Bu çalışma NS042228 MCH Olympus FV1000 konfokal UW Zooloji Bölümü (PI Bill Bement) için bir NIH paylaşılan enstrümantasyon hibe S10RR023717 ile satın alındı ​​R01 NIH tarafından desteklenmiştir. Erica Andersen, Namrata Asuri ve Matthew Kil bu kağıt eşit katkıda bulunmuştur.

1. Enjeksiyon slaytlar ve görüntüleme slaytlar Meclisi Enjeksiyon slaytlar: <ol><li> Sylgard silikon elastomer üreticinin talimatlarına göre hazırlayın. </li><li> Bond üç standart cam mikroskop Sylgard kullanın kesiştiği uzun kenarları yan yana düzenlenmiş olan diğer iki slaytlar, üstüne bir slayt istifleme birlikte slaytlar. Üst slayt dik açılı bir köşesinde ya da üst ve alt slaytlar arasında &quot;duvar&quot; oluşturur. </li><li> Sylgard gecede ayarlanmış olsun. Bu slaytları yeniden kullanılabilir. </li></ol> Görüntüleme slayt: <ol><li> Odasına mikroskop sahnede slayt sahibinin uygun şekilde standart bir cam mikroskop lamı (75mm x 25mm x 1mm) aynı boyutta kesilmiş paslanmaz çelik dikdörtgenler kullanın. 1,5 cm delik dikdörtgenin merkezinde kesilir. </li><li> Sylgard metal dikdörtgen yapıştırmayın 22 mm 2 lamel. Iyice lamel mühür yeterli Sylgard kullanın. Inverted mikroskop görüntü, embriyolar bu lamel üzerine monte edilebilir ve alttan görüntülenmiş. </li><li> Sylgard gecede ayarlanmış olsun. Bu slaytları yeniden kullanılabilir ve kullanımlar arasında% 70&#39;lik alkol ile temizlenmelidir. </li></ol> 2. DNA yapılarının hazırlanması Transgen bir doku belirli bir şekilde ifade etmek için, faiz gen hücre özel bir organizatörü arkasında klonlanmış. Biz zebrafish neurogenin1 gen (3.1ngn1) bir cis-düzenleyici element 2 ya da duyusal nöronların veya nöral krest hücrelerinden ifade sürücü sox10 gen (-4.9sox10) 3, sırasıyla kullanın. Hücre ve membran dinamikleri görselleştirmek için, sitoplazmik ifade veya membran lokalize fluorophores. F-aktin dağıtım için, kodlayan DNA mCherry (UtrCH-mCherry) erimiş utrophin calponin homoloji etki alanı ifade eder. UtrCH mCherry biyosensör prob aktin dinamikleri 1 ile müdahale olmadan F-aktin görselleştirme sağlar. Biz rekombinasyon tabanlı klonlama stratejisi 4 ve zebrafish Tol2kit 5 sağlanan vektörler Invitrogen Gateway kullanarak bu DNA yapıları oluşturur. <ol><li> Zebrafish Tol2kit 5 Multisite-Gateway Teknolojisi ve vektörler kullanarak ifade vektör (ler) oluşturun. Plazmidler pT2KXIGDin gelen Tol2 omurgası içerir. </li><li> QIAfilter Plazmid Midi Hazırlık kiti (QIAGEN A.Ş.) ve steril su Zehir ile plazmid DNA arındırın. DNA enjeksiyon için lineerleştirilmiş olması gerekmez. </li></ol> 3. DNA yapıları Enjeksiyon <ol><li> Su,% 0.2 fenol kırmızısı (enjeksiyon sırasında görünüm için) nihai konsantrasyonu 10-50 mg / ml DNA DNA sulandırınız. </li><li> Iğne doldurun ve kalibre: yaklaşık 1 ul DNA çözümü ile çekilmiş bir kılcal iğne Geri dolgu ve iğne tutucu (bir Picospritzer) enjeksiyon aparat takın. , Ince bir forseps kullanarak, iğne ucu açıklığı yaklaşık 10 mm çapında olduğu gibi kapalı ucunu kırmak. Oküler mikrometre ile mineral yağ damlacık çapı ölçülür. Nl hacmi yaklaşık 1 Bir damla almak için baskı süresi ayarını ayarlayın. </li><li> E3 embriyo orta (5mM NaCl, 0.17mM KCl, CaCl 2 0.33mM, 0.33mM MgSO 4 * 7H 2 O, pH 7.0) 1-hücreli aşamada embriyoların toplayın. </li><li> Enjeksiyon slayt ve konumu, üst ve alt slaytlar arasında oluşan dik açı köşesinde embriyoların yaklaşık 30-60 embriyoların transferi. Embriyoların, yüzey gerilimi tarafından slayt düzenlenen E3 böylece en çıkarın. </li><li> Bükülmüş bir diseksiyon pin hücre enjeksiyonu slayt duvara böylece embriyolar döndürmek için kullanın. </li><li> Yumurta sarısı ve embriyo tek bir hücreye, embriyonun koryon üzerinden iğne kılavuzu mikromanipülatör kullanın. 0.5-1 nl DNA çözümü hücre içine enjekte edilir. </li><li> 28.5 E3 ve inkübe Petri kapları içine embriyo transferine ° C Gerekirse, geliştirme, düşük sıcaklıkta embriyolar inkübe edilmesiyle önlenebilir. </li></ol> 4. Görüntüleme için Montaj embriyolar <ol><li> Embriyolar görüntüleme (amaçlar için yaklaşık 14-17 saat sonrası döllenme) için istenilen yaş ulaşmadan önce yaklaşık bir saat ince forseps ile embriyoların chorions çıkarın. </li><li> Floresan ile donatılmış bir diseksiyon mikroskobu kullanılarak etiketli hücreler embriyo seçin. Biz uzun çalışma mesafesi ve yüksek büyütme kombinasyonu nedeniyle Nikon AZ100 kapsamı 4X objektif (40X büyütme) kullanın. </li><li> Bir embriyo yaklaşık 50 ul E3% 0.2 Tricaine anestezi (10X konsantrasyonda) içeren bir mikrofuge&#39;de tüp içine yerleştirin. </li><li> Tricaine seyreltilmesi% 0.02 nihai konsantrasyonu 10 mM HEPES (42 ° C sıcaklıkta tutulan), 500 ul E3% 1 düşük erime noktası agaroz ekleyin. </li><li> Hemen bir agaroz açılan embriyo transferigeniş bir delik cam pipet kullanarak görüntüleme slayt lamel. </li><li> Agaroz sertleşir önce etiketli hücreleri ile bölgenin alt lamel (nöral krest hücreleri ve spinal duyusal nöronlar için aşağı dorsolateral sırt aşağı) karşı aşağı bakacak şekilde, embriyonun konumu. </li><li> Görüntüleme odasının birleştirin: Coat bir tarafı plastik, metal görüntüleme slayt silikon vakum yağ ve yapıştırmayın ile bir halka (2 cm dış çapı 3 mm, yüksek). Gres yağıyla halka diğer tarafında ceket. </li><li> E3 içeren 10 mM HEPES ve% 0.02 Tricaine odasının doldurun. Halka iyi yağlanmış yüzey 22 mm 2 lamel iliştirilmesi odasının üst Seal. </li></ol> 5. Olympus FV1000 konfokal IX81 mikroskobu ile hücre davranışlarının 4D görüntüleme Görüntü elde detayları mikroskop sistemi özelliklerine bağlıdır. Burada Olympus FV1000 konfokal mikroskop için geçen zaman satın alma süreci tanımlamak. Zeiss LSM 510 konfokal sistemi 6 time-lapse edinimi için önceki vallahi protokol bakın. <ol><li> Mikroskop sahnede slayt sahibinin görüntüleme odasına yerleştirin ve iletilen ışık altında 10X veya 20X objektif kullanılarak embriyo odaklanmak. </li><li> 60X objektif (1.2 veya daha yüksek NA) geçin ve Epifloresans ile etiketli hücre odaklanmak. Biz ya immersiyon yağı (UPLSAPO 60xO NA 1.35) ya da su altında kalacak (UPLSAPO 60xW NA 1.20) lens kullanabilirsiniz. </li><li> FV yazılımı (Sür 1.7c), lazer tarama XY tekrar başlamak için tıklayın. </li><li> Satın alma ayarları ve görüntü elde etme denetimleri (lazer çıkış, tarama hızı, HV, kazanç ve ofset seviyeleri) en az (% 25 veya daha düşük), doku hasarı önlemek ve photobleaching azaltmak için lazer güç tutarak sinyal optimize etmek için ayarlayın. </li><li> Z-serisi elde etmek için, adım büyüklüğü ile birlikte, z yığının alt ve üst sınırları belirler. Derinlik simgesi XY Sweep simgesini XY ve Z vurgulanan o kadar meşgul olduğundan emin olun. XYZ Sweep simgesini tıklayarak satın alma başlayın. </li><li> Hızlandırılmış bir zaman serisi elde etmek için, Toplama Ayarlar penceresinde TimeScan başlığı altında bir zaman aralığı ve zaman noktalarının sayısını ayarlamak. XY Sweep simgesini XY (Z) ve T vurgulanan böylece Image Acquisition Kontrol Pencerede Zaman Simgesi tıklayın. XY (Z) T Sweep simgesini tıklayarak satın alma başlayın. </li><li> Alternatif olarak, TimeController bir zaman atlamalı kurmak için kullanılabilir. Büyük veri kümeleri oluştururken bellek ayırma aşmadığı Bu programlama, yararlı olur. Aygıt altında, TimeController pencere açın. Yeni bir görüntüleme Görev oluşturun. Görüntü parametre penceresi açılacaktır. Satın alma ayarlarını güncellemek için FV Durum düğmesini tıklatın. Emin olun Kaydet ve Sil sonlandırın başlığı altında kontrol edilir. Çıktı dosyası adı ayarlayın ve ayarları kaydetmek ve pencereyi kapatmak için Tamam&#39;ı tıklatın. Loop fonksiyonu, zaman noktalarında istenen zaman aralığı ve sayı ayarlamak için kullanın. Hazır tıklayın ve satın alma işlemini başlatmak için Başlat. Duraklat ve Z-shift fonksiyonları ise görüntüleme yeniden odaklanabilmek için kullanılabilir. </li></ol> 6. Temsilcisi Sonuçlar Rakamlar ve film örnekleri, membran hedefli fluorophores veya aktin biyosensör probu ile etiketlenmiş duyusal nöronlar ve nöral krest hücrelerinden tasvir. <img src="/files/ftp_upload/1726/1726fig1.jpg" alt="Şekil 1" /> Şekil 1 bir Tg (ngn1: GFP-caax) Konfokal görüntüleri (z-projeksiyonları): mCherry-caax DNA 25 pg ngn1 ile enjekte edilen transgenik embriyo. MCherry CAAX etiketler yeşil etiketli tüm Rohon Sakal duyusal nöronlar ile arka planda bir nöron kırmızı (A) (B) yeşil ve kırmızı kanalları C) Birleştirilmiş görüntü. Ölçek çubuğu = 50 mm. <img src="/files/ftp_upload/1726/1726fig2.jpg" alt="Şekil 2" /> Şekil 2 Konfokal z projeksiyonları ~ 20 pg ngn1 ile enjekte edilen embriyolar nöronların:. MCherry-UtrCH DNA A) 16,5 hpf embriyo Rohon Sakal duyusal nöron. F-aktin sinyal büyüme konisi (ok başları) ve yeni kurulan aksonlar boyunca çıkıntılar güçlü B) Nöron 20 hpf embriyonun büyüme konileri dallı periferik akson (ok başları) ve daha olgun merkezi zayıf sinyal güçlü F-aktin sinyali gösteren aksonlar (oklar). Ölçek çubuğu = 50 mm.

<ol>
	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li></ol>

DNA enjekte optimum konsantrasyon organizatörü büyüklüğü ve gücü oluşturmak ve ampirik olarak tespit edilmelidir bağlı olarak değişecektir. Transgen ifade enjekte embriyo çok küçük bir oranı çok az neden olurken çok fazla DNA enjeksiyonu, geniş hücre ölümü ile sağlıksız embriyolar yol açabilir. DNA ekspresyon seviyesi hücreden hücreye değişir fluorofor sinyal gücü ile ilişkilidir. Epifloresans altında embriyolar sıralama iken, son derece yüksek seviyede ifade olanlar dahil değild...

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<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5040-250G</td>
<td>&nbsp;</td>
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<td>Sylgard Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
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<td>QIAfilter Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen Inc.</td>
<td>12243</td>
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<td>Low melting point agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-014</td>
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<td>Picospritzer</td>
<td>&nbsp;</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

Zebra balığı, in vivo geliştirme sırasında görüntüleme hücre davranışları için ideal bir model . Zebra balığı embriyolar dışarıdan döllenmiş ve gelişimin her aşamasında böylece kolayca ulaşılabilir. Ayrıca, doğal ortamda sağlam embriyonun hücre ve moleküler dinamiği yüksek çözünürlüklü görüntüleme, optik netlik sağlar. Biz nöral krest hücrelerinin göç ve nöron akson akıbet ve rehberlik sırasında hücre davranışlarını analiz etmek için canlı bir görüntüleme yaklaşımı kullanıyor. Canlı görüntüleme, hücre hareketi süreçleri düzenleyen mekanizmalar anlamak için yararlıdır. Gibi çıkıntılı aktivite ve moleküler dinamik olarak hücre hareketi detayları, görselleştirmek için, tek tek hücrelerin etiketlemek için avantajlıdır. Zebra balığı, plazmid DNA enjeksiyonu, geçici bir mozaik ifade desenini verim ve diğer hücre etiketleme yöntemleri üzerinde farklı avantajlar sunuyor. Örneğin, transgenik çizgiler genellikle tüm hücre popülasyonlarının etiket ve böylece tek bir hücrenin ince çıkıntılar (veya moleküler dağılımında değişiklikler) görselleştirme gizleyebilir. Buna ek olarak, tek hücreli aşamada DNA enjeksiyonu, daha az invazif ve daha sonraki aşamada boya enjeksiyonu çok daha hassas. Burada bireysel gelişmekte olan nöronların veya nöral krest hücreleri ve görüntüleme in vivo olarak davranışlarını etiketleme için bir yöntem açıklanmaktadır . Biz mozaik transgen ifadesi sonuçları 1-hücreli dönem embriyoların, plazmid DNA enjekte edilir. Vektörlerin hücre özel rehberleri içeren duyusal nöronların veya nöral krest hücrelerinin bir alt kümesi olan ilgi bir gen sürücü ifadesi. Biz membran hedef GFP ile veya canlı hücreleri 1 F-aktin görselleştirme sağlayan bir biyosensör probu ile işaretli hücrelerin örnekler sunuyor. Erica Andersen, Namrata Asuri ve Matthew Kil, bu çalışmaya eşit katkıda bulundu.

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

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

Bu protokol zebrafish embriyolar yaşayan tek tek nöronların veya nöral krest hücreleri görüntüleme açıklar. Bu yöntem, floresan konfokal zaman atlamalı mikroskopi kullanılarak cep davranışlar ve aktin lokalizasyonu incelemek için kullanılır.

Zebra balığı Embriyolar Bireysel Nöronlar ve Sinir Crest Hücreler Aktin Sitoiskelet Hücre Motilite ve Canlı Görüntüleme

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

live-imaging-cell-motility-actin-cytoskeleton-individual-neurons

Research

JoVE Journal

Biology

13.3K Görüntüleme.  University of Wisconsin-Madison. Bu protokol zebrafish embriyolar yaşayan tek tek nöronların veya nöral krest hücreleri görüntüleme açıklar. Bu yöntem, floresan konfokal zaman atlamalı mikroskopi kullanılarak cep davranışlar ve aktin lokalizasyonu incelemek için kullanılır.

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

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Fare Epidermal Sinir Crest Kök Hücre (EPI-NCSC) Kültürler

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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 Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Floresan üzerinden F-aktin Dynamics Canlı Hücre Görüntüleme speckle Mikroskopi (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Canlı Zebra balığı Embriyolar iki-Foton-Tabanlı fotoaktivasyon

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplantasyon GFP of-Kimerik Zebra balığı Embriyolar Retina ve Beyin Gelişimi Canlı Görüntüleme blastomer ifade

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Gelişmekte olan Zebra balığı Epidermis Hücre Ekstrüzyon Canlı Görüntüleme

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

GFP-Aktin ifade Canlı Endotel Hücreleri Aktin Sitoiskelet Çalışması

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Bireysel hazırlanması Drosophila</emCanlı Görüntüleme için> Yumurta Chambers

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

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.