This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1. Numune Hazırlama <ol><li> % 10 fetal sığır serumu ve penisilin streptomisin 50 ng / ml, 100 IU / ml ile takviye edilen DMEM kullanılarak doku kültürü şişelerinde CHOK1 hücreleri korumak. 37 ° C sıcaklıkta 5% CO2 nemlendirilmiş inkübatör hücreleri inkübe edin. </li><li> Hasat daha sonra 0.16 mm kadar bir kalınlığı olan bir yüzey 14 mm çaplı mikro çukuruna CHOK1 hücreleri ve plaka. 60-70% konfluansa çevresinde görüntüleme için en uygun yoğunluk için, tohum hücreleri. </li><li> 37 ° C&#39;de gece boyunca inkübe hücreleri,% 5 CO2. Yeşil ve tübülin izci yeşil 150 nM Lysotracker DND26 50 nM ihtiva eden bir çözelti içinde hücreleri HBSS (Hank Tamponlu Tuz Çözeltisi) içindeki hücreleri üç kez yıkayın ve inkübe edilir. 37 ° C&#39;de 1 saat boyunca hücrelerin inkübe edin. İsteğe bağlı adım: kuluçkalamadan önce, bir mitokondrial matris boyama boyasının (25 nM) ilave bir leke gerçekleştirir. </li><li> Herhangi bağlanmamış boya kaldırmak için HBSS&#39;de hücreleri üç kez yıkayın. Imag öncesinde taze DMEM büyüme ortamı ilave edinhücreleri ing. </li></ol> 2. Mikroskop Yapılandırmaları Video protokolde tarif parçacık takibi için mikroskop, bir ticari olarak temin edilebilir, ters çevrilmiş mikroskop (Şekil 4) şasisi üzerine monte edilir. 3D yörünge parçacık izleme Ancak ticari modülleri artık mevcuttur. <ol><li> 690 nm-1040 nm arasında ayarlanabilir bir çıkış dalga boyu aralığında, Sa femtosaniye lazer uyarım ışık kaynağı: Tutarlı Bir Bukalemun-Ultra II Ti kullanın. </li><li> Lazer ışını IR-kaplı aynalar kullanılarak mikroskop arka limanında hizalanmış olduğundan emin olun. Lazer ışını, bir kalsit doğrusal polarizörün ardından dönen yarım dalga plakası kullanılarak azaltılır. Örnek olarak, ortalama güç 0.5 mW 2 arasında olacak şekilde ışını zayıflatır. NOT: Lazer ışını, örnek düzlemde odaklanmış demetin konumunda ve yörüngesini kontrol etmek için kullanılır galvano-motor tahrikli bir çift ayna tarafından yansıtılır. Birkısa geçişli renkli aynadan yansıması sonrasında mikroskop objektif arka girmeden önce, bir ışın genişletici geçen galvanometre aynalar çıkan dengesi ayarlanmış lazer ışını genişletilmiş tipik yapılandırma (10X). </li><li> Işık yoluna yüksek sayısal açıklık su hedef (60X NA 1.2) yerleştirerek numuneden floresan ışığı toplamak. Bir ya da iki kanal konfigürasyonunda, istenen emisyon dalga boyuna göre, bir floresan filtre küpü seçin. Ayrıca yayılan floresan spektral aralığını seçmek için emisyon band geçiren filtreler kullanır. </li><li> Motorlu bir sahneye örnek yerleştirin ve objektif bir piezo-kumandayı kullanarak mikroskop objektif ince hareketini ayarlayın. </li><li> Sinyal tefrik ve dijital I / O veri toplama kartı gönderilir photomultiplier tüpler içine filtre küp ışık yönlendirin. NOT: Bilgisayar dalga I / O kartının analog çıkış ve provid vardır oluşturulantarayıcılar için ed elektronik kontrol. Fotomultiplikatörler tarayıcılar için çıkış sinyalinden foton sayma girişi ölçülür ve Floresan Dynamics SimFCS yazılımı Laboratuvarı tarafından I / O kartı üzerinden kontrol edilir. </li></ol> 3. Görüntüleme <ol><li> Sahnede hücreleri yerleştirin ve iletilen ışık aydınlatma kullanarak odaklanın. </li><li> Boya dahil olan hücreleri tespit etmek ve altında yatan mikrotübülleri görselleştirmek için raster tarama görüntüleme geçin. Vezikül hareketi varsa ilk alan tanımlayın. </li><li> Izole bir kese tespit edildikten sonra, yörünge takibi için aşağıdaki parametreleri ayarlayın: <ol><li> Parçacığın boyutu izleniyor göre dairesel tarama boyutunu tanımlamak için yörüngenin yarıçapını seçin. Bir nokta Verici için, duyarlılık ve tepki maksimize etmek uyarma kirişin Noktası Yayılması Fonksiyonu (PSF) bel eşit yörüngenin yarıçapı ayarlayın. </li><li> Eksenel ayarlamaeksenel yön boyunca parçacık konumunun belirlenmesi için gerçekleştirilen iki yörünge arasındaki mesafeyi belirlemek için mesafe. 1,5-3 kat PSF bel eksenel mesafeyi ayarlayın. </li><li> Ayrıca fotoğraf ağartmak oranını belirleyecek yörüngesinin her noktasında harcanan zamanı ayarlamak için parçacığın parlaklığına göre bekleme süresini tanımlayın. 10-100 mikro-saniye arasında bir bekleme süresi kullanın. </li><li> 4-32 msn sırayla yörünge dönemleri verim ve parçacık konumunu belirlemek için bir yüksek temporal çözünürlük sağlamak için 64 veya 128, her yörüngede noktalarının sayısını ayarlayın. </li></ol></li><li> DC raster taranan görüntüde bir imleç seçim yoluyla grafiksel kullanıcı arabirimi yoluyla parçacık üzerindeki kiriş ortalamak için aynalara gönderilen ofset sinyali değiştirin. </li><li> Yörünge tarama tarama-görüntüleme gelen mikroskop moduna geçerek izlemeye başlayabilirsiniz. </li><li> Geribildirim ve veri toplama etkinleştirin. </li></ol> 4. TrajectORY Analizi <ol><li> Yörünge boyunca ve bir &quot;yoğunluk halı&quot; şeklinde, her bir zaman noktasında her bir noktada toplanan floresan görüntülemek için yazılımı kullanın. Halının boyunca alınmış yoğunluğu başka parlak nesneler ile parçacığın etkileşimi hakkında bilgi sağlar. Hem de zaman serisi şeklinde zaman foto-çoğaltıcı tüp toplanan floresan yoğunluğu (x, y ve z tarayıcılar -piezo arasında zamanla DC yerinden yani) yörünge bilgileri hem görüntülemek için yazılımı kullanın. </li><li> Yörünge ilgi sadece bir bölgeyi seçmek için zaman serisi temsil ve &#39;şiddeti halı&#39; bilgileri kullanın. </li><li> Partikül yörünge seçilmiş bir bölümünün 2D çıkıntısının görüntülemek için yazılımı kullanın. Parçacık floresan yoğunluğuna göre renk kodu yörüngesini uygun kontrolleri seçiniz. </li><li> S görüntülemek için seçeneğini seçinfloresan yoğunluğuna göre 3D makale yörünge ve renk-kod. Mikrotubullerin boyunca lizozom hareket özelliklerini görselleştirmek için denetimlerini kullanarak 3D yörünge döndürün. </li></ol>

<ol>
	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <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+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. C., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+nanometer+images+of+biological+fibers+by+directed+motion+of+gold+nanoparticles.">3D nanometer images of biological fibers by directed motion of gold nanoparticles.</a> Nano Lett. 11, 4656-4660 (2011).</li><li>Kis-Petikova, K., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+measurement+by+circular+scanning+of+the+excitation+beam+in+the+two+photon+microscope.">Distance measurement by circular scanning of the excitation beam in the two photon microscope.</a> Microscopy Research and Technique. 63, 34-49 (2004).</li><li>Levi, V., Ruan, Q., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+particle+tracking+in+a+two+photon+microscope+application+to+the+study+of+molecular+dynamics+in+cells.">3 D particle tracking in a two photon microscope application to the study of molecular dynamics in cells.</a> Biophys J. 88, 2919-2928 (2005).</li><li>Lund, F. W., Wustner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+single+particle+tracking+and+temporal+image+correlation+spectroscopy+for+quantitative+analysis+of+endosome+motility.">A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility.</a> Journal of Microscopy. 252, 169-188 (2013).</li><li>Nath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+Neurodegenerative+Pathology+via+Neuron+to+Neuron+Transmission+of+beta+Amyloid.">Spreading of Neurodegenerative Pathology via Neuron to Neuron Transmission of beta Amyloid.</a> Journal of Neuroscience. 32, 8767-8777 (2012).</li><li>Balint, S., Vilanova, I. V., Alvarez, A. S., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+live+cell+and+superresolution+microscopy+reveals+cargo+transport+dynamics+at+microtubule+intersections.">Correlative live cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 3375-3380 (2013).</li><li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> Embo Journal. 30, 3481-3500 (2011).</li><li>Ragan, T., So, P. T., Kwon, H. S., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+particle+tracking+on+the+two+photon+microscope.">3D particle tracking on the two photon microscope.</a> Multiphoton Microscopy in the Biomedical Sciences. 2, 247-258 (2001).</li><li>Konig, K., So, P. T. C., Mantulin, W. W., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+near-infrared+femtosecond+laser+pulses+in+two+photon+microscopes.">Cellular response to near-infrared femtosecond laser pulses in two photon microscopes.</a> Optics Letters. 22, 135-136 (1997).</li><li>Caviston, J. P., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+motors+at+the+intersection+of+trafficking+and+transport.">Microtubule motors at the intersection of trafficking and transport.</a> Trends Cell Biol. 16, 530-537 (2006).</li><li>Cardarelli, F., Lanzano, L., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+directed+molecular+motion+in+the+nuclear+pore+complex+of+live+cells.">Capturing directed molecular motion in the nuclear pore complex of live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 9863-9868 (2012).</li></ol>

The authors have nothing to disclose.

Yüksek zamansal çözünürlüğe sahip üç boyutlu floresan parçacıkların yörüngeleri elde son yıllarda floresan mikroskopi teknikleri ve instrumentations, muazzam gelişmelere rağmen alanında bir meydan okuma kalmıştır. Yüksek zamansal çözünürlüğü, iki boyutta izleme parçacıkları elde edilmişse, eksenel yöne uzantısı genellikle, sistemin 10 bir frekans yanıtı şiddetli bir azalma getirir. Bu video protokolde iki foton mikroskop kullanılarak yüksek...

Yaklaşımlar çok sayıda, bir mikroskop kullanılarak üç boyutlu olarak floresan parçacıkların izlemek için bugüne kadar geliştirilmiştir. En ideal yaklaşımlar, tipik olarak, eksenel yönde izleme elde etmek için mikroskop emisyon optik özel modifikasyonları ile birlikte, iki boyutlu izlemek için uygun, hızlı kameraların kullanımına dayanmaktadır. Bu işlem, uygun bir zaman çözünürlüğü (10 Hz), tipik olarak zaman alıcı ve verir, ancak geleneksel bir partiküldür izlenen halinde, Z yığınlarının bir zaman dizisi gerçekleştirerek, floresan partikül izleyebilir (konfokal ya da iki ya da foton) lazer tarama mikroskobu Etkin bir geri besleme mekanizması 2 ile küçük bir raster görüntüleme bölgenin merkezinde tuttu. Kilitleme-in parçacığa fikri yörünge izleme yönteminin temelidir. Bunun yerine, bir raster dairesel bir yörünge floresan de yaklaşık tarama gerçekleştirilir. Boyunca floresans yoğunluğuyörünge tam parçacık pozisyonunu 4 lokalize. Parçacığın lokalize konumu daha sonra mikroskop tarayıcıları ve yeniden merkezi partikül yörünge konumu üzerinde harekete geçirmek için kullanılabilir. Mikroskop galvanometre tarayıcılar bir analog voltaj ile tahrik edilmektedir. Bu gerilimin AC bileşeni, bir sinüs ve dairesel bir yörünge performans sağlayacak X ve Y, tarama ayna uygulanan bir kosinüs dalgası, yani, odaklı lazer ışını ile bir yörüngeye da yapılabilir. Sinyalinin offset DC yörünge merkezinin pozisyonunu değiştirerek kolaylaştırır. Bir yörünge süresi belirlenir ve AC sinyali için dalga yapılandırıldıktan sonra, geri besleme sistemi bir sinyalin DC bileşenini güncellemek gerekir. Dedektörler toplanan floresan sinyalini okumak mümkün bir yazılım DC kapalı, tarayıcılar ve dedektörleri kontrol etmek parçacığın konumunu hesaplamak için ve güncelleme gereklidirayarlayın. Yörünge parametrelerine (boyut ve zamanlama) dikkatli bir seçim görüntüleme başarılı bir geribildirim için gerekli ve bu parametreler o izlemek için gerekli olan floresan parçacıkların özelliklerine dayalı ayarlanması gerekiyor edilir. Tekniğinin fiziksel ve matematiksel temelleri 2D ve 3D uygulamaları için geçmiş 4,5&#39;den tarif edilmiştir. Bu protokolde kısaca kurulum ana donanım bileşenlerini tanımlamak ve daha ayrıntılı parametrelerin seçimi ve numune hazırlama canlı bir hücrenin içinde yüksek bir zamansal çözünürlükte bir lizozomun yerinden takip etmemizi sağlayan tipik bir deney için gerekli.

<table border="0" cellpadding="0" cellspacing="0" width="525">
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			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
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3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

Bu Video protokolün amacı, bir modifiye iki-foton mikroskop 1 kullanarak üç boyutlu floresan yörünge parçacık izleme deneyi gerçekleştirmek ve analiz nasıl tartışmaktır. Geleneksel yaklaşımlar (raster tarama veya kare bir yığın tabanlı geniş alan) aksine, 3D yörünge izleme lokalize ve yüksek uzaysal (10 nm doğruluk) ve temporal çözünürlük (50 Hz frekans tepkisi) 3D deplasman ile takip sağlar mikron 2 yüzlerce uzunluğu-ölçeklerde hareketli bir floresan parçacık. Bu yöntem takip edilecek nesnenin etrafında dairesel bir yörünge gerçekleştirmek için bir iki-fotonlu lazer tarama mikroskobu donanım kontrol eden bir geri besleme algoritmasına dayanır: geri besleme mekanizması yer değiştirme kontrol ederek merkezinde floresan nesne koruyacaktır tarama ışın 3-5. Bu tekniğin avantajlarını göstermek için, biz al içinde hızlı bir hareket organelini, lizozom, takipiving hücre 6,7. Hücreler, standart protokollere göre kaplama, ve bir ticari olarak lizozom boya ile boyandı. Biz yaşayan hücrelerin içinde bir 3D yörünge izleme deneyi gerçekleştirmek için, kısaca donanım yapılandırması ve daha detaylı kontrol yazılımı tartışmak. Ayrıntılı olarak tarama mikroskobu kontrol ve parçacık etrafında kapalı bir yörüngede kirişin hareketi mümkün kılmak için gerekli parametreleri tartışırlar. Bu yöntem etkili bir şekilde canlı hücre içinde 3D mikrotübüller boyunca bir etiketli lizozom hızlı hareketi izlemek için nasıl kullanılabileceğini gösteren sonucuna varmaktadırlar. Lizozomlar tipik olarak mikrotübül ağı 8 boyunca yönlendirilmiş hareketini görüntüleyen, 0.4-0.5 mm / sn aralığında hızlarla hareket edebilir.

Bu protokole göre hızlı 3D tek parçacık izleme floresan etiketli lizozomların değiştirmesini izlemek için değiştirilmiş iki-fotonlu mikroskop kullanılarak canlı hücreler içinde gerçekleştirilebilir. Yapılan deney endozom olgunlaştırma işlemi 9 sonra hücre içinde hareket eden, izole edilmiş bir lizozom izleme oluşur. Lizozomlar floresan yeşil boya kullanılarak boyandı ve 2-foton uyarma istismar 930 nm&#39;de heyecanlandırdı. Bizim veriler 32 milisaniye (Şekil 1a)</strong...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Modifiye İki foton mikroskop 3D Orbital Takip: Hücre içi veziküllerin İzlemeye Bir Uygulama

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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Research

JoVE Journal

Bioengineering

10.1K Görüntüleme.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

Video: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Encapsulated Hücre Agregalar içinde Takip Hipoksik Sinyalizasyonu

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Biyomühendislik

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Partikül izleme Microrheology kullanarak 3D Tümör Modelleri Ekstrasellüler Matriks Rijitliğinin Boyuna Ölçümü

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Parçacık izleme Microrheology tarafından Biyofilmler Mekanik Özelliklerinin Yerinde Haritalama

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Bir Mikro Kuyruklu Dizi Platformunda Mikrobik Topluluk Geliştirmenin Montajı ve Takibi

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Gerçek zamanlı 3D tek parçacık izleme için bir iletişim kuralı

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Memeli hücre kültür Tekdüzen toplamları üretmek için O şeklindeki damarlarının sallayarak bir Orbital

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Çoklu görüntüleme modları bir floresan mikroskop ile iletken

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Miyokard Enfarktüsü'nün Murine Modelinde 3Boyutlu Biyobaskılı Yama Transplantasyonu

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.