فإن الكتاب أود أن أشكر البروفيسور نيكولاس سميرنوف من جامعة اكستر لنصيحته ، وجامعة اكستر مرفق Bioimaging. وقدم التمويل من جانب المملكة المتحدة الحيوية والعلوم الحيوية مجلس البحوث (منحة مرجع BB/E002682/1).

بروتوكول الواردة أدناه توضح طريقة بسيطة لاستخدام PFD كوسيط الارتشاحي المتصاعدة في أوراق thaliana Arabidopsis ، لكننا نتوقع أن تكون قد استخدمت هذا الأسلوب في مجموعة متنوعة من التطبيقات حيث هو المطلوب التصوير الجوي الغنية الأنسجة. 1. عينات نبات المتصاعد في PFD <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد شريحة المجهر مع طوقا من الغاز نفاذية polydimethylsiloxane (PDMS ، كارولينا مراقبة جل). PDMS هو بوليمر اللزجة ويمكن مصبوب لتوفير غرفة مصممة خصيصا لمتطلبات التجريبية. </li><li style=";text-align:right;direction:rtl"> PFD تتوازن مع الهواء. ويمكن تحقيق هذا عن طريق محتدما مع الهواء أو عن طريق هز وحدة تخزين صغيرة من PFD في زجاجة مليئة بالهواء. </li><li style=";text-align:right;direction:rtl"> PFD صب في طبق بيتري المفتوحة وتعويم الشتلة كليا أو رفعه على أوراق PFD لمدة 5 دقائق. يجب أن تصبح الأنسجة شفافة ، تشبه أنسجة المزجج (الشكل 2 (أ)). قد يبدو أكثر قتامة أو أوراق أخف من ذي قبل التعرض لPFD ، depenقرع على استخدام ظروف الإضاءة وعمر الأنسجة. </li><li style=";text-align:right;direction:rtl"> تملأ الغرفة PDMS مع الهواء ومعايرتها PFD نقل بعناية عينات الأنسجة من الحضانة طبق بتري إلى غرفة PDMS. ختم الشريحة مع ساترة والصورة وفقا لمتطلبات التجريبية. </li><li style=";text-align:right;direction:rtl"> ملاحظة : PFD ينفذ بشكل جيد أيضا في غرفة مفتوحة بنيت على ساترة أو في غرفة نضح ومتوافق مع الشحوم السيليكون. PFD غير متوافق مع مكونات تفلون كما يذوب فيها. </li></ol> 2. ممثل النتائج : التفتيش المجهري لعينات أوراق PFD - المحتضنة يظهر أن غمرت المياه معظم أجوائها. الشكل 2 (ب) يبين أجوائها غمرت مع وقف التنفيذ في المؤتلف GFP PFD. فمن الواضح أن غمرت المياه على ورقة مع PFD الحضانة ، ولكن من حين لآخر جيوب الهواء قد تبقى. مياه الفيضان لا ورقة في ظل هذه الظروف. LSCM عينات المحتضنة والتي شنت في<html> الأشعة تحت الحمراء ، يظهر الماء أو PFD PFD أن يعطي ميزة على الماء والهواء في عمق التصوير المحتملة (الشكل 3). المثال الوارد في الشكل 3 يبين الهواء والماء وشنت PFD Arabidopsis يترك التعبير عن فينوس المترجمة cytosolically ، وهو البديل من YFP ال 11 التي يتم التعبير constitutively تحت المروج 35S. يمكننا أن نرى تقريبي 2 أضعاف زيادة في عمق التصوير عند مقارنة PFD والمياه. وتجدر الإشارة إلى أنه ، مع ذلك ، أن تحسين دقة المشاهدة عند استخدام PFD يختلف مع الفتحة العددية للعدسة ، ونوع من النسيج المستخدمة. لقد شهدنا تدفق حشوية والجذرية استطالة الشعر في PFD الشتلات المعالجة ، كل الإرشادية من النباتات الصحية. بالإضافة أظهرنا (2) أن FV / FM ، وهو مقياس لأداء التمثيل الضوئي للنباتات لا يزال في حدود 12 تحمله خلال فترات زمنية المستخدمة في التصوير. 3394fig1.jpg &quot;/&gt; الشكل 1. أوراق النبات التعقيد والآثار البصرية Diagrammatical التمثيل تبين الخصائص التشريحية للألف أوراق thaliana فيما يتعلق البصرية انشاء. المختصرات المستخدمة هي الكائنات. = الهدف العدسة ، IMM. = السائل الغمر ، COV. = ساترة ، MNT. = mountant ، قطع. = بشرة ، الإعلانية. الجيش الشعبي. = متجه نحو المحور البشرة ، ش. = المسام فوهي ، س. = mesophyll الاسفنجية ، كما = الفضاء الجوي ، PAL. = الحسيكة mesophyll ، VB = حزمة الأوعية الدموية ، والإعلان. الجيش الشعبي. = متجه نحو المحور البشرة. يشار إلى جدران الخلايا بواسطة خطوط سوداء. <img alt="الشكل 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> الشكل 2. PFD تخترق بسهولة يترك Arabidopsis (أ) أوراق المحتضنة في الهواء أو الماء أو PFD لمدة 5 دقائق والمصورة باستخدام كاميرا رقمية لايكا DCF3000FX مع المجهر MZ16F ايكا (لايكا مايكروسيستمز (المملكة المتحدة) المحدودة ، وميلتون كينز ، المملكة المتحدة). وقد طبع الشعار على الفيلم إن الرب خلات ومضيئة جيئة وذهابامتر تحت الضوء مع مربع. كان زمن التعرض 89 مللي ، وجمعت كل الصور ومعالجتها بشكل مماثل. أشرطة النطاق تمثل 2 ملم. (ب) تحمل PFD المنقى المؤتلف البروتين الفلوري الأخضر (GFP) يحد أجوائها في الجسم الحي. GFP خضراء اللون كاذبة ، ويستخدم لإظهار الحمراء لصناعة السيارات في مضان الكلوروفيل الذي يفصل الخلايا mesophyll. (الشكل 2 (ب) مستنسخة بإذن والتفاصيل التقنية الكاملة المتاحة في ليتل وآخرون 2010 2). أشرطة النطاق تمثل 25 م <img alt="الشكل 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> الشكل 3. PFD يحسن التصوير مبائر في الأوراق التقط الصور يترك LSCM تظهر المترجمة cytoplasmically فينوس مضان (الأخضر) ، وتألق ذاتي الكلوروفيل (الحمراء) في thaliana ألف سليمة في الهواء أو الماء أو PFD. وكان طول الموجة 514 نانومتر ، والإثارة وسجلت الانبعاثات مضان في 518 نانومتر 604 نانومتر لفينوس وعلى 657 نانومتر679 نانومتر لالبلاستيدات الخضراء. كل لوحة هو مقطع واحد متحد البؤر مأخوذة من Z - 11 كومة من الصور مبائر المكتسبة في فترات 5 ميكرون. تم استخراج هذه من كومة - Z كامل من 59 صور حصلت على 1 ميكرومتر فترات ، والتي تم استخدامها لإنتاج الأفلام التكميلية. وترد قياسات العمق النسبي لسطح البشرة. وتظهر الصور التمثيلية ، والتي تم تجهيزها بشكل مماثل. تفاصيل التجريبية مثل إعدادات المجهر مماثلة لتلك الموجودة في ليتل وآخرون ، 2010 2. Scalebars تمثل نحو 20 ميكرومتر.

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	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, 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=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, 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+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

هذا هو أسلوب بسيط وسهل الاستخدام لتحسين الفحص المجهري للهواء أو معقدة مليئة بصريا الأنسجة. لقد أظهرنا أن لديه بعض مزايا تقنية قوية ، ونحن نأمل أن يتم استخدامها لتوضيح المسائل المتعلقة البيولوجية في الهواء الغنية الأنسجة. على سبيل المثال فإنه سيكون خيارا طبيعيا للدراسات من مهاجمة مسببات الأمراض النباتية في mesophyll أو في الرئتين. ونحن أيضا على بينة من القيود المفروضة على هذه التقنية. نحن نعمل على تحسين مطابقة معامل الانكسار ، مع استخدام وسائل أخرى للالمجهري ، وتجديد المشبعة mountant خلال التجارب الزمني الطويل. ونحن ندرك أيضا ، على الرغم من أن واحدة من المزايا الرئيسية لبالفلور ، ويجري وهي خاملة بيولوجيا والجانب الثاني. بالفلور لا تذوب بسهولة الجزيئات البيولوجية مما يعني أيضا أنه لا يمكن استخدامها بسهولة لتوفير مركبات ذات الأهمية مثل الهرمونات ، والمخدرات ، والجزيئات الصغيرة الأخرى أو الأيونات.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> Perfluorodecalin </td><td> F2 كيماويات </td><td> N / A </td><td> الهاتف لأجل. </td></tr><tr><td> كارولينا مراقبة جل </td><td> شفرات البيولوجي </td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

ومن المعترف به على نطاق واسع لمشكلة الحصول على صور عالية الدقة في عمق العينات البيولوجية 1. في الهواء مملوءة الأنسجة مثل mesophyll الاسفنجية من أوراق النبات أو الرئتين الفقاريات المزيد من الصعوبات الناجمة عن التحولات المتعددة في معامل الانكسار بين المكونات الخلوية ، وبين الخلايا وأجوائها ، وبين الأنسجة البيولوجية وبقية النظام البصري. علاوة على ذلك ، عدم التطابق معامل الانكسار يؤدي إلى توهين من الإثارة وانبعاث fluorophore في إشارة المجهري مضان. نحن هنا وصف تطبيق المشبعة ، perfluorodecalin (PFD) ، كوسيط التصوير الارتشاحي مما يحسن الليزر بصريا المسح المجهري متحد البؤر (LSCM) على عمق التصوير عينة ، من دون اللجوء الى الزيادات الضارة في قوة الليزر ، والحد الأدنى من التأثير الفيزيولوجي 2. نحن تصف البروتوكول لاستخدام PFD مع Arabidopsis thaliana الأنسجة ورقة ، وهو مجمع بصريا نتيجة لهاهيكل (الشكل 1). PFD لديها عدد من السمات التي تجعلها مناسبة لهذا الاستخدام 3. معامل انكسار PFD (1.313) هو مقارنة مع تلك المياه (1.333) ، وأقرب إلى العصارة الخلوية (حوالي 1.4) من الهواء (1،000). بالإضافة إلى ذلك ، PFD في متناول الجميع ، غير الفلورسنت وغير سامة. التوتر السطحي انخفاض PFD (19 سم dynes -1) هو أقل من المياه (72 سم dynes -1) ، وأيضا أقل من الحد (25 -- 30 سم داين -1) لاختراق فوهي 4 ، مما يسمح لها الفيضانات وأجوائها mesophyll إسفنجي دون تطبيق فراغ يمكن أن تكون مدمرة أو السطحي. أخيرا والأهم من ذلك PFD لديه قدرة كبيرة على تذويب CO 2 و O 2 ، والذي يسمح بالإبقاء تبادل الغاز في الأنسجة غمرت المياه ، مما يقلل من تأثير على العينة الفسيولوجية. وقد استخدمت هذه الخصائص في مختلف التطبيقات التي تشمل التنفس السائل جزئية وinfla الرئة5،6 نشوئها ، والجراحة 7 ، 8 الدم الاصطناعي ، الأوكسجين في نمو وسائل الاعلام 9 ، ودراسات لتكوين بلورات الجليد في النباتات 10. حاليا ، فمن الشائع أن يشن في الماء أو الأنسجة العازلة للتصوير المائي مبائر حية. نحن نعتبر أن استخدام PFD كوسيلة متزايدة يمثل تحسنا في الممارسة الحالية وتسمح للتحضير عينات بسيطة من العيش ورقة كاملة للتصوير.

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

نحن تصف استخدام perfluorodecalin كوسيط الارتشاحي المتزايدة. هذه هي طريقة بسيطة لتحسين عمق في التصوير<em&gt; thaliana Arabidopsis</em&gt; أنسجة نبات مع الحد الأدنى من التأثير الفيزيولوجي.

طريقة بسيطة للتصوير Arabidopsis يغادر طريق Perfluorodecalin كوسيط التصوير إختراقي

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K Views.  The University of Exeter. نحن تصف استخدام perfluorodecalin كوسيط الارتشاحي المتزايدة. هذه هي طريقة بسيطة لتحسين عمق في التصوير thaliana Arabidopsis أنسجة نبات مع الحد الأدنى من التأثير الفيزيولوجي.

Video: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : طريقة بسيطة لالتنميط الجيني دقيقة

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

و السابقين البيضة الثقافة الأجنة النظام الدجاج مناسبة للتطبيقات التصوير والجراحة المجهرية

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

تحليل خلية معينة من<em&gt; ل arabidopsis</em&gt; يترك طريق الإسفار خلية المنشط الفرز

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

إعداد بسيط ضوء مجهر ورقة ل<em&gt; في توتو</em&gt; التصوير من<em&gt; C. ايليجانس</em&gt; التنمية

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

تقنية تسلل الطرد المركزي لاستخراج Apoplastic السوائل من أوراق النبات عن طريق<em&gt; فاصيلوس الشائع</em&gt; كمثال

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

الحنق التصوير في الهلاميات المائية ثلاثية الأبعاد

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

التمييز وتوصيف السكان غير المتجانسة باستخدام تقنيات التصوير الكمي

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

طريقة موحدة للتعقيم الفائق من بذور نبات

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

لوسيفراس "التكامل بالتصوير الإنزيم" في أوراق نيكوتيانا بينثاميانا "تحديد عابر ديناميات التفاعل" البروتين-بروتين

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

تصوير فيتك-ديكستران كمراسل للرقابة الخاضعة للتنظيم

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...