وأيد هذا العمل من قبل الزمالة ويلكوم ترست باحث أول لديفيس أولا.

<ol>
	<li>Arias, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+and+the+development+of+biology+in+the+20th+century.">Drosophila melanogaster and the development of biology in the 20th century.</a> Methods Mol. Biol. 420, 1-25 (2008).</li><li>Parton, R. M., Valles, A. -. M., Dobbie, I. D., Davis, I., Goldman, R. D., Swedlow, J. R., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pushing+the+Limits+of+Live+Cell+Imaging+in+Drosophila.">Pushing the Limits of Live Cell Imaging in Drosophila.</a> Live Cell Imaging: A Laboratory. , 387-418 (2010).</li><li>Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+messages:+the+intracellular+localization+of+mRNAs.">Moving messages: the intracellular localization of mRNAs.</a> Nat. Rev. Mol. Cell Biol. 6 (5), 363-375 (2005).</li><li>Jansen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+Localization:+Message+on+the+Move.">mRNA Localization: Message on the Move.</a> Nat. Rev. Mol. Cell Biol. 2, 247-256 (2001).</li><li>Van De Bor, V., Hartswood, E., Jones, C., Finnegan, D., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gurken+and+the+I+factor+retrotransposon+RNAs+share+common+localization+signals+and+machinery.">gurken and the I factor retrotransposon RNAs share common localization signals and machinery.</a> Dev. Cell. 9 (1), 51-62 (2005).</li><li>Delanoue, R., Herpers, B., Soetaert, J., Davis, I., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Squid&#47;hnRNP+helps+Dynein+switch+from+a+gurken+mRNA+transport+motor+to+an+ultrastructural+static+anchor+in+sponge+bodies.">Drosophila Squid&#47;hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies.</a> Dev. Cell. 13 (4), 523-538 (2007).</li><li>Jaramillo, A. M., Weil, T. T., Goodhouse, J., Gavis, E. R., Schupbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+fluorescently+labeled+endogenous+gurken+mRNA+in+Drosophila.">The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila.</a> J. Cell. Sci. 121 (Pt. 6), 887-894 (2008).</li><li>Forrest, K. M., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+endogenous+mRNA+reveals+a+diffusion+and+entrapment+mechanism+for+nanos+mRNA+localization+in+Drosophila.">Live imaging of endogenous mRNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila.</a> Curr. Biol. 13, 1159-1168 (2003).</li><li>Bertrand, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol. Cell. 2 (4), 437-445 (1998).</li><li>Spradling, A. C., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. 1, 1-70 (1993).</li><li>MacDougall, N., Clark, A., MacDougall, E., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+gurken+(TGFalpha)+mRNA+localizes+as+particles+that+move+within+the+oocyte+in+two+dynein-dependent+steps.">Drosophila gurken (TGFalpha) mRNA localizes as particles that move within the oocyte in two dynein-dependent steps.</a> Dev. Cell. 4 (3), 307-319 (2003).</li><li>Tekotte, H., Tollervey, D., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+migrating+border+cell+cluster+in+living+Drosophila+egg+chambers.">Imaging the migrating border cell cluster in living Drosophila egg chambers.</a> Dev. Dyn. 236 (10), 2818-2824 (2007).</li><li>Weil, T. T., Parton, R., Davis, I., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+bicoid+mRNA+anchoring+highlight+conserved+mechanisms+during+the+oocyte-to-embryo+transition.">Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition.</a> Curr. Biol. 18, 1055-1061 (2008).</li><li>Prasad, M., Jang, A. C., Starz-Gaiano, M., Melani, M., Montell, D. 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+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nat. Protoc. 2 (10), 2467-2473 (2007).</li><li>Morris, L. X., Spradling, A. 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+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138 (11), 2207-2215 (2011).</li></ol>

ليس لدينا ما يكشف.

<html> حي التصوير الخلية هو فحص قوي لفحص العمليات الخلوية في الوقت الحقيقي. بالإضافة إلى المراقبة الميدانية بسيط مشرق، وبالإضافة إلى ذلك من تسميات فلوري إلى RNAs والبروتينات والاهتمام قد يؤدي إلى اختراقات كثيرة. هنا وقد أوجزنا بروتوكول للالبويضات التصوير المعيشة الفردية التي يمكن استخدامها في تركيبة مع فحوصات الجينية والبيوكيميائية. في هذا البروتوكول، ونحن أيضا شرح كيفية التعامل مع التجربة الحية البويضات عن طريق الحقن. هناك احتمالات كثيرة للمادة لحقن، بما في ذلك في المختبر توليفها فلوري الحمض النووي الريبي وصفت لفحص قدرة هيكل الثانوي لتوطين RNA مباشرة (الكرة وديفيس، غير منشورة)، والأجسام المضادة التي تمنع وظيفة من البروتينات 6. والعمل في المستقبل على الارجح ادخال مكونات غيرها من العلامات والآلات الخلوية إلى البويضات المعيشة مما يمكن اختبار الآليات الجزيئية. تي &quot;&gt; الحفاظ على سلامة وصحة الأنسجة أمر ضروري عند العمل مع الخلايا الحية. في هذا البروتوكول، ونحن نشير إلى عدد من الخطوات التي يمكن أن تؤدي إلى الإجهاد للغرفة البيض. على سبيل المثال، على الرغم من كونها متفوقة النفط للتصوير، يمكن للثقافة موسعة في مجال النفط أن تؤدي إلى التأكيد على غرفة بيضة. هذا يمكن رصدها بسهولة في ظل التعرض لمجال مشرق من خلال دراسة مورفولوجية النووية والموقف، وغشاء الخلية البيضية التي من شأنها أن تشوه وفقاعة تحت الضغط (قارن 7E الشكل (بهيج) لFigure7F ( وأكد)). المرحلة غرف بيضة 9 مشاهدة RNA الترجمة والحدود هجرة الخلية أكثر من 12 ساعات عدة. ومع ذلك، فإن مرحلة 7/8 غرفة بيضة ستبدأ في معرض الآثار السيئة في مجال النفط الكربون هالة بعد نحو 40 دقيقة من المبيض يتم إزالتها من ويمكن للإناث. بيضة في المرحلة المتأخرة الغرف، المرحلة 11 إلى 14 تتطور بشكل طبيعي سواء في النفط أو وسائل الإعلام مائي بسبب إفراز قشرة البيضة من الخلايا المسام في هذه المراحل 13. لقد كان الريبوrted أن إضافة الأنسولين والمتوسطة الحشرات المائية قادرة على المحافظة على مرحلة 9 البويضات لمدة تصل الى 6 ساعات 14 و germaria تصل إلى 14 ساعات و 15. في جميع الحالات، ينبغي تجنب المناورات العدوانية للغرفة البيض، كما أنه يؤكد على البويضات ويقلل من قدرتها على البقاء. التصوير أقل من البيض وغرف العناية لعلاج كل واحد بلطف أفضل وسيلة لضمان بقاء الأمثل.

وينبغي أن الأدوات المستخدمة في تشريح تكون نظيفة ولكن لا يجب أن تعقم. مسح الأدوات مع ETOH والسماح لهم لتجف قبل البداية. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> غرامة فرشاة الدهان الرؤوس (فنانون السمور، جولة، حجم 00 أو 0) </td><td> متاح من معظم وسائل الفن الامدادات </td><td></td><td> فرشاة ذات نوعية جيدة من المهم </td></tr><tr><td> الهالوكربون منتجات الشركة، السلسلة 95 </td><td> الهالوكربون منتجات شركة، 887 Kinderkamack الطريق، نهر حافة NJ 07661 </td><td></td><td> الأوروبي الموزع: شركة فرنسة سولاوادي (شركة محدودة) تأكد من الأوكسجين من قبل فقاعات الهواء عن طريق النفط. </td></tr><tr><td> تغطية زلة لا. 1.5، 22 خ 50mm </td><td> دولي NO1-VWR القطة = 631-0137-22-50mm </td><td> منزل، جلاسر-22-40mm-400-070T MNJ </td><td></td></tr><tr><td> دومون No.5 - Dumostar </td><td> أدوات العلم غرامة </td><td> 11253-20 </td><td> &quot;البيولوجيا&quot; معلومات سرية هي أيضا جيدة لكنها هشة أكثر </td></tr><tr><td> تشريح مسبار </td><td> أدوات العلم غرامة </td><td> 10140-01 </td><td></td></tr><tr><td> تشريح المجهر </td><td> أوليمبوس </td><td> SZ61 </td><td></td></tr><tr><td> CO2 وسادة </td><td> متاح من خلال <a href="http://www.flystuff.com">http://www.flystuff.com</a> / (A تقسيم العلمية جينيسي) </td><td> 59-114 (789060 Dutscher) </td><td> الأوروبي الموزع: على شبكة الاتصالات العالمية. dutscherscientific. com</a> / كما أنه من السهل نسبيا على مخصصة لبناء منصات الخاص ذبابة الخاصة </td></tr><tr><td> KimCare </td><td> Kimbery كلارك، KimCare-الطبية مناديل </td><td> KLEW3020 </td><td></td></tr><tr><td> مجهر DeltaVision </td><td> التطبيقية الدقة </td><td> DC-CORE </td><td></td></tr><tr><td> Micromanipulator </td><td> Micromanipulators بيرلي من التجويف شركة مجموعة حيوية. 2260 Argentia الطريق، ميسيسوجا، أونتاريو، كندا L5N 6H7 </td><td> بيرلي PCS-5000 سلسلة، TS-5000-300 </td><td> <a href="http://www.ldgi-burleigh.com">http://www.ldgi-burleigh.com</a> / (Exfo سابقا) </td></tr><tr><td> جهاز حقن </td><td> Tritech أبحاث، وشركة 2961 المخضرم افي لوس انجليس، كاليفورنيا 90064 </td><td> MINJ-1 </td><td> تعديل مع حامل لاتخاذ إيبندورف Femptotips </td></tr><tr><td> حقن إبرة </td><td> إيبندورف العقيمة Femtotips الأول والثاني </td><td> 930000043 </td><td> نصائح مختلفة هي أفضل لتطبيقات مختلفة </td></tr><tr><td> تحميل نصائح 20μl </td><td> إيبندورف </td><td> 5242956.003 </td><td></td></tr><tr><td> نشط خميرة جافة </td><td> فلايشمان الخميرة </td><td> # 2192 </td><td></td></tr></tbody></table>

preparing individual drosophila egg chambers for live imaging

حي التصوير الخلية هو أسلوب مهم ينطبق على عدد من الأنسجة ذبابة الفاكهة المستخدمة كنماذج للتحقيق في مواضيع مثل مواصفات المحور، تمايز الخلايا وتوالد 1. إعداد الصحيح للنماذج تجريبية هي الحاسمة، وغالبا ما تهمل، خطوة. الهدف من إعداد هو التأكد من ملاءمة الفسيولوجية وإلى إيجاد ظروف التصوير المثلى. للمحافظة على سلامة الأنسجة، وهذا أمر بالغ الأهمية لتجنب، نقص الأكسجة الجفاف، وتدهور أو ارتفاع درجة حرارة متوسطة 2. غرفة البيض ذبابة الفاكهة هو نظام راسخ لدراسة المسائل ذات الصلة، ولكن ليس على سبيل الحصر، إلى تنميط الجسم، وتوطين مرنا وتنظيم هيكل الخلية 3،4. بالنسبة لغرف البيض في وقت مبكر، ومنتصف المسرح، وتصاعد في الهالوكربون النفط هو جيد للبقاء على قيد الحياة لأنه يتيح نشر خالية من الأكسجين، ويمنع الجفاف ونقص الأكسجة ولها خصائص بصرية رائعة للفحص المجهري. ايمالشيخوخة من البروتينات الفلورية هو ممكن من خلال إدخال الجينات المحورة في غرفة البيض أو حقن المادية من الحمض النووي الريبي وصفت، والبروتين أو الأجسام المضادة 5-7. على سبيل المثال، إضافة إلى البنى MS2 جينوم الحيوانات يتيح مراقبة الوقت الحقيقي من mRNAs في البويضة 8. هذه التركيبات تسمح في وضع العلامات المجراة من مرنا من خلال الاستفادة من التفاعل MS2 الحمض النووي الريبي عاثية حلقة الجذعية مع بروتين معطف 9 منه. هنا، نقدم لبروتوكول لاستخراج المبيض وكذلك عزل ovarioles الفردية والغرف البويضة من ذبابة الفاكهة من الإناث. للحصول على وصف مفصل من الكرسي ذبابة الفاكهة oogenesis Spradling جيم ألان (1993، طبعة 2009) 10.

Watch this Scientific Journal Video about Preparing Individual Drosophila Egg Chambers for Live Imaging at JoVE.com

Preparing Individual Drosophila Egg Chambers for Live Imaging

و<em&gt; ذبابة الفاكهة</em&gt; غرفة البيض هو نموذج ممتاز لدراسة آليات توطين مرنا. من أجل التقاط أحداث الحيوية التي ترتكز عليها عمليات التعريب، مطلوب السريع التصوير عالية الدقة من الأنسجة الحية. هنا، نقدم بروتوكول للتشريح والتصوير من عينات حية بأقل قدر من الانقطاع.

إعداد فردية ذبابة الفاكهة البيض غرف للتصوير لايف

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

preparing-individual-drosophila-egg-chambers-for-live-imaging

Research

JoVE Journal

Biology

Preparing Individual Drosophila Egg Chambers for Live Imaging

19.1K Views.  University of Oxford. و ذبابة الفاكهة غرفة البيض هو نموذج ممتاز لدراسة آليات توطين مرنا. من أجل التقاط أحداث الحيوية التي ترتكز عليها عمليات التعريب، مطلوب السريع التصوير عالية الدقة من الأنسجة الحية. هنا، نقدم بروتوكول للتشريح والتصوير من عينات حية بأقل قدر من الانقطاع.

Video: Preparing Individual Drosophila Egg Chambers for Live Imaging

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

يعيش التصوير للهجرة الخلايا الدبقية في القرص العين تخيلي ذبابة الفاكهة

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

لإعداد ذبابة الفاكهة الأجنة لايف التصوير باستخدام بروتوكول إسقاط الشنق

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

الحصاد وإعداد ذبابة الفاكهة الأجنة لتسجيل الكهربية وغيرها من الإجراءات

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

يعيش تصوير ذبابة الفاكهة السوداء البطن هجرات كرية دموية الجنينية

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

يعيش التصوير في القدرة على الحركة والهيكل الخلوي أكتين خلية من الخلايا العصبية الفردية والخلايا العصبية في الأجنة كريست اسماك الزرد

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

إعداد وتطوير الكبار ذبابة الفاكهة الأمخاخ والشبكية للتصوير لايف

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

يعيش التصوير من البروتينات التي تحمل علامات GFP في<em&gt; ذبابة الفاكهة</em&gt; البويضات

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

يعيش التصوير من تخليص الخلية أفكارك أثناء<em&gt; ذبابة الفاكهة</em&gt; مرحلة التطور الجنيني

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

والطماطم / GFP-FLP / FRT الطريقة للتصوير لايف من الفسيفساء الكبار<em&gt; ذبابة الفاكهة</em&gt; خلايا مستقبلة للضوء

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

تصور وتتبع mRNAs الذاتية في غرف البيض الحية Drosophila الميلانوجاستر البيض

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...