Yazarlar nazik Zebrafish sox10 organizatörü ayıracı paylaşmak için Robert Kelsh teşekkür ederim.

1.. Fotoçevrim için Embriyo Koleksiyonu <ol><li> Akşam 5 ve 6 arasında kaede transgenik zebra balığı: sox10 en az on çiftleri kurmak. </li><li> Ertesi sabah, bölücüler çekin ve öğle saatlerinde yumurta toplamak. Petri yemekleri aktarmak ve 28.5 ° C inkübatör içine koydu. </li><li> Yaklaşık 24 saat sonra döllenme de, ölü embriyolar kaldırarak Petri kapları temizleyin. Işık alan mikroskopisi ve gelişmiş yeşil flüoresan protein (EGFP) filtre ile herhangi bir floresan mikroskop kullanılarak embriyo 13 gelişim aşamasını kontrol edin. Kaede Bu filtre aracılığıyla görünür olacaktır. </li><li> Embriyolar ayrı bir Petri kabı içine 60 hpf aktarımı beş parlak floresan embriyoların ulaşmak ve gerekirse bunları dechorionate zaman. </li></ol> 2. Fotoçevrim için Embriyo montajı <ol><li> Normal bir floresan mikroskop kullanılarak embriyo monte edin. Konfokal mikroskop embriyo montajı son derece zordur. </li><li> Standı kullanınard E3 su ya da aşağıdaki tarifi kullanarak taze E3 hazırlamak: 1x E3L yapmak için aşağıdaki ekleyin distile su dH 2 O bir litre başına ekleyin: 0.292 g 5,0 mM NaCl 0.013 g 0.17 KCI 0.044 g 0.33 mM CaCl2 (kurutucu 96,% ACS ayıraç) 0.081 g 0,33 mM MgSO 4 (anhydrated) İsteğe bağlı: Bir mantar ilacı ve heyecan gibi 1X E3% 0.05 metilen mavisi 200 ul ekleyin. Orta 1 hafta oda sıcaklığında tutar. </li><li> Standart tricaine kullanın veya şu tarifi kullanarak tricaine hazırlamak: 0.2,% 5 L için aşağıdaki maddeleri karışımı tricaine: 45 ml tris-Cl (1 M pH 9.5) 5 LH 2 O 10 g tricaine </li><li> 46.25 ml E3 3.75 ml% 0.2 tricaine ekleyerek, &#39;film suyu&#39; (50 ml E3,% 0.015 tricaine çözelti) hazırlayın. </li><li> 50 ml cam bir şişe içinde 46.35 mg, düşük erime noktası (LMP) agaroz ile 46.25 mi E3 su karıştırılarak agaroz hazırlamak ve mikrodalga katı parçacıklar çözülür. Sonra eklemekAgaroz ve kullanıma kadar 50 ° C su banyosu içine agaroz ile cam bir şişeye koymak için 3.73 ml% 0.2 tricaine. </li></ol> 3. Hazırlık ve Embriyo Montaj <ol><li> Slaytı adım 1.1.2 ve transfer embriyo yapılan E3/0.015% tricaine çözeltisi ile embriyolar uyutmak. </li><li> Kağıt dokusu ve embriyo üzerine agaroz put ile tüm aşırı E3 içinize çekin. </li><li> Pozisyon embriyo mükemmel Microloader ipuçları ile dorsal. Bütün embriyo agaroz üstünde yüzen, ancak bütün vücudu aşağı itmek değil emin olun. Odası tamamen sıvıyla dolana kadar sonra agaroz gömülü embriyo üzerinde E3/0.015% tricaine çözüm dökün. </li></ol> 4. Eşodaklı Mikroskop Yazılım Ayarlarını <ol><li> Embriyo odaklama için; 20X hava dalgıç objektif (iğne deliği boyutu 20 sayısal açıklık 0.75) kullanın. Sonra mikroskop L100 ışık düğmesine basın ve yazılım kanalları seçmek. Simgeleri görünümünü açın / edinimi controls/A1settings ve clickonfokal kurulum düğmesine k. Sonraki &#39;otomatik&#39; tıklayın ve tekrar penceresini kapatmadan önce şu kanalları seçmek: Ch1: DAPI kanal 403.4 nm (350-410 nm arasında dalga boyları kullanılabilir) Ch2: 488.0 nm Ch3: 561.9 nm </li></ol> 5. Fotoçevrim <ol><li> Ch1 ve 3 kapatın ve kaldır kilit düğmesi ve tarama tıklamadan önce Ch2 açın. </li><li> Fotoçevrim için ilgi hücreleri üzerinde durulacak hangi 1frame/sec ve yüksek gerilim (HV) 200 ile başlayan ve daha sonra kare / sn kadar gidiyor ve buna çevresinde 1/32 kare / sn ve HV kadar ulaşan HV düşürerek tarafından yapılan en iyi 100-120, arka plan gürültü bağlı. </li><li> Embriyo odakta olduğunda, ekranın sağ kenarında bakmak yeşil jant kapmak ve Fotoçevrim ve fare ile sağ klik için ilgi alanı sığacak şekilde küçültebilir. Fotoçevrim geri döndürülemez çünkü küçük iyidir. </li><li> 1 ile 200 HV ve kare / sn çevirin ve vurdutarayın. Fotoçevrim alanında bir yakınlaştırılmış görüntü şimdi görünür. </li><li> Ch1&#39;den (403.4 nm) açarak Photoconvert hücreleri. GFP kanalı ile görüldüğü gibi, yeşil kaede neredeyse yok olana kadar bölge boyutuna bağlı olarak, herhangi bir 5-60 saniye photoconverting lazer Açığa. </li><li> Yeşil kaede sinyal neredeyse yok olduğunda, Ch1 kapatın. Ch2 ve 3 açın. Sonra A1 tarama alanı alanında penceresinin üzerine sağ tıklayın ve istenen hücreler photoconverted olsaydı kontrol etmek için &#39;orijinal boyutuna dönüşünü&#39; çarptı. </li></ol> 6. Z-yığını yapma <ol><li> Menü çubuğunda simge &#39;yakalama Z-serisi&#39; Bul ve başlamadan önce Z-yığını üst ve alt sınırları ayarlamak. LUT&#39;u ayarlayın. </li></ol> 7. Yazılım Ayarları Time-lapse <ol><li> A1Simple GUI kutusuna gidin ve aşağıdaki ayarları kene: 1/32 kare / sn boyutu 1024 ortalama 4 veya 8X bir döngü içinde alınması gereken Z-yığınlarının sayısına bağlı olarak, </li><li> : Gitview / kazanım kontrolleri / ND dizisi edinimi ve set zaman çerçevesi (8-30 dk), sürekli tıklayın ve Z-yığını tıklatın. Olarak test öncesi sınırlarını ayarlayın ve film başlar. </li><li> Gün boyunca E3/0.015% tricaine çözüm odasına slayt eklemeye devam. Geceleri, tamamen odasını doldurmak. Yani ertesi sabaha kadar sürecek. </li><li> Ertesi sabah, E3/0.015% tricaine çözüm dolum ve embriyo hala hayatta olup olmadığını kontrol edin. Floresans kaybı ölümünü göstermektedir. Gün boyunca doldurulmasına ve kontrolleri devam ediyor. </li><li> Yapı oluşturan bittikten veya embriyo ölünce, filmi bitirmek. </li></ol> 8. Post-prodüksiyon İşleme <ol><li> Araç çubuğunda &#39;show maksimum yoğunluk projeksiyonu&#39; ı tıklatın, sonra resmin üzerine sağ tıklayın ve yeni bir belge oluşturmak. Video şimdi mümkün olan en iyi kalitede görünür. Gerekli değildir kareleri silmek ve LUT&#39;u ayarlamak için gidin. Nihayet. Avi olarak kaydetmek. Bu biçim, Windows için iyi olacak. Mac indir yazılım to. mov veya. wmv dönüştürmek. </li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

İşte Zebrafish modelinde kraniofasiyal gelişme görüntülenmesi için yeni bir yöntem gösterilmektedir. Sox10: kaede transgenik zebra balığı hattı başarıyla ayrıntılı CNCC arasında göç desen tanımlamak için kullanılan bir model organizma 5 olarak kullanılır. Önceki çalışmalar hücrelerini hedef göz gibi brüt işaretlerini kullanmış ve kaede mRNA enjeksiyon, Fotoçevrim tahlillerde veya Fotoçevrim 10, 11, 14, 15 için kafesli floresein dek...

Orofasiyal yarıklar 1/700-1 ile, en yaygın kraniofasiyal deformitesi temsil, 000 teslimatlar 1 etkiledi. Erken embriyolojik kraniofasiyal gelişme bozulması yarık dudak ve damak (CL / P) oluşumuna yol açabilir. Sendrom yarık için nedenler büyük ölçüde gösterilmiş olmasına rağmen, orofasial yarıklaşmanın nonsendromik formlarının genetik ve epigenetik bazlar arasında hala 2-4 açık olması gerekir. Bu malformasyonların etyolojisi ve patogenezi anlamak için, bir hücresel bazda kraniofasial yapıların gelişmesini aydınlatmak için gereklidir. Tüm omurgalı türlerin kranial nöral krest hücreleri (CNCC) orofasiyal yapıların oluşumuna katkıda yutak kemerler, doldurmak için dorsal nöral tüp göç. Erken embriyolojik nöral krest gelişim bozulması CL / P 5-7 gibi kraniofasiyal malformasyonların oluşumuna yol açabilir. ReklamdaZebra balığı ve memeli kraniofasial geliştirme (CNCCs homolog bölgelerinde ikamet) arasındaki yapısal benzerlikler dition, gen düzenleyici ağ son derece muhafaza edilir. Ayrıca CNCCs Zebra balığı CL / P. gelişimsel ve genetik temelin çalışma için güçlü bir organizma haline amniote türler ve zebrabalıkları 8 ile aynı şekilde geliştirmek olduğu gösterilmiştir Bu küçük boyutlu, hızlı ve ex-utero embriyonik gelişim ve yüksek üreme oranları gibi birçok avantajı vardır. Ayrıca, embriyo mikroskop altında 9 karmaşık bir gelişimsel olayların gözlem it yatkın hale optik olarak transparandır. Bu göç ve kafatası nöral tepe hücreleri farklılaşma çalışma için ideal bir hayvan modelidir. Kaede transgenik model 5: önce basılmış olan çalışmaları 8, 10, 11,, CNCC of göç desen üzerinde genişletilmesi sox10 kullanılarak detaylı bir şekilde tarif edilmiştir. Kaede fotoğraf konvertibl protein olduğunu tufotoğraf aktivasyon sonra yeşilden kırmızıya rns ve mümkün tam CNCCs iz yapar. Bu dönüşüm sırasında, peptid omurgası dönüşüm hücrelerin nihai hedefe 12 için izlenebilir, yani stabil olduğunu düşündürmektedir bölünmektedir. Sox10 transkripsiyonel kontrolü altında kaede ile etiketlenmiş transjenik çizgiler amniote damak ve zebrafish etmoid levha Y şekilli birleşme dikiş arasında benzer olan frontonasal öne (FNP) ile ve iki taraflı maksiller çıkıntıların (MXP) füzyonu ile homolog meydana gösterdi türleri. Diğer uygulamalar arasında, sox10: kaede transgenik zebra balığı modeli normal ve anormal kraniofasial yapıların oluşumunu göstermek için farklı bir gelişim aşamalarında zebra balığı embriyoların videoları oluşturmak için kullanıldı. Hücrelerin spesifik grupların Fotoçevrim mümkün onların gelişimini takip etmek için yapar. Craniofac gelişen canlı görüntüleme oluşturmak için bu yöntemle bir yaklaşım ileZebrafish ial yapılar kolay görsel Bu karmaşık bir gelişimsel süreci göstermek için yapım tanıtıldı. Örnek olarak kaede transgenik zebra balığı: Bu protokol sox10 de etmoid plakanın normal gelişimini kullanarak bu videoları üreten deneyim paylaşımı amaçlanıyor. Bu protokol ayrıca zebrabalıkları kranial nöral krest hücrelerinden türetilen herhangi bir yapının zaman atlamalı videoları hale uygulanabilir.

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			<td>Company</td>
			<td>Catalog Number</td>
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		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
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		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
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		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
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			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
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			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
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			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
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			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
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			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
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visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

Omurgalı palatogenesis kranial nöral (CNC) hücreleri, yakınsama ve yüz çıkıntıların uzantısı ve kraniofasiyal iskeletin olgunlaşması geçişini içeren bir derece koreografi ve karmaşık bir gelişimsel süreçtir. Kaede transgenik Zebrafish hattı oluşturuldu: Zebrafish damak belirli bölgelerde bir sox10 için kranial nöral katkısını incelemek. Sox10 böylece geleneksel boya veya raportör mRNA enjeksiyon daha hassas süreci etiketleme hücreyi yapma, nöral için kaede raportör protein soy kısıtlama sağlar. Kaede fotoğraf aktivasyonu sonrası yeşilden kırmızıya döner ve mümkün hassas hücrelerin takip yapan bir fotoğraf konvertibl proteindir. Sox10: kaede transgenik çizgi çene elemanlarının karşı maksiller doğuran ve amniotes için yüz çıkıntıların homoloji göstermektedir CNC hücre popülasyonlarının belirginleştiren soy analizini gerçekleştirmek için kullanılmıştır. Bu protokol basamağını açıklayankaede Zebra balığı embriyosu: Bir sox10 canlı zaman atlamalı video üretmek için s. Etmoid plaka Kalkınma pratik bir örnek olarak hizmet verecek. Bu protokol, transjenik zebrabalıkları herhangi kaede veya benzeri photoconvertible raportör proteinin bir zaman atlamalı konfokal kayıt yapmak için uygulanabilir. Ayrıca, zebra balığı mutantlar kraniofasial yapıları, normal, aynı zamanda anormal değildir gelişimi yakalamak için kullanılabilir.

Sox10 in: kaede transgenik hattı, göçmen ve sonrası göç CNCCs floresan yeşil etiketli. Yeşil floresan ile etiketlenmiş kaede CNCC hücreleri, endojen sox10 mRNA ekspresyonunu 5 özetlemek. Diğer uygulamalar arasında, bu hayvan modeli daha iyi CNCC bağımlı kraniofasial yapıların gelişimini görselleştirmek için kullanılmıştır. Özel yapılar ve aynı zamanda kraniofasiyal malformasyonların patolojik gelişimi, özellikle yarık dudak ve damak normal gelişim...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Deneysel verilerin görselleştirilmesi bilimsel topluluk sonuçların sunulması önemli bir unsur haline gelmiştir. Büyüyen embriyoların canlı time-lapse kayıt Üretimi karmaşık bir gelişimsel süreçlerinin daha iyi tanıtımı ve anlaşılmasına katkıda bulunmaktadır. Bu protokol Zebrafish kaede protein Fotoçevrim yoluyla hücre etiketleme için bir adım-adım bir kılavuzdur.

Sox10 içinde Kraniyofasiyal Kalkınma Görselleştirme: kaede Transgenik Zebra balığı Hattı Time-lapse konfokal mikroskopi kullanılarak

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K Görüntüleme.  Massachusetts General Hospital.  Deneysel verilerin görselleştirilmesi bilimsel topluluk sonuçların sunulması önemli bir unsur haline gelmiştir. Büyüyen embriyoların canlı time-lapse kayıt Üretimi karmaşık bir gelişimsel süreçlerinin daha iyi tanıtımı ve anlaşılmasına katkıda bulunmaktadır. Bu protokol Zebrafish kaede protein Fotoçevrim yoluyla hücre etiketleme için bir adım-adım bir kılavuzdur. 

Video: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Zaman atlamalı Mikroskopi Sökme Aktin İzlenmesi

Biyoloji

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

İki foton axotomy ve zaman atlamalı konfokal görüntüleme canlı zebrafish embriyolar

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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.

Konfokal Mikroskop Zebra balığı Embriyonik Beyin Görüntüleme Canlı

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 ...

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.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

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

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Transgenik Zebra balığı Multicolor Time-lapse Görüntüleme: Hedefli Nöronal Hücre Ablasyon aktifleşir görselleştirme Retina Kök Hücreler

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Kraniofasial mezenşimin, Elektroporasyon

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Uzun süreli, yüksek çözünürlüklü Konfokal Time Lapse Görüntüleme<em&gt; Arabidopsis Kotiledon</emÇimlenme sırasında&gt; Epidermis

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D konfokal mikroskopi kullanılarak Zebra balığı Kranyofasiyel morfogenez Analizi

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...