس السكان استخدام واسعة مجال-من-عرض "تتبع النظام الأساسي"

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

الأساسية<em&gt; Caenorhabditis ايليجانس</em&gt; طرق: التزامن والمراقبة

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

تتبع وقياس العمليات التنموية في

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انجذاب كهربي المستندة ميكروفلويديك للتحليل الكمي بناء على الطلب

C. ايليجانس تتبع وقياس السلوكية

المجهر الضوئي ورقه الخفيفة والمؤتمتة الخلايا النسب تحليلات لكتالوج Caenorhabditis الاجنه الجنينية مع القرار تحت الخلوية

تحليل مقارن للطرق التجريبية لقياس النشاط الحيواني في نماذج Elegans Caenorhabditis من مرض الميتوكوندريا

طريقه التصوير شبه عاليه الانتاجيه ومجموعه أدوات التصور البيانات لتحليل التنمية الجنينية c. اليجس

Microchambers الاغاروز لفترة طويلة التصوير الكالسيوم من

التصوير C. ايليجانس الأجنة باستخدام مجهر Epifluorescent وبرمجيات المصدر المفتوح

تحليل الآلي لل

الآلي التحليل السلوكي لكبير C. ايليجانس السكان استخدام واسعة مجال-من-عرض "تتبع النظام الأساسي"

الأساسية