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

<ol>
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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

19.2K 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 ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

يعيش تصوير الدماغ الجنينية اسماك الزرد بواسطة الميكروسكوب متحد البؤر

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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 ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

سبين الإلكترون التصوير بالرنين الصغرى ، من الأنواع الحية لرسم خرائط الأوكسجين

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

تستقيم تصوير ذبابة الفاكهة الأجنة

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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.