Bu araştırma, RDW için bir Ulusal Bilim Vakfı Kariyer ödülü (MCB-0.746.066, acil Davranış düzenler transkripsiyonel Aktivatörler Karakterizasyonu) mümkün olmuştur LJ Shimkus, BS Goldman, G. Suen, M. Singer, LG Welch, KA Murphy, ve HG Taş el yazması üzerinde yararlı tartışmalar ve yorumlar için teşekkür ediyoruz.

Malzemeleri gerekli: <ul><li> Klett metre </li><li> Pipet ve ipuçları </li><li> 2.5 ml mikrosantrifüj tüpler </li><li> Mikrosantrifüj </li><li> CTTYE medya: <ul><li> % 1.0 Casitone (Difco Laboratories),% 0.5 maya özü (Difco Laboratories), </li><li> 10.0 mM Tris-HCl (pH 8.0), 1.0 mM KH 2 PO 4, 8.0 mM MgSO 4 </li></ul></li><li> TPM medya: <ul><li> 10.0 mM Tris-HCl (pH 8.0), 1.0 mM KH 2 PO 4, 8.0 mM MgSO 4 </li></ul></li><li> Agaroz </li><li> Su banyosu, 55 ° C </li><li> Cam mikroskop lamı </li><li> Büyük cam lamelleri </li><li> 2 x 2 cm, 0.5-mm kalınlığında silikon kauçuk conta (Grace Bio-Lab Inc.) </li><li> Parafilm (ya da etiketleme bant) </li><li> Forseps </li><li> Küçük bağlayıcı klipleri </li><li> Micro-örnekleme pipet (Balıkçı) </li><li> 100 ul cam tek ucu (Balıkçı) </li><li> Kimwipes </li></ul> Bölüm 1: Hücre Hazırlama Steril bir ortam oluşturarak başlayın. <ul><li> Temiz çalışma alanı, don eldiven ve ışık brülör. </li></ul><ol><li> CTTYE suyu içeren bir balona aşılama ve dinç dönen ile 32 ° C de karanlıkta inkübe bir gecede hücre kültürü başlayın. </li><li> Sonra kültür 5 x 10 8 hücre / ml, 2.5 ml mikrosantrifüj tüp içine pipetle 1 ml hücre yoğunluğu ulaştı. </li><li> 16.000 xg (veya maksimum hız) 2 dakika süreyle santrifüj Pelet hücreleri. </li><li> Durusu ve supernatant atın. </li><li> 1 ml TPM (tuz dengeli, besin ücretsiz medya) ile hücre pelletini yıkayın. <ul><li> yeniden askıya alma ve vorteks </li></ul></li><li> 16.000 xg (veya maksimum hız) az 2 dakika için iplik Re-pelet hücreleri. </li><li> Durusu ve supernatant atın. KRİTİK ADIM </li><li> Pipetleme ve vorteks bir arada kullanarak 100 ul TPM medya tamamen pelet yeniden askıya. ÖNEMLİ NOT: Bu adım, tüp içinde kalan hücreleri hiçbir kümeleri olduğunu sağlar. Bu biraz zaman alabilir. </li><li> Izleme tahlil plakalar hazırlarken oda sıcaklığında kenara hücreleri ayarlayın. </li></ol> Bölüm 2: Agar hazırlanması <ol><li> TPM (testi medya) ve CTTYE (besleyici disk) medya hem de 50 ml hazırlayın. </li><li> Her (agaroz agar daha az difraktif) 0.5 g agaroz ekleyin. </li><li> Sterilize etmek Otoklav. </li><li> Sterilizasyon sonrasında, 55 medya korumak ° C (bir inkübatör veya su banyosu) izleme tahlil plaka kompleksleri inşa ederken. </li></ol> Bölüm 3: Besin Disk İnşaat <ol><li> Oluşturan küçük bir iyi, bir alev steril cam mikroskop lamı üst 0.5 mm kalınlığında steril bir silikon kauçuk conta yerleştirin. </li><li> Pipet ~ 300 ul 55 ° C de slaytta conta tarafından oluşturulan ve besin disk içeren bir CTTYE medya / agaroz. CTTYE medya / agaroz kadar höyük. </li><li> CTTYE medya / agaroz üst alev sterilize slayt (conta ile) yerleştirin. ÖNEMLİ NOT: Bu slayt şekillendirme kabarcıklarını engellemek için bir açıyla aşağı doğru ayarlanmış olması gerekir. </li><li> Slayt yerleştirildikten sonra, mini bağlayıcı klipleri ile birlikte karmaşık kelepçe - her iki tarafta. </li><li> Kırpılır kompleksi, 4 ° C ve CTTYE medya / agaroz ayarlamak için izin yerleştirin. Bu genellikle yaklaşık 5 dakika sürer. </li></ol> Bölüm 4: Takip Testi Hazırlık - Plaka Kompleksleri Kurma Parçaları bir araya toplamak, slayt kompleksleri hazırlamak, besin disk yerleştirin, TPM medya / agaroz, ayrı ve kuru plaka kompleksleri, plaka hücreleri ve tahlil plaka kompleksleri izleme monte dökün. Plaka karmaşık parçaları bir araya <ol><li> Her bir kompleks için, alev, bir cam mikroskop lamı sterilize </li><li> Otoklavlanmış conta slayt üstünde bir koyun ve bir kenara koyun (yüzü yukarı sterilize). Bu testin alt oluşturacaktır. </li><li> Alev bir cam kapak kayma ve yer etiketleme bant (veya Parafilm) ile sarılmış bir ikinci cam mikroskop lamı üstüne (yüzü yukarı sterilize) bir kenara sterilize edin. Bu çatlama lamel tutmak için bir destek slayt olarak kullanılır. Bu testin en oluşturacaktır. </li><li> Alev üçüncü bir cam mikroskop lamı sterilize ve kenara koyun (yüzü yukarı sterilize). Bu agaroz düzleştirmek için kullanılır. ÖNEMLİ NOT: contalar, forseps ile aşağı basarak cam bir mühür formu emin olun, aksi takdirde medya / agaroz kurumasına neden olabilir. </li></ol> P<html> besleyici diske dantel ÖNEMLİ: 10 ile 5 Adımda bir defada bir slayt kompleksi yapılması gerekir, aksi takdirde medya / agaroz film kalitesi kötü sonuçlanan katılaşmaya başlar. <ol start="5"><li> Besleyici disk kompleksi formu çıkarın 4 ° C (bölüm 3 adım 5) ve şu anda katılaşmış CTTYE / agaroz açığa slaytları ayrı gözetlemek. </li><li> 100 ul cam tek ipucu, bir mikro-örnekleme pipet kullanarak yukarıda açıklanan CTTYE medya / agaroz 1 mm çapında &#39;besleyici disk&#39; çıkarın ve kapak kayma (yukarıdaki adım 3) conta tarafından oluşturulan . </li></ol> Plakalar dökün KRİTİK ADIM <ol start="7"><li> Pipet ~ 300 ul 55 ° C TPM medya / agaroz ve kapak kayma ve besin disk içeren conta tarafından oluşturulan içine. TPM medya / agaroz kadar höyük. KRİTİK ADIM </li><li> TPM medya / agaroz üstünde herhangi bir conta (3 adım) alev sterilize slayt yerleştirin. ÖNEMLİ NOT: Bu slayt şekillendirme kabarcıklarını engellemek için bir açıyla aşağı doğru ayarlanmış olması gerekir. </li><li> Slayt (5 adım) yerleştirildikten sonra, mini bağlayıcı klipleri ile birlikte karmaşık kelepçe - her iki tarafta. </li><li> Kırpılır kompleksi, 4 ° C ve medya / agaroz ayarlamak için izin yerleştirin. Bu genellikle yaklaşık 5 dakika sürer. </li></ol> Ayrı ve kuru plakalar ÖNEMLİ: medya / agaroz kurumasını önlemek için, 20 üzerinden 11, bir defada yalnızca bir slayt kompleksi yapılmalıdır adımları. ÖNEMLİ: En iyi sonuç için, 13 ile 11 adımda 4 yapılmalıdır ° C <ol start="11"><li> Medya / agaroz ayarlandıktan sonra, bağlayıcı klipleri kaldırmak ve bant sarılmış slayt gevşetmek için kompleks sonuna sıkmak. </li><li> Bant sarılmış bir slayt çıkarın ve daha sonra kullanmak için tezgah üzerine yerleştirin. KRİTİK ADIM </li><li> Destek slayt (conta), bir kama olarak forseps kullanma kapak slip / conta / media / agaroz kompleksi ayrı ve destek slayt atın. ÖNEMLİ NOT: kapak kayma / conta kompleksi ayrı bir meraklı hareket etmeyin. Bu kapak kayma kırılması ve / veya medya / agaroz destek slayt yapışmasını neden olabilir. </li><li> Bant sarılmış slayt kapak kayma / conta / media / agaroz kompleksi (conta tarafı yukarı) yerleştirin ve 4 kaldırmak ° C. Tüm görünür nem yeni maruz medya / agaroz yüzeyinden buharlaşması izin vermek için bir yazıcı için bu karmaşık sonraki yerde en fazla 1 dakika. ÖNEMLİ NOT: M. medya / agaroz kuru bu kadar çok uzun süre izin vermeyin etkileyebilir xanthus sürüsünün davranış. </li></ol> Plaka hücreleri KRİTİK ADIM <ol><li> Sonra aşırı nem, besin disk uzak medya / agaroz yaklaşık 1 mm üzerine konsantre hücreleri (part 1 adım 8) 0.5 ul pipet buharlaştı. ÖNEMLİ NOT: Bu hücreler, düz aşağı ve sonra doğruca yukarı pipet hareket ettirerek tevdi olduğundan emin olmak için son derece önemlidir. Bu sürüsü yuvarlak olmasını sağlar. ÖNEMLİ: Bu medya / agaroz pipet ucu dokunmak için son derece önemlidir. Bu yüzey üzerinde bir depresyon ve M. davranış değişikliği xanthus sürüsü. İPUCU: pipet medya / agaroz yaklaşmadan önce sıkıp. Bu hücreler bir damla pipet ucu alt görünür ve hücreleri daha kolay yatırmak için yapmak için izin verecektir. </li><li> Hücrelerin yatırılır sonra, brülör sonraki karmaşık hücre nokta kurumasını bekleyin yerleştirin en fazla 20 sn. </li></ol> Tahlil birleştirin <ol><li> Hücre nokta kuruduktan sonra, kapak kayma / conta / media / agaroz / hücre kompleksi (13 adım) contası ile slayt / conta kompleksi (adım 1) hizaya getirin ve yavaşça bir mühür formu ile birlikte basın. KRİTİK ADIM </li><li> Slayt yüzeyleri temizleyin ve Parafilm bıraktığı kalıntı kaldırmak için bir Kimwipe kayma kapsayacak. Tamamlanan test Şekil 1 benzer. </li><li> , 32 ° C&#39;de muhafaza ısıtılmış aşaması tamamlanan slayt kompleksi Place (slayt aşağı yukarı kayma kapak) yoğuşmaya oluşturan önlemek için (adım 15) hemen sonra aşağı silin. Varsa, yoğunlaşma oluşan görüntü elde etme yazılımı başlamadan önce birkaç saniye boyunca ısıtmalı sahnede slayt karmaşık bekletin. </li><li> (2X, 4X, 10X) uygun bir hedef seçin. </li></ol> Bölüm 5: Film Hazırlığı t &quot;&gt; KRİTİK ADIM <ol><li> Açın, fotoğraf makinesi ve mikroskop ve ışık düzeylerini kontrol (ışık çok yoğun olmadığından emin olun). Bilgisayarı başlatın ve görüntü elde etme programını açın. </li><li> Canlı görüntü penceresi tıklayın ve mikroskop odak. Görüntü Şekil 3&#39;te görüldüğü gibi görünmelidir. Düzgün agar yüzeyinden dikkat edin. </li><li> Görüntüler elde başlayın. KRİTİK ADIM </li><li> Medya / agar odak kayması neden yerleşmek eğilimi, ilk bir saat boyunca odağı düzenli olarak kontrol edin. </li><li> Çalışma alanını temiz. </li><li> Görüntü alımı tamamlandıktan sonra, transfer görüntüleri depolama için satın almış ve bir gecede% 90 etanol iliklerine slayt kompleksi yıkmak. </li><li> Yeniden Otoklav conta. </li></ol><img src="/files/ftp_upload/2038/2038fig1.jpg" alt="Şekil 1" /> Şekil 1 TM plaka kompleks Karikatür resimde. (A) patladı görünümü ve kesit temel TM plaka kompleksi gösterir. (B) büyük contaların kullanımını gösterir. <img src="/files/ftp_upload/2038/2038fig2.jpg" alt="Şekil 2" /> Şekil 2 Mikroskop küme. Her mikroskop düğüm (inset) Nikon E400 mikroskop, hedefleri, ısıtılmış bir aşamada, bir Insight kamera ve dizüstü bilgisayar oluşur. Her düğüm birlikte ağ ve bir ana denetleyici bilgisayara bağlı. Düğümlerin İki EXFO ışık kaynağı ve iki Uniblitz kepenkler oluşur floresan yetenekleri ile ayarlanır. <img src="/files/ftp_upload/2038/2038fig3.jpg" alt="Şekil 3" /> Şekil 3. 20X görüntü izleme analiz aygıtının bir anda = 0. Ölçeği bar, 1 mm. <img src="/files/ftp_upload/2038/2038fig4.jpg" alt="Şekil 4" /> Şekil 4 TM plaka kompleks Uyarlanabilirlik. (A) M. 100X görüntü % 1.0 agar CTTYE xanthus kayma motilite. (B) P. 20X görüntü aeruginosa motilite seğirmesi. (C) 20X görüntü S. marcescens motilite swarming. Her ikisi de (B) ve (C),% 1.0 agar LB ölçüldü. (D) M. 40X görüntü smegmatis% 0.5 agar LB motilite kayar. Bu görüntü Şekil 1B görülen alternatif tahlil yapılandırmayı kullanarak ele geçirildi. Video 1 izleme tahlil tabi bir sürüsü bir zaman atlamalı video. Bir M. Video 2. bir zaman atlamalı video xanthus sürüsü hücrelerin% 1 floresan ile işaretlenmiş. Alternatif faz kontrast ve floresan görüntüler yakalanır ve floresan hücrelerinin sürüsü içindeki konumunu aydınlatmak için üst üste. Bu video% 1.0 agar CTTYE üzerine yakalandı. M. Video 3. bir zaman atlamalı video xanthus kayma motilite. Bu video% 1.0 agar CTTYE üzerine yakalandı. Video 4 P. bir zaman atlamalı video aeruginosa motilite seğirmesi. Bu video% 1.0 agar LB yakalandı. Video 5 S. bir zaman atlamalı video marcescens motilite swarming. Bu video% 1.0 agar LB yakalandı. Video 6 M. A time-lapse video smegmatis motilite kayar. Bu video Şekil 1B görülen alternatif tahlil yapılandırmayı kullanarak% 0.5 agar LB yakalandı.

<ol>
	<li>Taylor, R. G., Welch, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+as+an+emergent+property+of+a+swarm.">Chemotaxis as an emergent property of a swarm.</a> J Bacteriol. 190, 6811-6816 (2008).</li><li>Curtis, P. D., Taylor, R. G., Welch, R. D., Shimkets, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Myxococcus+xanthus+During+Fruiting+Body+Formation.">Spatial Organization of Myxococcus xanthus During Fruiting Body Formation.</a> J Bacteriol. 189, 9126-9130 (2007).</li><li>Mignot, T., Merlie, J. P., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+pole-to-pole+oscillations+of+a+bacterial+gliding+motility+protein.">Regulated pole-to-pole oscillations of a bacterial gliding motility protein.</a> Science. 310, 855-857 (2005).</li><li>Stoodley, P., Hall-Stoodley, L., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+surface+migration,+and+other+dynamic+behavior+in+bacterial+biofilms+revealed+by+digital+time-lapse+imaging.">Detachment surface migration, and other dynamic behavior in bacterial biofilms revealed by digital time-lapse imaging.</a> Methods Enzymol. 337, 306-319 (2001).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu Rev Microbiol. 54, 49-79 (2000).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+and+twitching+motility+are+necessary+for+Pseudomonas+aeruginosa+biofilm+development.">Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.</a> Mol Microbiol. 30, 295-304 (1998).</li><li>Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substratum-induced+morphological+changes+in+a+marine+bacterium+and+their+relevance+to+biofilm+structure.">Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.</a> J Bacteriol. 176, 6900-6906 (1994).</li><li>Dworkin, M., Eide, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxococcus+xanthus+does+not+respond+chemotactically+to+moderate+concentration+gradients.">Myxococcus xanthus does not respond chemotactically to moderate concentration gradients.</a> J Bacteriol. 154, 437-442 (1983).</li><li>Dworkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactic+behavior+of+Myxococcus+xanthus.">Tactic behavior of Myxococcus xanthus.</a> J Bacteriol. 154, 452-459 (1983).</li><li>Wu, S. S., Kaiser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+expression+of+the+pilA+gene+in+Myxococcus+xanthus.">Regulation of expression of the pilA gene in Myxococcus xanthus.</a> J Bacteriol. 179, 7748-7758 (1997).</li><li>Bustamante, V. H., Mart&#237;nez-Flores, I., Vlamakis, H. C., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Frz+signal+transduction+system+of+Myxococcus+xanthus+shows+the+importance+of+the+conserved+C-terminal+region+of+the+cytoplasmic+chemoreceptor+FrzCD+in+sensing+signals.">Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals.</a> Mol Microbiol. 53, 1501-1513 (2004).</li></ol>

Time-lapse microcinematography (TM), prokaryotik motilite 2-7 okuyan bir standart yaklaşım haline gelmiştir . Geleneksel olarak, TM yüzeylerde 8-11 gibi filtre kağıdı fitil, ince agar pedleri, veya agar plaka kullanılarak yapılır. Bu yöntemler bakteri hareketin genel çizimler için görüntü dizileri oluşturmak için kullanıldığı zaman etkili, yeterli ve maliyet. Ancak, görüntü sıralarını tekrarlanabilir ve nicel titiz veri üretimi sonucu gerekiyorsa, bu yöntemlerin zaman ...

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		<th>Catalogue Number</th>
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<td>1.0% Casitone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.5% yeast extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Micro-sampling pipette</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>100 &mu;l glass disposable tip</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2 x 2 cm, 0.5-mm-thick silicone rubber gasket</td>
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<td>Grace Bio-Lab Inc.</td>
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recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography

Δ-proteobacterium Myxococcus xanthus sürüsü milyonlarca sinyalleri bir dizi çevresel uyaranlara yanıt olarak karmaşık, dinamik desenler oluşturmak için kolektif bir, koordine bir hareket olarak hareket hücre içerir . Bu modeller, kendini organize ve acil bireysel hücrelerin davranışlarını gözlemleyerek tahmin edilemez. Bir zaman atlamalı microcinematography izleme testi kullanılarak, M. ayrı bir acil desen tespit xanthus kaynak 1 doğru bir besin degrade kadar bir sürüsü yönlendirilmiş bir hareket olarak tanımlanan, kemotaksis olarak adlandırılır. Time-lapse microcinematography yoluyla kemotaksis verimli karakterize etmek için, bir çok değiştirilebilir plaka kompleksi geliştirdi (Şekil 1) ve 8 mikroskoplar (Şekil 2), zaman atlamalı videos yakalama yetenekli her bir küme inşa edilmiş. Testin ölçülebilir verilerin tutarlı çoğaltma yetecek kadar titiz, ve ortaya çıkan videolar ince sürüsü davranış değişiklikleri gözlemlemek ve izlemek için izin verir. Bir kez yakalanan, videolar, binlerce videoları işlemek ve depolamak için yeterli belleğe sahip bir analiz / depolama bilgisayara aktarılır. Bu kurulum esnekliği kanıtlanmıştır M. birkaç üyelerine faydalı xanthus toplum.

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Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Çalışmak için<em&gt; Myxococcus xanthus</em&gt; Sürüsü davranış, biz farklı deneyleri için modifiye edilebilir bir zaman atlamalı microcinematography protokol tasarladık. Mikroskopi için uygun standart büyüme istihdam ve ucuz, yeniden silikon conta kullanımı tekrarlanabilir sonuçlar verir. Biz çok hücreli kemotaksis ölçmek için bu yöntemi kullanmıştır.

Hücreli Davranış kaydetme Myxococcus xanthus</emZaman atlamalı Microcinematography kullanarak> Biyofilmler

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

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Biology

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

12.2K Görüntüleme.  University of South Carolina (USC). Çalışmak için Myxococcus xanthus Sürüsü davranış, biz farklı deneyleri için modifiye edilebilir bir zaman atlamalı microcinematography protokol tasarladık. Mikroskopi için uygun standart büyüme istihdam ve ucuz, yeniden silikon conta kullanımı tekrarlanabilir sonuçlar verir. Biz çok hücreli kemotaksis ölçmek için bu yöntemi kullanmıştır.

Video: Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

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.  ...

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 ...

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 ...

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 ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Tek ve Çok hücreli Bağlamlarda Invadopodia aracılı Ekstrasellüler Matriks Proteoliz Kantitatif Ölçümü

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

In Oksijen yetersizliği kaynaklı Asma Animasyon Visualize&#39;a Time Lapse Mikroskopi kullanın<em&gt; C. elegans</em&gt; Embriyolar

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Tomurcuklanma Maya Floresan Time-lapse Filmler Kazanılması ve GREFTLERİ kullanarak Tek hücreli Dynamics analiz

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

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

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

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, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Hücreli Davranış kaydetme Myxococcus xanthus Biyofilmler