We thank the MDI Biological Laboratory animal core service facility for zebrafish maintenance. Research reported in this publication was supported by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM104318 (for COBRE) and P20GM103423 (INBRE) and Department of Defense &#8211; USAMRAA (W81XWH-BAA-1) grant.

<ol>
	<li>San Miguel-Ruiz, J. E., Garc&#237;a-Arrar&#225;s, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+cellular+events+occur+during+wound+healing+and+organ+regeneration+in+the+sea+cucumber+Holothuria+glaberrima.">Common cellular events occur during wound healing and organ regeneration in the sea cucumber Holothuria glaberrima.</a> BMC Dev Biol. 7, 115 (2007).</li><li>Poss, K. D., Keating, M. T., Nechiporuk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tales+of+regeneration+in+zebrafish.">Tales of regeneration in zebrafish.</a> Dev Dyn. 226, 202-210 (2003).</li><li>Akimenko, M. A., Mar&#237;-Beffa, M., Becerra, J., Old G&#233;raudie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+questions,+new+tools,+and+some+answers+to+the+mystery+of+fin+regeneration.">Old questions, new tools, and some answers to the mystery of fin regeneration.</a> Dev Dyn. 226, 190-201 (2003).</li><li>Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+research+today.">Regeneration research today.</a> Dev Dyn. 226, 162-166 (2003).</li><li>Seifert, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+shedding+and+tissue+regeneration+in+African+spiny+mice+(Acomys).">Skin shedding and tissue regeneration in African spiny mice (Acomys).</a> Nature. 489, 561-565 (2012).</li><li>Goss, R. J., Grimes, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+downgrowths+in+regenerating+rabbit+ear+holes.">Epidermal downgrowths in regenerating rabbit ear holes.</a> J Morphol. 146, 533-542 (1975).</li><li>Williams-Boyce, P. K., Daniel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ear+tissue+regeneration+in+mammals.">Comparison of ear tissue regeneration in mammals.</a> J Anat. 149, 55-63 (1986).</li><li>Allan, C. H., 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=Tissue+response+and+Msx1+expression+after+human+fetal+digit+tip+amputation+in+vitro.">Tissue response and Msx1 expression after human fetal digit tip amputation in vitro.</a> Wound Repair Regen. 14, 398-404 (2006).</li><li>Borgens, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+regrow+the+tips+of+their+foretoes.">Mice regrow the tips of their foretoes.</a> Science. 217, 747-750 (1982).</li><li>Han, M., Yang, X., Lee, J., Allan, C. H., Muneoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+regeneration+of+the+neonatal+digit+tip+in+mice.">Development and regeneration of the neonatal digit tip in mice.</a> Dev Biol. 315, 125-135 (2008).</li><li>Muneoka, K., Allan, C. H., Yang, X., Lee, J., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+regeneration+and+regenerative+medicine.">Mammalian regeneration and regenerative medicine.</a> Birth Defects Res C Embryo Today. 84, 265-280 (2008).</li><li>Takeo, M., 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=Wnt+activation+in+nail+epithelium+couples+nail+growth+to+digit+regeneration.">Wnt activation in nail epithelium couples nail growth to digit regeneration.</a> Nature. 499, 228-232 (2013).</li><li>Akimenko, M. A., Johnson, S. L., Westerfield, M., Ekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+induction+of+four+msx+homeobox+genes+during+fin+development+and+regeneration+in+zebrafish.">Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish.</a> Development. 121, 347-357 (1995).</li><li>Reginelli, A. D., Wang, Y. Q., Sassoon, D., Muneoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digit+tip+regeneration+correlates+with+regions+of+Msx1+(Hox+7)+expression+in+fetal+and+newborn+mice.">Digit tip regeneration correlates with regions of Msx1 (Hox 7) expression in fetal and newborn mice.</a> Development. 121, 1065-1076 (1995).</li><li>Brien, G. 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=Coordinate+development+of+skin+cells+and+cutaneous+sensory+axons+in+zebrafish.">Coordinate development of skin cells and cutaneous sensory axons in zebrafish.</a> Journal of Comparative Neurology. 520, 816-831 (2012).</li><li>Kawakami, A., Fukazawa, T., Takeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+fin+primordia+of+zebrafish+larvae+regenerate+by+a+similar+growth+control+mechanism+with+adult+regeneration.">Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.</a> Dev Dyn. 231, 693-699 (2004).</li><li>Yoshinari, N., Ishida, T., Kudo, A., Kawakami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+and+functional+analysis+of+zebrafish+larval+fin+fold+regeneration.">Gene expression and functional analysis of zebrafish larval fin fold regeneration.</a> Dev Biol. 325, 71-81 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Detrich, H. W., Westerfield, M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish:+Cellular+and+Developmental+Biology,+part+A.+Preface.">The Zebrafish: Cellular and Developmental Biology, part A. Preface.</a> Methods Cell Biol. 100 (13), (2010).</li><li>Dahm, N. -. V. 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: A Practical Approach, Issue 975. , 303 (2002).</li><li>Detrich, H., Westerfield, M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: 2nd Edition Genetics, Genomics and Informatics. , (2005).</li><li>Concha, M. L., Adams, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+cell+divisions+and+cellular+morphogenesis+in+the+zebrafish+gastrula+and+neurula:+a+time-lapse+analysis.">Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis.</a> Development. 125, 983-994 (1998).</li><li>Distel, M., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+time-lapse+imaging+of+zebrafish+embryonic+development.">In vivo time-lapse imaging of zebrafish embryonic development.</a> CSH Protoc. 2007, (2007).</li><li>Kulesa, P. M., Kasemeier-Kulesa, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+Heated+Incubation+Chamber+around+a+Microscope+Stage+for+Time-Lapse+Imaging.">Construction of a Heated Incubation Chamber around a Microscope Stage for Time-Lapse Imaging.</a> CSH Protoc. 2007, (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bitplane.">Bitplane.</a> Imaris V 6.1.0 Reference Manual. , (2008).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Image Processing with ImageJ. , 36-42 (2004).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9, 676-682 (2012).</li><li>Mateus, 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=In+vivo+cell+and+tissue+dynamics+underlying+zebrafish+fin+fold+regeneration.">In vivo cell and tissue dynamics underlying zebrafish fin fold regeneration.</a> PLoS One. 7, e51766 (2012).</li><li>Jones, E. 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=et+al.Dynamic+in+vivo+imaging+of+postimplantation+mammalian+embryos+using+whole+embryo+culture.">et al.Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture.</a> Genesis. 34, 228-235 (2002).</li><li>Rieger, S., Senghaas, N., Walch, A., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-2+controls+directional+chain+migration+of+cerebellar+granule+neurons.">Cadherin-2 controls directional chain migration of cerebellar granule neurons.</a> PLoS Biol. 7, e1000240 (2009).</li><li>Langenberg, T., Brand, M., Cooper, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+development+and+organogenesis+in+zebrafish+using+immobilized+embryonic+explants.">Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.</a> Dev Dyn. 228, 464-474 (2003).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139, 3242-3247 (2012).</li><li>Tadrous, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+PSF+generation+for+3D+brightfield+deconvolution.">A method of PSF generation for 3D brightfield deconvolution.</a> J Microsc. 237, 192-199 (2010).</li><li>Distel, M., Babaryka, A., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+in+vivo+time-lapse+imaging+at+cellular+resolution+by+stereomicroscopy.">Multicolor in vivo time-lapse imaging at cellular resolution by stereomicroscopy.</a> Dev Dyn. 235, 1100-1106 (2006).</li></ol>

الكتاب ليس لديهم ما يكشف.

يسمح الطريقة المعروضة لرصد التئام الجروح وترميم الأنسجة في العيش اليرقات الزرد في الجسم الحي مع مرور الزمن التصوير على stereomicroscope brightfield، وذلك باستخدام بسيط نسبيا مجموعة المتابعة. يتطلب هذا الإجراء بعض الجوانب الهامة التي اختبرناها، والتي سوف تحسين نتائج: 1) تركيزات منخفضة الاغاروز (~ 0.5٪) وتقليل العوائق نمو اليرقات الزرد تزايد مستمر، 2) إزالة الاغاروز حول زعنفة مهمة لا لإخفاء عملية الشفاء، 3) محاصرة الاغاروز في شبكة بلاستيكية تحتفظ الاغاروز والحيوانية في وضع مستقر في جميع أنحاء الداخلي، و4) بيئة تسيطر عليها درجة الحرارة المناسبة، وهو أمر ضروري لبقاء اليرقات. تكيفنا حضانة غرفة ساخنة 23،24، الذي يستخدم التفاف فقاعة التي سجلت على الورق المقوى، وسخان قبة سلكي للسيطرة على درجة الحرارة ودوران الهواء السليم مع تقلبات طفيفة خلالإجراء التصوير. هذه الغرفة بسيطة وفعالة من حيث التكلفة يمكن أن تكون على استعداد لتناسب أي المجهر. وقد استخدمت وغرفة الحضانة ساخنة مماثلة أيضا بالنسبة للفئران التصوير والتنمية فرخ 24،29. ونقترح أن اليرقات بترت قبل هي التي شنت لصورة ما قبل البتر، demounted للبتر، وremounted للتصوير مرور الزمن. على الرغم من أنه من الممكن لتنفيذ هذه الخطوات في خطوة واحدة في غرفة التصوير النهائية، في تجربتنا وجدنا أن بتر زعنفة الذيل على ساترة الزجاج ليس الأمثل، لأنه يمزق الأنسجة و لا يؤدي إلى قطع نظيفة. طريقة بتر القائم على الاغاروز باستخدام إبرة حقنة ووصف في الأصل من قبل كواكامي وزملاؤه (2004) 16 وأيضا، في تجربتنا، مثالية لأداء بتر الأطراف. وهكذا، فإن سلسلة معقدة بدلا من الخطوات التي قدمناها له ما يبرره جيدا ويضمن نتيجة تجديد المثلى. أظهرنا أن larvآل الزرد في 2 DPF ولا يمكن تصوير ما يصل إلى 1.5 أيام في الاغاروز وحل تريكين. كنا تريكين (PH7) الحل الأمثل درجة الحموضة أعد مع الملح المحيط الفوري، والتي لا تتعارض مع صحة العينة للفترة التصوير المقدمة. ونحن مع ذلك سابقا أظهرت أيضا أن استخدام تريكين في Danieau المتوسطة يسمح الوقت الفاصل بين التصوير من 2.5 DPF الزرد اليرقات على المجهر متحد البؤر لا يقل عن 2 أيام 30. وهكذا، يمكن أن الظروف المثلى عازلة تمتد الصحة اليرقات وطول التصوير. بدلا من ذلك، يمكن استخدام تركيزات أقل تريكين للتخدير، أو 2-فينوكسييثانول، التي وجدنا جيد التحمل خلال مراحل اليرقات والكبار عند 28 درجة مئوية لمدة لا تقل عن 60 ساعة. لتجنب عيوب في تجديد الزعانف، ونحن إزالة الاغاروز من زعنفة الذيل قبل التصوير. بياناتنا تظهر أنه في غضون 1.5 أيام تمت إعادة إنشاء الزعنفة إلى حوالي 60٪. هذا المعدل تجديد يتسق مع دراسة سابقة تحديد 3 أيام فيليالي متوسط ​​الوقت للزعنفة الذيل تجديد في يرقات الزرد تصل إلى 6 DPF 16. بيد أنه يمكن استخدام أساليب بديلة لالاغاروز لتركيب الأسماك للتصوير. على سبيل المثال، جلطات البلازما رقيقة 31 أو المفلورة الإثيلين والبروبيلين (محطة إثراء الوقود) أنابيب المغلفة مع ميثيل ومليئة تركيزات الاغاروز منخفضة جدا (0.1٪) وقد أوصى للضوء ورقة المجهري 32 و قد تكون مناسبة لأسلوبنا المقدمة. ومع ذلك، فإننا لا ننصح ميثيل و 0.1٪ الاغاروز، لأنها تتطلب أن العينة هي التي شنت على الجزء السفلي من الغرفة نظرا لعدم وجود تصلب هذه الوسائط. سوف تركيزات عالية جدا من ميثيل تولد علاوة على ذلك جيوب الهواء بناء على خبرتنا، وهذه قد تتداخل مع إجراء التصوير. إذا فضلت وسائل الإعلام هذه مع استخدام الغرفة السفلى، من المهم أن المسافة العمل المناسبة بين عدسة موضوعية والعينة موجودة. وتجدر الإشارة إلى أن مويوصى إيثيل سيللولوز كوسيلة تصاعد فقط لمدة تصل إلى 1 في اليوم، لأنها قد تتداخل مع صحة اليرقات 32. تركيب العينة في الغطاء قد يؤدي إلى بطء الانجراف نحو الانخفاض الجاذبية. لذا فمن المستحسن أن صورة مقاطع متعددة في كل نقطة زمنية، والتي يمكن أن تكون إما المتوقعة في طائرة واحدة أو الصور فقط التي هي في المستوى البؤري يمكن استخراجها لتجميع الفيلم النهائي. يمكن تصوير العينة في الغرفة السفلى تكون منهجية بديلة لتجنب الانجراف نحو الانخفاض المحتمل. جلطات البلازما يمكن أن تكون مفيدة لتجنب الانجراف، والبلازما ستتمسك طبقة يغلف الخارجية (EVL، محيط بالأدمة) 31، وبالتالي قد استقرار العينة. ولكن هذا يحتاج لفحصها، وكذلك كيف يمكن الحفاظ على الزرد اليرقات طويلة في تجلط البلازما دون التدخل في الصحة اليرقات أو تجديد الزعانف. تم تجميعها دينا فيلم استخدام المقاطع الفردية (26 ميكرون)من سجلت ض المكدس، الذي غطى سمك الكامل للزعنفة (~ 10 ميكرون)، والتي تمثل المحتملين ض الانجراف الزعنفة أثناء إجراء التصوير. من أجل الحفاظ على المعلومات 3-D، فمن الممكن أيضا أن المشروع مداخن-Z في الصور واحدة. لأن هذا قد يؤدي إلى التشويش من الصورة، قد يكون المطلوب brightfield إزالة التفاف. البرامج، مثل Deconvolve أو X3 Autoquant يمكن استخدامها لهذا الغرض. بدلا من ذلك، خوارزميات رياضية (الموصوفة في تادرس 33) يمكن تطبيقها للحصول على وظيفة نقطة انتشار نسبة عالية إشارة إلى الضوضاء (SNR). الحصول على SNR عالية تمثل واحدة من العقبات الرئيسية في brightfield إزالة التفاف. على الرغم من أن هذه الطريقة يتطلب التباين العالي ورقيقة سمك العينة، سيكون من المناسب للتصوير من زعنفة الذيل نتيجة لانخفاض عرضه. وهناك ميزة واضحة عن طريقة التصوير قدمت هو أنه قابل للتكيف بسرعة إلى أي stereomicroscope مجهزة كاميرا CCDد البرمجيات الوقت الفاصل بين ويقدم بديلا منخفض التكلفة لأنظمة التصوير متحد البؤر أكثر تكلفة. في حين أن هذا الأسلوب لا تستخدم مضان للكشف عن الخلايا، ويمكن تمديدها لهذه التطبيقات من خلال استخدام نظام آلي للتحكم في مصراع وبعد التصوير إزالة التفاف البرمجيات 34. وهذا من شأنه تمكين المستخدمين لمواصلة مراقبة عمليات إصلاح الجرح وتجديد مع خلية واحدة أو قرار التحت خلوية على مدى فترات زمنية أطول. وضوح بصري والسهولة التي الجنينية والزرد اليرقات يمكن التعامل معها، والقدرة على التكيف من هذا الأسلوب لأي stereomicroscope يجعلها مناسبة لتدريس علم الأحياء الفقارية الأساسي في إعداد الفصول الدراسية. يمكن لهذا الأسلوب تزويد الطلاب بفهم أفضل للعمليات البيولوجية الأساسية الكامنة وراء إصلاح الأنسجة وتجديدها. العمليات البيولوجية الأخرى التي تم التقاطها باستخدام طريقة مماثلة هي التطور الجنيني الزرد 23،34 والقلبوظيفة (غير منشورة). هذا الأسلوب يوفر أيضا إمكانية لإصلاح مراقبة الجرح وتجديد في اليرقات التي تم التلاعب بها وراثيا ودواء.

The ability of an organism to orchestrate tissue repair processes after injury is crucial for its survival 1. While all animals have the capacity to heal their wounds, the extent to which tissues regenerate differs greatly among species. Vertebrate species such as zebrafish, salamanders and frog tadpoles have the remarkable ability to regenerate lost tissues, including their appendages, portions of their eyes, heart, and central nervous system 2-4. Mammalian species, such as the African spiny mouse and rabbits, are capable of regenerating holes in their pinnae 5-7, and humans and mice regenerate portions of their liver as well as their digit tips during fetal and juvenile stages 8-12. Although it is not well understood yet why and how certain species regenerate tissues more effectively than others, the presence of similar genetic pathways suggests that these mechanisms may lie dormant in species without great regeneration potential 13,14. Thus elucidating tissue repair and regeneration mechanisms in species with satisfactory regeneration outcomes will benefit regeneration in humans.
We have chosen the larval zebrafish tail fin as a paradigm to demonstrate its regeneration with time-lapse brightfield stereomicroscopy. The zebrafish larval tail fin is anatomically simple as compared to the more complex adult structures, consisting of a two-layered, infolded epithelium with somatosensory axons innervating the skin that surrounds medially located mesenchymal cells 15. Despite the anatomical differences, larval tail fin regeneration is somewhat comparable to adult fin regeneration in terms of the molecular signatures and the outgrowth responses 16,17. As compared to the adult fin, imaging larval tail fin regeneration has however several advantages: 1) larval fin regeneration is completed within just 2-3 days 16, 2) larvae can be mounted in low-melt agarose, and 3) larvae do not require feeding until ~ 5 days post fertilization (dpf) due to the presence of the yolk sac. This makes zebrafish larvae ideal for observing tissue repair dynamics in vivo.
The presented method enables the capture of detailed dynamics underlying the early processes of fin regeneration. Many studies have utilized fluorescence-based confocal microscopy to study cellular and subcellular biological processes in embryonic and larval zebrafish. Sophisticated confocal imaging setups are however often not accessible to everyone and highly expensive as compared to other imaging techniques. In contrast, the presented methodology utilizes a Discovery V12 stereomicroscope equipped with Axiovision software and a time-lapse module, thus providing a more affordable alternative to expensive imaging equipment to examine tissue behaviors. We demonstrate that this method can be utilized for imaging tissue regeneration with high temporal resolution at a minimal cost. The implications for this method could extend beyond basic biology to advance mammalian regeneration studies using organ cultures, for therapeutic development through pharmacological and genetic screens, and it can serve as a teaching tool in a classroom setting.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bullseye Agarose (MidSci, Cat. No. BE-GCA500)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melt agarose (Fisher BioReagents, Cat No. BP1360-100)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea [Alfa Aesar, Cat No. L06690]&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean Aquarium Salt (Pet store)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Blue (0.1% solution) (Sigma, Cat. No. M9140)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate, Sigma-Aldrich, Cat. No. E10505)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Phenoxyethanol (Sigma-Aldrich, Cat. No. 77699)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 35 x 15 mm (BD Falcon, Cat. No 351008)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 60 x 15 mm (BD Falcon, Cat. No 351007)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 100 x 25 mm (BD Falcon, Cat. No 351013)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5.75 inch boroschillate glass pipets (Fisher)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Glass Top Glass Bottom Dish (MatTek Corporation, Cat No. D35-20-0-TOP) Glass: 0.085-0.115mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost/Plus microscope slides (Fisherbrand, Cat No. 12-550-15)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips (Electron Microscopy Services, Cat No. 72191-75)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips (Warner Instruments, Cat. No. CS-18R15)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phifer Phiferglass Insect Screen Charcoal - 48&#34; (Home Depot)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DOW CORNING&#174; HIGH VACUUM GREASE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipette tips 20 &#181;l (Eppendorf, Cat. No. 930001007)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharply Angled Up (Fine Science Tools, Cat. No. 14037-10)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Luer-Lok&#8482; disposable syringe&#160;(BD, Cat. No. 309657)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Luer-Lok&#8482; disposable syringe (BD, Cat. No. 309653)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>23-gauge syringe needles (BD, Cat. No. 305145)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps (Fine Science Tools, Cat. No. 11295-00)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabDoctor Mini Dry Bath (MidSci)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Discovery.V12 compound microscope&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Plan Apo S 3.5X objective</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioCam MRm&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axiovision software, Release 4.8.2SP1 (12-2011)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

تقنية عرض مناسبة لتوضيح ديناميات إصلاح الأنسجة ردا على بتر. يوضح الفيلم الذي بتر الزعنفة مشغلات البداية تأثير محفظة سلسلة، وتتميز تقلصات عبر كابلات الأكتين الميوسين التي تكون موجودة في الزعنفة أضعاف 28 (الشكل 5 A، B). وفي الوقت نفسه، تنبثق الخلايا من الجرح (انظر الفيلم). وبالتالي قد تقلص يكون وسيلة لطرد الخلايا التي من المرجح الموجهة للخضوع لموت الخلايا. تظهر نتائجنا أيضا أن النمو التنموي لليرقة يحدث بشكل مستقل عن تجديد (فيلم)، في حين لا بدء تجديد الزعانف حتى حوالي 14 ساعة بعد بتر مقاسا طول الزعانف والمنطقة على مدار الساعة من 36 ساعة بعد بتر (الشكل 5C ، D). وبلغ إجمالي النمو زعنفة التجدد بعد 1.5 أيام حوالي 60٪ من طول الزعانف الأصلي (الشكل 5E). أخذت معا، وهذه النتائج تثبت أن آر البتر<html> iggers انكماش الزعانف، وقذف من الخلايا من الجرح، واستجابة التجدد تأخر زمنيا. في حين أن الخلايا مقذوف من المرجح متجهة للخضوع لموت الخلايا، وطبيعة هذه الخلايا تحتاج إلى مزيد من التوضيح. <img alt="الشكل (1)" src="/files/ftp_upload/52654/52654fig1.jpg" /> الشكل 1. التصوير التجمع حلقة الغرفة (A) يظهر هو حلقة من البلاستيك التي يتم تركيبها على ساترة مع الشحوم السيليكون. ومرفق شبكة بلاستيكية إلى داخل الغرفة مع أربعة نقاط صغيرة من الشحوم السيليكون. (B) يتم تعبئة غرفة تحتوي على يرقة مزودة حل تريكين ويتم إرفاق شريحة زجاجية إلى الأعلى. (C) والتي شنت يوم 2-القديم يرقة (السهم) ويظهر في أعلى التكبير لتصوير حجمه بالنسبة للشبكة.&quot;_blank&quot;&gt; اضغط هنا لمشاهدة نسخة أكبر من هذا الرقم. <img alt="الشكل 2" src="/files/ftp_upload/52654/52654fig2.jpg" /> الشكل 2. التصوير غرفة جعلت الجمعية من أطباق بتري. (A) يظهر هو الزجاج التجاري الزجاج العلوي طبق بتري السفلي مع شبكة بلاستيكية تعلق على ساترة الزجاج. (ب) يظهر هو طبق بتري الغرفة التي شيدت الذاتي مع وجود ثقب حفر في غطاء وساترة المرفقة من الخارج مع الشحوم السيليكون. شبكة واليرقة هي التي شنت داخل غرفة تحتوي على حل تريكين. لختم الغرفة، يتم تطبيق الشحوم السليكون إلى العليا، الحافة الخارجية للغرفة السفلى وغطاء رأس المرفقة. <a href="https://www.jove.com/files/ftp_upload/52654/52654fig2large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <i<html> بديل ملغ = &quot;الشكل 3&quot; SRC = &quot;/ الملفات / ftp_upload / 52654 / 52654fig3.jpg&quot; /&gt; الشكل 3. مخطط البتر وتصاعد ليرقة للتصوير. (A) لبتر، وضع اليرقة تخدير على أي طبق بتري المغلفة الاغاروز وبتر زعنفة الذيل مع إبرة حقنة. (B) للتركيب، ونقل اليرقة مع ماصة نقل في أنبوب 1.5 مل مليئة 42 ° C الاغاروز السائل وماصة قطرة تحتوي على اليرقة إلى غرفة التصوير، توجيه الأسماك وتغطية عزز الاغاروز مع الجنين المتوسطة. (C) كشط قبالة الاغاروز من زعنفة الذيل باستخدام microloader ماصة توج أو أداة مماثلة، واستبدال الجنين المتوسطة مع المتوسطة الطازجة. (D) صورة زعنفة الذيل تحت stereomicroscope. <a href="https://www.jove.com/files/ftp_upload/52654/52654fig3large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> ithin صفحة = &quot;دائما&quot;&gt; <img alt="الشكل (4)" src="/files/ftp_upload/52654/52654fig4.jpg" /> الرقم 4. غرفة الحضانة ساخنة-شيدت الذاتي. (AC) يظهر هو غرفة الحضانة ساخنة مصنوعة من الورق المقوى، والتفاف فقاعة والفيلكرو. وترد سخان قبة سلكي (المصممة أصلا لبيضة الدجاج الحضانة) إلى غرفة باستخدام شريط الألمنيوم. <a href="https://www.jove.com/files/ftp_upload/52654/52654fig4large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 5" src="/files/ftp_upload/52654/52654fig5.jpg" /> الرقم 5. ديناميات تجديد مؤشرات مالية (A) مخطط الذيل تستخدم الزعانف فحص البتر والكمي طريقة لتحديد طول الزعانف (السهم الأحمر) والمنطقة (مخطط الحمراء للزعنفة). (B) الذيل بتر الزعنفة مشغلات البداية انكماش الزعنفة، تليها REGEثمرة الأنسجة nerative. يخضع الزعنفة أيضا النمو التنموي، كما يتضح من زيادة حجم الجانبية. (C) يظهر هو طول الزعانف بوصفها وظيفة من الزمن، وكشف عن انطلاق النمو التجديدي الخطي في ~ 14 HPA. (D) الكمي لمنطقة الزعانف يكشف عن انخفاض في البداية في الحجم، والتي يمكن أن تعزى إلى تقلص الزعنفة. بعد ~ 14 ساعة، ويزيد من حجم الزعانف بمعدل خطي. (E) مقارنة طول الزعنفة قبل بتر وبعد 36 ساعة معارض ~ 60٪ إعادة نمو. شريط مقياس: 100 ميكرون المختصرات: قبل أمبير، قبل بتر. آخر ساعة البتر، HPA. مغذي، وتجديد. أمبير، بتر <a href="https://www.jove.com/files/ftp_upload/52654/52654fig5large.jpg" target="_blank">الرجاء النقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <a href="https://www.jove.com/files/ftp_upload/52654/movie1.mp4">فيلم</a> . تجديد زعنفة على مدار الساعة من 36ساعة. المعروضة زعنفة ذيل يرقة القديمة 2.5 يوما خلال التجدد. وبدءا تصوير 30 دقيقة بعد بتر النمو التجديدي في 30 دقيقة فترات على stereomicroscope باستخدام عدسة الهدف 3.5X.

Watch this Scientific Journal Video about Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy at JoVE.com

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

التقاط إصلاح الأنسجة في الزرد اليرقات مع الوقت الفاصل بين Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

capturing-tissue-repair-zebrafish-larvae-with-time-lapse-brightfield

Research

JoVE Journal

Developmental Biology

10.6K Views.  MDI Biological Laboratory. We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Video: Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

تحليل الزرد اليرقات الهيكل العظمي النزاهة العضلات مع ايفانز الأزرق صبغ

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

التصوير الهياكل التحت خلوية في الأجنة الحية اسماك الزرد

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

تحليل<em&gt; في فيفو</em&gt; الهجرة الخلية باستخدام الزرع خلية والتصوير الوقت الفاصل في الزرد الأجنة

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

تحليل التنمية الكلى الزرد مع الوقت الفاصل بين التصوير باستخدام مجهر تشريح مجهزة لباجتزاء البصري

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

بيوسنسينغ موتور الخلايا العصبية الغشاء المحتملة في الأجنة الزرد الحية

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

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

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

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

تصوير مرور الوقت فوتون متعددة لتصور التنمية في الوقت الحقيقي: التصور لترحيل الخلايا العصبية كريست في الأجنة الزرد

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

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

تحليل ديناميات Actomyosin في جداول الخلوية والأنسجة المحلية باستخدام أفلام الفاصل الزمني من غرف البيض Drosophila المستزرعة

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

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

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

طريقه تركيب طبقات لتمديد المجهر البؤري المجهري لأجنه الزبرافيش الكاملة بفواصل زمنيه طويلة

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

كامل جبل المناعة الكيمياء في الأجنة حمار وحشي واليرقات

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

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

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

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

تصور الدماغ النامية في سمك الحمار الوحشي المعيشة باستخدام Brainbow والوقت الفاصل Confocal التصوير

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