جيم Vinegoni يقر بدعم من المعاهد الوطنية للصحة (NIH) منحة - 1 - RO1 EB006432.

ومخزونات melanogaster ذبابة الفاكهة المستخدمة في هذه التجربة هي التالية : <ul style="list-style-type:none;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> elav - Gal4 (FBti0002575) </li><li style=";text-align:right;direction:rtl"> AP - Gal4md544 (FBal0051787) </li><li style=";text-align:right;direction:rtl"> UAS - srcEGFP (FBti0013990) </li><li style=";text-align:right;direction:rtl"> w1118 (FBal0018186) </li></ul> لجميع التجارب ، تم استخدام نظام UAS - Gal4 لGFP overexpress في أنسجة الفائدة (أي الغدد اللعابية أو أقراص الجناحين). بشكل أكثر تحديدا ، وعبرت الذباب الإناث وراثيا مع Gal4 المناسبة لزملائه وراثيا UAS - EGFP. وقد تربى الصلبان في 25 درجة مئوية في حاضنة مرطب. وعندما تبدأ الشرانق ملء قارورة (حوالي 5-6 أيام بعد بدء الصليب) ، اختاروا لمضان GFP في هذه المرحلة prepupal البيضاء (0-1 ساعة بعد تشكيل puparium) وجمعت للتصوير المقطعي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> انشاء تقاطعات والخلفية المناسبة عند 25 درجة مئوية </li><li style=";text-align:right;direction:rtl"> 5-6 أيام بعد بدء الصليب ، وتحديد النسل نيون للتصوير. استخدام الفرشاة المبللة لإزالة طيف من prepupae بيضاء من الجانبين من القارورة. </li><li style=";text-align:right;direction:rtl"> وضعت وضعت prepupae في قطرة ماء أو برنامج تلفزيوني في طبق بتري لإزالة جزيئات الطعام والغراء والتي تغطي حالة العذراء ويجعل العينة autofluorescent. نظيفة بلطف مع فرشاة الرسم. إذا شيخوخة الشرانق ضروري ، ومكان المحدد prepupae الأبيض في طبق بتري بمنشفة مبللة ورقة (غرفة الرطبة) أو على جانبي قنينة نظيفة مع الطعام الطيران. وهذا ضمان وجود ما يكفي من الرطوبة ، للسماح للتنمية السليمة للحيوانات. </li></ol> تركيب ذبابة الفاكهة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الغراء في الجزء السفلي من حالة العذراء في أنبوب زجاجي الشعرية (800 ميكرون قطر) ان هذه الذبابة الأمامي / الخلفي توجه المحور الموازي للأنبوب. </li><li style=";text-align:right;direction:rtl"> إصلاح الأنابيب الشعرية عموديا على مرحلة التناوب. </li><li style=";text-align:right;direction:rtl"> ضبط محور يميل للمرحلة التناوب من أجل محور الدوران لتكون موازية للأعمدة بكسل من اتفاقية مكافحة التصحر التصوير. </li></ol> تقرير إعادة توجيه النموذجي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إصلاح خادرة في كوب الشعرية وجبل كما هو موضح قبل </li><li style=";text-align:right;direction:rtl"> ملء أنبوب السيليكا الشعرية (100 ميكرون ، OD) مع صبغة الفلورسنت (Cy5.5) </li><li style=";text-align:right;direction:rtl"> إدراج الأنابيب الشعرية داخل الشرنقة الى 45 درجة فيما يتعلق المحور الأمامي / الخلفي </li><li style=";text-align:right;direction:rtl"> تضيء خادرة مع الإثارة شعاع الليزر مع فتحة عدسة رقمية منخفضة. جمع الصور تضوء مضان أكثر من 360 درجة تناوب على طول خادرة محورها العمودي. </li><li style=";text-align:right;direction:rtl"> احتواء البيانات التجريبية مع موديلا لأحد قدما المناسبة </li></ol> الغدد اللعابية التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شن GFP خادرة الإعراب في الغدد اللعابية كما هو موضح قبل </li><li style=";text-align:right;direction:rtl"> شعاع يضيء مع الإثارة وجمع تضوء في إشارة مضان GFP أكثر من 360 درجة تناوب على طول خادرة محورها العمودي. </li><li style=";text-align:right;direction:rtl"> إعادة بناء البيانات التي حصل عليها الاستفادة من النموذج إلى الأمام </li></ol> الوقت الفاصل بين التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شن GFP prepupa الأبيض في التعبير عن الأقراص وتخيلي الجناح </li><li style=";text-align:right;direction:rtl"> الحفاظ على البيئة الرطبة ، والتصوير في درجة حرارة الغرفة لتجنب الجفاف للخادرة. </li><li style=";text-align:right;direction:rtl"> وضع مصراع الكاميرا لتجنب الإضاءة المستمرة </li><li style=";text-align:right;direction:rtl"> شعاع يضيء مع الإثارة وجمع تضوء في إشارة مضان GFP أكثر من 360 درجة تناوب على طول خادرة محورها العمودي. </li><li style=";text-align:right;direction:rtl"> إعادة بناء البيانات التي حصل عليها الاستفادة من النموذج إلى الأمام. </li></ol> الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تشريح الغدد اللعابية CNS / أقراص العين في برنامج تلفزيوني 1X. </li><li style=";text-align:right;direction:rtl"> إصلاح الأنسجة في فورمالديهايد 4 ٪ ، 1X بيم (مواسير 100mM ، 1MM EGTA ، 1MM MgCl 2) ل20min في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إزالة التثبيت وجبل في Vectashield مع دابي (ناقلات) </li><li style=";text-align:right;direction:rtl"> صورة مع المجهر الفلورسنت. </li></ol> الوقت الفاصل بين الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع البيض ومرحلة prepupae منهم في غرفة رطبة. </li><li style=";text-align:right;direction:rtl"> جمع الشرانق في مراحل تنموية مختلفة </li><li style=";text-align:right;direction:rtl"> وخز حالة العذراء مرتين (0 ساعات بعد تشكيل Puparium) مع غرامة إبرة (دبوس الحشرات ، 000 FST) قبل التثبيت. </li><li style=";text-align:right;direction:rtl"> الإصلاح الشرانق في فورمالديهايد 8 ٪ ، 1X لمدة 6-8 ساعات بيم في درجة حرارة الغرفة مع الانفعالات لطيف. </li><li style=";text-align:right;direction:rtl"> بعد إزالة التثبيت والشطف مع 1X PBS ، قطع خادرة الثابتة بشفرة حادة في اثنين قطع على محور عمودي A / P ؛ </li><li style=";text-align:right;direction:rtl"> غمر قطعتين في Vectashield مع دابي (ناقلات) وجبل على الشرائح ساترة (لاب تيك) للتصوير. </li><li style=";text-align:right;direction:rtl"> الحصول على صور مع مجهر متحد البؤر مضان. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="الشكل 1" border="0" /> الشكل 1. الرجاء <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">انقر هنا</a> لرؤية بنarger نسخة من الشكل 1.

<ol>
	<li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light:+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light: the evolution of whole-body photonic imaging.</a> Nat. Biotechnol. 23, 313-320 (2005).</li><li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat. Methods. 4, 331-336 (2007).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Nat. Methods. 5, 45-47 (2008).</li></ol>

The authors have nothing to disclose.

داخل الجسم الحي اعادة البناء في القضية العذراء والغدد اللعابية GFP - معربا عن وجود دال هو مبين في الشكل رقم 1 (أ) ؛ melanogaster prepupa (المقياس بار ، 500 ميكرون) مع الأنسجة المقابلة (الأخضر والأزرق GFP مضان ، تلوين دابي). الوقت الفاصل بين التصوير سلسلة من دال ويرد melano...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

تصور النامية تشكيل هيئة وكذلك progession والعلاج من المرض بشكل كبير في كثير من الأحيان يعتمد على القدرة على استجواب بصريا التغيرات الجزيئية والوظيفية في الكائنات الحية سليمة. معظم طرق التصوير الضوئي الموجودة ليست كافية للتصوير في الأبعاد التي تقع بين حدود تغلغل المجهر الضوئي الحديثة (0.5 - 1MM) والحدود التي يفرضها الانتشار من الفحص العياني البصرية (&gt; 1cm) [1]. وهكذا ، كثير من الكائنات الحية نموذجا هاما ، مثل الحشرات والأجنة الحيوانية أو الأطراف الحيوانات الصغيرة ، وتظل غير قابلة للوصول لداخل الجسم الحي التصوير الضوئي. على الرغم من أن هناك اهتماما متزايدا نحو تطوير نانومتر القرار أساليب التصوير الضوئي ، لم تكن هناك الكثير من الجهود الناجحة في تحسين عمق الاختراق التصوير. القدرة على أداء في الجسم الحي ، والتصوير المجهري وراء الحدود هو في الواقع اجتمع مع الصعوبات المرتبطة نثر فوتون موجودة في الأنسجة. الجهود المبذولة مؤخرا لأجنة الصورة بأكملها على سبيل المثال [2،3] خاصة تتطلب المعالجة الكيميائية للعينة ، لحملهم على مغادرة نثر ، وهو الإجراء الذي يجعلها مناسبة فقط لتصوير ما بعد الوفاة. لكن هذه الأساليب دليل على الحاجة إلى عينات أكبر من تلك التصوير يسمح عادة عن طريق الفحص المجهري أو ثنائي الفوتون مبائر ، ولا سيما في البيولوجيا التطورية ، واكتشاف المخدرات. لقد قمنا بتطوير تقنية التصوير الضوئي الجديد المسمى جرحشاعثزرحشرتاد الإسفار التصوير المقطعي [4] ، والتي مناسبة لتصوير داخل الجسم الحي غير الغازية في أبعاد 1MM - 5mm. القرار التبادلات طريقة لعمق الاختراق ، ولكن عروض لم يسبق لها مثيل الأداء والتصوير تصوير الشعاعي الطبقي تم تطويره لإضافة الوقت وبعدا جديدا في الملاحظات البيولوجيا التطورية (وربما غيرها من مجالات البحوث البيولوجية) عن طريق نقل القدرة على صورة تطور مضان. الموسومة الردود على مر الزمن. على هذا النحو فإنه يمكن تسريع الدراسات المورفولوجية أو التبعيات الوظيفية على الطفرات الجينية أو مؤثرات الخارجية ، ويمكن أن الأهم من ذلك ، التقاط صورة كاملة للتنمية أو وظيفة الأنسجة من خلال السماح طولية الوقت الفاصل بين التصور للكائن ، نفس النامية. تقنية تستخدم المجهر المختبر المحورة والإسقاط متعددة الإضاءة لجمع البيانات على 360 درجة التوقعات. وينطبق ذلك على تبسيط فيرمي إلى حل فوكر بلانك ، من المعادلة النقل الفوتون ، جنبا إلى جنب مع مبادئ البصريات الهندسية من أجل بناء نظام انعكاس واقعي مناسب للمجموعة جرحشاعثزرحشرتاد. هذا يسمح في الجسم الحي ، كامل الجسم التصور هياكل ثلاثية الأبعاد غير شفافة في عينات تصل إلى عدة مليمترات في الحجم. لقد أثبتنا أداء داخل الجسم الحي بواسطة تقنية التصوير ثلاثي الأبعاد هياكل الأنسجة النامية ذبابة الفاكهة في الجسم الحي ، وذلك من خلال اتباع التشكل من الأجنحة في ذبابة الفاكهة الشرانق مبهمة في الوقت الحقيقي أكثر من ست ساعات متتالية.

Watch this Scientific Journal Video about Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila at JoVE.com

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

جرحشاعثزرحشرتاد مضان التصوير المقطعي يعمل خارج حدود اختراق الأنسجة sectioning المجهري مضان. وتستند هذه التقنية على التعددية إسقاط الإضاءة والنقل وصفا الفوتون. علينا أن نظهر في الجسم الحي ، كامل الجسم التصور 3D من التشكل من GFP - معربا عن أقراص تخيلي في الجناح<em&gt; ذبابة الفاكهة السوداء البطن.</em

جرحشاعثزرحشرتاد الإسفار عن التصوير المقطعي في الجسم الحي. تصوير النامية ذبابة الفاكهة</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

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Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K Views.  Massachusetts General Hospital. جرحشاعثزرحشرتاد مضان التصوير المقطعي يعمل خارج حدود اختراق الأنسجة sectioning المجهري مضان. وتستند هذه التقنية على التعددية إسقاط الإضاءة والنقل وصفا الفوتون. علينا أن نظهر في الجسم الحي ، كامل الجسم التصور 3D من التشكل من GFP - معربا عن أقراص تخيلي في الجناح ذبابة ...

Video: Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

وحيد الخلية في الجسم الحي Electroporation داخل الدماغ سليمة النامية

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

ولد لتطبيع الإسفار التصوير المقطعي الإسقاط الضوئية للتصوير القلب الجامع

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

الوقت الفاصل بين التصوير من الانقسام الميتوزي بعد ترنسفكأيشن سيرنا

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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 ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

داخل الجسم الحي الطرد المركزي الجنين ذبابة الفاكهة

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

في التصوير من يرقات ذبابة الفاكهة فيفو سليمة الفرعية الخلوية في القرار

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

التحليل السريع واستكشاف الإسفار الميكروسكوب الصور

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

إعداد ذبابة الفاكهة والتصوير الطولي من وظيفة القلب في فيفو باستخدام البصرية بالاتساق المجهري (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

التوليف من 68Ga جسيمات نانوية أكسيد الحديد يخدر الأساسية للتصوير المقطعي بانبعاث البوزيترون المزدوج/التصوير الرنين المغناطيسي (تي1)

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.