Bu araştırma, Ulusal Bilim Vakfı, Nörobilim McKnight Endowment Fund, Christopher ve Dana Reeve Vakfı ve Amerisure Vakfı tarafından desteklenmiştir.

<ol>
	<li>Hammarlund, M., Nix, P., Hauth, L., Jorgensen, E. M., Bastiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+regeneration+requires+a+conserved+MAP+kinase+pathway.">Axon regeneration requires a conserved MAP kinase pathway.</a> Science. 323, 802-806 (2009).</li><li>Steinmeyer, J. 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S., Ma, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+cavitation+dynamics+during+pulsed+laser+microsurgery+in+vivo.">Plasma and cavitation dynamics during pulsed laser microsurgery in vivo.</a> Physical review letters. 99, 158104-158104 (2007).</li><li>Venugopalan, V., Guerra, A., Nahen, K., Vogel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laser-induced+plasma+formation+in+pulsed+cellular+microsurgery+and+micromanipulation.">Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation.</a> Physical review letters. 88, 078103-078103 (2002).</li><li>Bourgeois, F., Ben-Yakar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+nanoaxotomy+properties+and+their+effect+on+axonal+recovery+in+C.+elegans.">Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans.</a> Opt Express. 16, 5963-5963 (2008).</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), e1129-e1129 (2009).</li><li>Tsai, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-mediated+ablation:+an+optical+tool+for+submicrometer+surgery+on+neuronal+and+vascular+systems.">Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems.</a> Curr. Opin. Biotechnol. 20, 90-99 (2009).</li><li>Chung, S. H., Clark, D. A., Gabel, C. V., Mazur, E., Samuel, A. 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Çıkar çatışması ilan etti.

3,5-11 farklı lazer sistemleri ile lazer mikrocerrahi çeşitli tartışmalar vardır. Femtosecond kızılötesi lazerler, 12 ve rahat bir görüntüleme tesisi ile ilişkili hücre içi lazer ablasyon için &quot;altın standart&quot;, ama bireysel kullanıcılar için genellikle çok pahalı . Görüntüleme derinliği nedeniyle görüntüleme örnek için femtosecond kızılötesi lazer ihtiyacınız varsa o zaman muhtemelen bir lazer axotomy için ihtiyacınız olacak. Yüzeyinin 30-50 um içinde hedef aksonlar Şeffaf dokular muhtemelen yüksek na daldırma objektifi aracılığıyla hedeflenen 20 UJ / nabız aralığında, mavi ve yeşil darbe lazerler ile mümkün. Özellikle hedef akson derinliği artar olarak, femtosecond lazer ile karşılaştırıldığında nano ve pico ikinci lazerler daha çok hasar var. C. elegans şeffaf ve tüm aksonlar yüzeyinin 20-30 um içinde. Biz rutin yüzeyinin 5 um içinde motor aksonlar kesti. Ayrıca kolayca balta kestikmanikür yüzeyi yaklaşık 20 um sinir halkası içinde ons. Biz yetişkin bir solucan çapı ile yaklaşık 30-50 um aksonlar kesme sınırı bulundu. Burada açıklanan lazer ablasyon sistemi, şeffaflık ve derinliği hedef akson kriterlere uygun birçok farklı hazırlıklarını iyi iş olasıdır. Yine de, bu biraz şaşırtıcı IR femtosecond lazerler, nano ve pikosaniye 355 nm ve 532 nm lazerler teorik avantajları göz önüne alındığında, C. lazer axotomy için çok iyi bir performans elegans 6,13. Biz nanosaniyelik 440 nm, 532 nm nanosaniyelik ve femtosecond kızılötesi lazerler lazer axotomy yanıt akson rejenerasyonu açısından herhangi bir farklılık görüyoruz. Katı hal 355 nm UV lazerler 532 nm pasif Q-anahtarlı katı hal lazer pompalanır aynı maliyet ile ilgili, ancak bu kısa dalga boyuna verimli geçmesi ya da daha yüksek güçler veya optik gerektirir. Çoğu optik görünür 400 ile iyi bir performans için tasarlanmıştır.700 nm ışık. 355 nm lazerler 6 gibi düşük plazma eşik, küçük spot büyüklüğü, ve ablasyon lazer hedef% 100 verimli bir şekilde doğrudan ve örnek sinyal kaybı olmadan GFP eş zamanlı görüntüleme sağlayacak uzun bir pas dikroik gibi bazı avantajları sunacak. Mavi 440 nm lazerler, görünür ışık lazer ile (standart optikler ve güvenliği ile iyi bir performans) çalışma avantajlarını tutacak. Ne yazık ki, bir DPP-Q-anahtarlı katı hal 440 nm lazer maliyet, şu anda benzer bir güç 355nm veya 532 nm lazer 3 kez maliyetidir. C için yeni bir sistem tasarımı olsaydı elegans, hedef derinlik az olduğu, 1 UV şeffaf lens (355nm az% 50-70 geçirgenliği ile maliyet 40X 1.3na petrol lens için yaklaşık 3000 $) ve 355 nm lazer 5-10 UJ / darbe üreten bir tercih olur -20 kHz. Axon ablasyon plazma oluşumu nedeniyle olduğu düşünülmektedir. Kısa bakliyat, düşük güç ve plazma hacimleri w plazma eşikleri üretecek<html> hasta 6 adet daha küçük olabilir. Amaç, darbe enerjisi en düşük güç ayarlayarak küçük ve kısa ömürlü plazma üretmek için. Büyük uzun ömürlü plazmalar kavitasyon baloncukları üretecektir hücreleri çevreleyen zarar verebilir. Biz genelde, yüksek frekans (yani 2.5kHz) 50-100 hakkında bakliyat kullanarak o ablasyon bulduk en iyi sonucu verir. Bu hasar yavaş yavaş bakliyat hasarın boyutunu daha iyi kontrol etmek için bir dizi üzerinden entegre edilebilir göstermektedir. Demeti hizalı ve doğru genişletilmiş ise, burada açıklanan lazer ablasyon sistemi, çok bağışlayıcıdır. Biz,% 10 lazer gücü (ortalama 0.3 mW 2500 Hz&#39;de ölçülür ve tahmini 1 UJ / pulse) yetişkin solucan aksonlar kesme ve% 14 güç ile sınırlı lokalize hasar kesebilir. Hasar daha büyük alanlar% 15-39 lazer güç izleyebilirsiniz, ancak sadece% 40 güç (yaklaşık 1 mW ortalama 2500 Hz&#39;de ölçülen) Yukarıdaki solucanlar büyük kavitasyon baloncukları ve solucan manikür zarar nedeniyle patlayabilir. e_content &quot;&gt; Steinmeyer ve ark. 2010 2 kağıt, lazer ablasyon sistemi oluşturmak için mükemmel bir kaynak. Leon Avery de ucuz bir N2 gaz 337 nm lazer tabanlı bir lazer ablasyon sistemi güzel bir pratik tanımı ve bazı büyük pratik sağlar tavsiye (elegans.swmed.edu / Worm_labs / Avery /) kendi sistemi tasarlarken ilk mikroskop hedefi nasıl ablasyon lazer hedef belirlemek için mikroskop temsilcisi ile konuşmak gerekir. mikroskop bir titreşim izolasyonu masaya monte edilmelidir (Newport, TMI, Thor). breadboard en optimal düzeyde, ancak sağlam bir çelik üst hala manyetik konumlandırıcılı kullanılabilir tüm optik elemanlar yer bırakılmalıdır çünkü dik bir kapsamda özel bir ara modül güzel Ancak, daha az pahalı ve kamera bağlantı noktası (ters) veya epi-floresan bir bağlantı noktasına bağlı bir &quot;birleştirici&quot; kullanmak daha uygun olabilir (bir arka diyafram ve geçirgenliği boyutunu bilmek gerekir<html> blation lazer dalga boyu) objektif lens kullanmak niyetinde. Bir kez bir lazer (örneğin, Crylas, teem, Kristal, ya da CRC) Thor ya da Newport doğru optik elemanlar seçerek, donanım ve güvenlik donanımları ile ilgili yardım için başvurmalısınız karar verir. Biz yer kazanmak için bir çift Galile ışın genişletici karar verdi, ancak basit bir Kepler genişletici bir seçenek (f2/f1 = genişleme faktörü). Özel bir ışın genişletici en pahalı olacak, ama Newport, Thor, ve çeşitli lazer üreticileri mükemmel kalitede ticari ışın genişleticiler sunmaktadır. Bazı lazer üreticileri de uygun maliyetli ve yerden tasarruf edilebilir, manuel ve elektronik kontrollü zayıflatıcılar sunuyoruz, ancak Glan-Thompson polarize ve yarım dalga plakası gibi çok yönlü değil. Ayrıca lazer nasıl kontrol etmek için karar vermeniz gerekir. Lazer şirket tarafından sağlanan ticari bir TTL jeneratör, ya da yazılım kullanmak, bir LabView tabanlı denetleyici tasarımı. Özel lazer üreticisi ile seçenekleri tartışın. çadır &quot;&gt; Biz biz genel bir kılavuz olarak sadece kullanılan var donanımın listesini sağlanan var. Bu sistem bizim mevcut görüntüleme sistemi eklendi zaman hakkında ABD doları 15.000 maliyet burada anlatılan. Sizin yerel Fizik bölümü çoğu zaman için bir pratik giriş sağlamak için istekli birine sahip olacak serbest ışını lazerler ile güvenli bir şekilde çalışıyor. Immobilizasyon ve zaman atlamalı görüntüleme C için alternatif yöntemler elegans son zamanlarda tarif edilmiştir. 14 Sorun Giderme En zor ve kritik bir adım mikroskop optik eksen merkezli genişletilmiş ışın uyum. Bir kere ışın merkezi nihai uyum için direksiyon aynalar kullanarak ÖNCE 5 um mikroskop optik eksen içinde hizalanır almak için çok çalışması gerektiğini Galile lensler ile merkezli ve genişletilmiş var. Mikroskop ışını hizalamak için direksiyon aynalar aşırı kullanımı, ışın ile eksen hareket edecekgenişletici. EXTREME DİKKAT KULLANARAK uygun ND filtre ile lazer ışını zayıflatır ve doğrudan bir yansıtıcı hedef (ön yüzeyi ayna) lazer ışın profili görebilirsiniz. (Demiryolu yüksekliğini ayarlamak için yukarı / aşağı aralığı merkezli edilememesi halinde) tam merkezinde 60X objektif alanında kiriş mikroskop hafifçe dürtmek yapabilirsiniz. Işın profili tam ortalanmış bir dairesel kiriş ve simetrik &quot;işaret fişekleri&quot; vermek için mikroskop optik eksene hizalamak için kullanılabilir. Asimetrik bir parlama mikroskop ekseni mükemmel uyumlu değildir (ya da raylı düz değil) olduğunu bir göstergesidir. Son olarak, L3 ayarlamak ve kiriş ve hatta simetrik bir genişleme ve daralma görmek gerekir. Sonra lazer ışını küçük bir noktaya odaklanmak için L3 ayarlamak Odak mikroskop ve hedef yüzeyinde tam. Artık lazer ışını LSCM veya CCD sistemi üzerinden görüntülü ve ablasyon nokta içinde 5 um merkezinin içinde olduğunu bulmak gerekirZ odak 1um. Eğer solucanlar &quot;havaya uçurmak&quot; ya da sürekli sorun büyük olasılıkla ablasyon lazer ince ayara aksonlar kesmek için büyük kavitasyon baloncukları üreten. En az (&lt;500nm) ablasyon yerinde Z odaklama (L3 lens ile yakınsama ayarlamak) ve XY güvene dayalı spot (son kinematik monte ayna ile ayarlamak) lokalize emin olun. Yüzeye yakın aksonlar Hedefleme daha az lazer güç gerektiren. Benzer şekilde, küçük hayvanlar (L1 ve L2) yetişkinler de aynı aksonlar hedeflemesine göre axotomy için daha az lazer güç gerektirir. Vahşi tip aksonlar rejenere veya aksonları çamaşır suyu yoksa, görüntüleme lazer gücünü azaltarak veya örnekleme oranı azalan denemelisiniz. Solucanları ölmek ya da kalırsanız sickly 1% adımlarla yüzde Agaroz azaltmayı deneyin. Başına yüksek kullanırken bu genellikle onları ölüme neden olacak gibi montaj solucanlar sonra lamel hareket etmiyor emin olun.Burada belirtilmesi gereken agaroz ve mikroküreler. Solucanlar time-lapse oturumu sırasında patlaması ya da ölürseniz nedeniyle: 1. Yüzde agaroz çok yüksek. 2. Hasar ablasyon lazer tarafından kütikülü. 3. Monte edildikten sonra lamel Hareketi. Manikür zarar hata ise sadece ablasyon lazer hedef olmuştur monte solucanlar etkileyecektir. Solucan time-lapse oturumu sırasında çok fazla hareket ederse,% 0.5 &#39;lik adımlarla yüzde agaroz artırmayı deneyin. Sağlıklı solucanlar Sağlıklı aksonlar her zaman &quot;aktif&quot; ve floresans tutarlı bir seviye göstergesi. , Floresan ya da aktivite ani bir düşüş görürseniz solucan ölüyor ya da öldü. Çoklu boncuklu veya parçalanmış aksonlar, ölüyor ya da ölü bir solucanın bir işaretidir. Tüm zaman atlamalı oturumu sırasında odak aksonlar tutmak sorun varsa birkaç farklı sorunları olabilir. İlk 10 saat boyunca bir bardak slayt görüntüleme inert bir örnek sahne sürüklenme kontrolrs. Birkaç mikron sürüklenme termal istikrarsızlık nedeniyle olabilir, ancak muhtemelen 5&#39;inden um mikroskop ile mekanik sorunları nedeniyle. Montaj ile ilgili sorunlar daha sık görülür. Zaman atlamalı başlamadan önce monte solucanlar 30 dakika için mikroskop aşaması ile muvazene edelim. Vazelin mühür zaman atlamalı oturumu sırasında sızıntıları geliştirmek olmadığını kontrol edin.

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> 535 nm Lazer </td><td> Crylas </td><td> FDSS532Q3 </td><td> (Teem, Kristal ve CRC de karşılaştırılabilir lazerlere) </td></tr><tr><td> Tüm optik ve donanım </td><td></td><td></td><td> (Thor karşılaştırılabilir optik ve donanım sunuyor) </td></tr><tr><td> Suprema Optik Mount, çapı 1.0 </td><td> Newport </td><td> SS100-F2KN </td><td> 2 </td></tr><tr><td> 1 &quot;çap mesaj 1&quot; height </td><td> Newport </td><td> PS-1 </td><td> 1 </td></tr><tr><td> 1 &quot;çap yazılan çatal </td><td> Newport </td><td> PS-F </td><td> 1 </td></tr><tr><td> Sıfır Sipariş Akromatik Wavel Tabak, yarım dalga Retardasyon, 400-700nm </td><td> Newport </td><td> 10RP52-1 </td><td> 1 </td></tr><tr><td> Rotasyon Sahne, 1 &quot;Diyafram </td><td> Newport </td><td> RSP-1T </td><td> 1 </td></tr><tr><td> 1 &quot;çap mesaj 1&quot; height </td><td> Newport </td><td> PS-1 </td><td> 1 </td></tr><tr><td> 1 &quot;çap yazılan çatal </td><td> Newport </td><td> PS-F </td><td> 1 </td></tr><tr><td> Glan-Lazer Kalsit Polarize, 430-700nm </td><td> Newport </td><td> 10GL08AR.14 </td><td> 1 </td></tr><tr><td> Polarize Rotasyon Dağı </td><td> Newport </td><td> RM25A </td><td> 1 </td></tr><tr><td> Çapı yazılan ¼ 1 &quot;ayırıcı&quot; </td><td> Newport </td><td> PS-0.25 </td><td> 1 </td></tr><tr><td> ½ &quot;height, 1&quot; çap Mesaj </td><td> Newport </td><td> PS-0.5 </td><td> 1 </td></tr><tr><td> Çatal, 1 &quot;çap Mesaj </td><td> Newport </td><td> PS-F </td><td> 1 </td></tr><tr><td> Işın Dökümü </td><td> Newport </td><td> PL15 </td><td> 1 </td></tr><tr><td> Mikroskop Amaç Lens Bayoneti </td><td> Newport </td><td> LH-OBJ1 </td><td> 1 </td></tr><tr><td> 1 &quot;çap mesaj 1&quot; height </td><td> Newport </td><td> PS-1 </td><td> 1 </td></tr><tr><td> 1 &quot;çap yazılan çatal </td><td> Newport </td><td> PS-F </td><td> 1 </td></tr><tr><td> Yüksek Enerji Nd: YAG Lazer Ayna, 25,4 mm çap, 45 °, 532 nm </td><td> Newport </td><td> 10Q20HE.2 </td><td> 4 </td></tr><tr><td> Suprema Optik Mount, 1,0 inç çap, temizleme montaj deliği </td><td> Newport </td><td> SN100C-F </td><td> 2 </td></tr><tr><td> Yüksek Hassas Knob Ayar Vidası, 12.7mm seyahat, 100TPI </td><td> Newport </td><td> AJS100-0.5K </td><td> 4 </td></tr><tr><td> Konu adaptörü, ¼ -20 erkek, 8-32 kadın </td><td> Newport </td><td> SS-1-B </td><td> 2 </td></tr><tr><td> 1,5 inç çaplı çubuk için Rod Kelepçe </td><td> Newport </td><td> 340-RC </td><td> 2 </td></tr><tr><td> Çubuk, uzun boylu 14 inç (35,5 cm) </td><td> Newport </td><td> 40 </td&gt; <td> 1 </td></tr><tr><td> Raylı taşıyıcı X26, kare 40mm uzunlukta </td><td> Newport </td><td> CN26-40 </td><td> 4 </td></tr><tr><td> Çelik Ray, 384mm (15 &quot;) uzunluk </td><td> Newport </td><td> X26-384 </td><td> 1 </td></tr><tr><td> 1,5 inç çaplı çubuk Çubuk Platformu </td><td> Newport </td><td> 300-P </td><td> 2 </td></tr><tr><td> Çubuk, uzun boylu 14 inç (35,5 cm) </td><td> Newport </td><td> 40 </td><td> 2 </td></tr><tr><td> Plano-Konkav Lens, 12.7 mm çap, 25mm EFL, 430-700nm </td><td> Newport </td><td> KPC025AR.14 </td><td> 2 </td></tr><tr><td> Plano-Konveks Lens, 25,4 mm çap, 50.2mm EFL, 430-700nm </td><td> Newport </td><td> KPX082AR.14 </td><td> 1 </td></tr><tr><td> Plano-Konveks Lens, 25,4 mm çap, 62.9mm EFL, 430-700nm </td><td> Newport </td><td> KPX085AR.14 </td><td> 1 </td></tr><tr><td> Sabit Lens Mount, 0.5 &quot;çapında, 1.0&quot; Eksen yüksekliği </td><td> Newport </td><td> LH-0.5 </td><td> 2 </td></tr><tr><td> Lens Mount, 1.0 &quot;çapında, 1.0&quot; Eksen yüksekliği </td><td> Newport </td><td> LH-1 </td><td> 2 </td></tr><tr><td> Mikroküreler 0.1um </td><td> Polysciences </td><td> 00876 </td><td></td></tr><tr><td> Agaroz </td><td> RPI </td><td> A20090 </td><td> EEO konularda </td></tr><tr><td> Muscimol </td><td> Sigma </td><td> M1523 </td><td></td></tr></tbody></table>

constructing a low-budget laser axotomy system to study axon regeneration in c. elegans

Time-lapse mikroskobu ile takip Lazer axotomy C ile akson rejenerasyonu fenotipleri için hassas bir testtir elegans 1. Bu testte temel zorluk, bir lazer ablasyon sistemi 2,3 uygulanması için gerekli olan algılanan maliyet ($ 25-100K) ve teknik uzmanlık. Ancak, mütevazı maliyetleri (&lt;$ 10K) solid-state darbe lazerler lazer ablasyon hedef aksonlar doku yüzeyine &quot;yakın&quot; şeffaf hazırlıkları için sağlam bir performans sağlayabilir. İnşaat ve hizalama bir sistem, bir gün içinde yapılabilir. Odaklanmış kondenser ablasyon lazer ışığı tarafından sağlanan optik yol, uygun bir uyum kılavuzu sağlar. Kaldırılır tüm optik ile bir ara modül ablasyon lazer adanmış ve lazer ablasyon oturumu sırasında herhangi bir optik elemanlar hareket gerekirse sağlar. Ara modül dikroik, eş zamanlı görüntüleme ve lazer ablasyon sağlar. Lazer ışını t Merkezlemeo odaklı mikroskop yoğunlaştırıcı lens ışın sistemi ilk uyum yönlendirir. Lensler çeşitli durum ve seçilen objektif lens arka diyafram doldurmak için kullanılan lazer ışını genişletmek. Final ön yüzeyi aynalı cam slayt hedefi ile uyum ve test yapılır. Lazer gücü minimum boyut ablasyon spot (&lt;1um) vermek için ayarlanır. Ablasyon nokta son kinematik monte ayna görüntüleme penceresinde sabit çapraz kıllar ince ayar ile ortalanır. Lazer güç axotomy için minimum hedef slayt (Bu kullandığınız hedef değişebilir) ablasyon nokta için gerekli olandan daha yüksek yaklaşık 10X olacaktır. Worms agaroz pedleri (veya mikroakışkan odaları 4) montaj, lazer axotomy ve time-lapse görüntüleme için immobilize olabilir . Agaroz pedleri mikrodalga erimiş dengeli tuz çözeltisi içinde% 10 agaroz ile kolayca yapılır. Bir damla erimiş agaroz cam bir slayt üzerine yerleştirilen ve başka bir ile düzleştirilmişbir yastık içine cam slayt kalınlığında yaklaşık 200 um (komşu slaytlar zaman bant, tek katmanlı bir boşluk bırakan olarak kullanılır). &quot;Sharpie&quot; kap, 13mm üniformalı bir çaplı dairesel bir ped kesmek için kullanılır. Anestezik (1ul Muscimol 20mm) ve Mikroküreler (Chris Fang-Yen kişisel iletişim) (1ul 2.65% Polistiren 0,1 um su), sol tarafta yatan böylece odaklı 3-5 solucanlar tarafından takip pad merkezine eklenir. Bir cam lamel uygulanır ve daha sonra vazelin lamel mühür ve örnek buharlaşmasını önlemek için kullanılır.

Watch this Scientific Journal Video about Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans at JoVE.com

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Lazer axotomy time-lapse görüntüleme mutasyonların etkileri de tahlil için hassas bir şekilde<em&gt; C. elegans</em&gt; Akson rejenerasyonu. Yüksek kaliteli, ama ucuz, lazer ablasyon sistemi en mikroskoplar kolayca eklenebilir. Time lapse görüntüleme 15 saat boyunca, solucan dikkatli immobilizasyon gerektirir.

Axon Rejenerasyon Eğitim Düşük bütçeli Lazer Axotomy Sistemi oluşturulması C. elegans</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

constructing-low-budget-laser-axotomy-system-to-study-axon

Research

JoVE Journal

Bioengineering

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

15.4K Görüntüleme.  University of Utah. Lazer axotomy time-lapse görüntüleme mutasyonların etkileri de tahlil için hassas bir şekilde C. elegans Akson rejenerasyonu. Yüksek kaliteli, ama ucuz, lazer ablasyon sistemi en mikroskoplar kolayca eklenebilir. Time lapse görüntüleme 15 saat boyunca, solucan dikkatli immobilizasyon gerektirir.

Video: Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Biyomühendislik

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Fetal Akciğer Hücrelerinde Mechanotransduction Okumak Deneysel Bir Sistem

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Için basit Mikroakışkan Cihazlar<em&gt; In vivo</emVe&gt; Görüntüleme<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Ve Zebra balığı

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

Serbestçe Mikro-organizmalar Yüzme Kantitatif Locomotion Eğitim Lazer Kırınım kullanma

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

Lazer Bağlı Akson Lezyon sırasında Gerilim Film ölçümü Piconewton ve Milisaniye Çözünürlük de Yüzey için aksonal yapışma değerlendir

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Vasküler Doku Rejenerasyon için Mühendislik 3D cellularized Kollajen jeller

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Gradient Difüzyon Sistemi 3D Kollajen Matrix Kemotaksis Araştırma

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Tanımlı İnsan Engineered Kalp Dokuların inşaatı Kardiyak Hücre Tedavisinin Mekanizmaları Eğitim için

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Sıvılardaki Darbeli Lazer-Ablasyonuyla Metal Nanopartikül Üretimi: Nanopartiküllerin Antibakteriyel Özelliklerini İncelemek İçin Bir Alet

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Astrocyte ağlar gelişimsel mekanizmaları özetlemek ve sinir sistemi yeniden oluşturma işlemi kolaylaştırmak için tasarlanmış üç boyutlu doku uyumlu

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...