Name: fluorescence imaging with one-nanometer accuracy (fiona)
Uploaded: 2014-09-26T15:00:00
Description: Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

Bu çalışma NIH Hibe 068.625, NSF Bağış 1063188 ve 0822613. Özel teşekkür tavşan gözlerinde hediye için İleri Bilim ve Teknoloji Beckman Enstitüsü&#39;nün Dr Marina Marjanovic gitmek Yaşayan Hücrelerinin Fizik Merkezi tarafından desteklenmiştir.

Etik Açıklama: tavşanlardan Kornea dokusu Illinois Kurumsal Hayvan Bakımı ve Kullanımı kılavuzların Üniversitesi doğrultusunda toplanmıştır. 1. TIRFM Kur NOT: lazer güvenlik gözlük Tüm zaman giyin. <ol><li> Malzeme Listesi&#39;nde yer gerekli tüm optik bileşenleri mevcuttur ve uyum için hazır olduğundan emin olun. Gerekirse, diğer şirketlerden eşdeğer fonksiyonları ile değiştirmeleri kullanın. Aynalar ve lensler kullanımı lazer ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics international. 11 (7), 36-42 (2004).</li><li>Enderlein, J., Toprak, E., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+effect+on+position+accuracy+of+fluorophore+localization.">Polarization effect on position accuracy of fluorophore localization.</a> Optics Express. 14 (18), 8111-8120 (2006).</li><li>Cheezum, M. K., Walker, W. F., Guilford, W. 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Fiona 1 msn 4 8 aşağı nanometre hassas ve zamansal çözünürlüğe sahip bir floresan yayıcı (organik flüorofor ya da kuantum nokta) konumunu lokalize bir tekniktir. Yeterli fotonlar toplandığı zaman, bu tekniğin çok daha doğru kırılma sınırı (~ 200 nm) daha floresan emitörün konumunu belirlemek için izin verir ve bu nedenle bu tekniği geleneksel / konvansiyonel optik mikroskopi 4 ile görülmemiştir ne gözlemlemek için bir yol açar - 8. Onun icadından bu yana...

1882 civarında, Ernst Abbe bir görünür ışık mikroskobu çözünürlüğü olduğu bulundu ~ λ / 2NA, (λ dalga boyu ve NA sayısal açıklık olduğu) 1,2 ~ 200 nm veya. Bu nedenle, bu boyutundan daha küçük bir amacı, bir optik mikroskop bir kırınım sınırlı bir leke olarak görülecektir. Bununla birlikte, daha yüksek bir hassasiyet ile 3, bir nokta merkezini, nesnenin konumunu belirlemek mümkündür. Bir nanometre doğruluk (Fiona) ile floresan görüntüleme xy düzleminde 4 nanometre hassasiyetle tek florosforlar lokalize için basit ama kullanışlı bir tekniktir. Lokalizasyon hassasiyet, σ μ (yani, ortalamanın standart hatası) toplandı, fotonların sayısına bağlıdır <img alt="Denklem 1" fo:content-width="1in" src="/files/ftp_upload/51774/intro_eqn.jpg" width="100" /> N foton sayısı olduğu, s floresan noktanın standart sapmasıdır, a,piksel görüntü detektörünün büyüklüğü, ve b, 3,4 arka standart sapmasıdır. ~ 10.000 fotonları yayan bir flüorofora için, Fiona ~ 1 nm hassasiyet 4 elde edebilirsiniz. FIONA hassas bir şekilde sabit bir yayıcı konumunu, ya da (yeterince hızlı alınabilir görüntü varsayılarak) hareketli bir belirlemek için kullanılabilir. Fiona filmin kareleri sırayla uygulanan ve böylece tek bir molekülün 4 8 hareketini takip edilebilir. Foto-koruyucu reaktifler örnek ışıkla olmamasını sağlamak için gerekli olabilir. Ayrıca, flüoresan nesnenin kendisi herhangi bir boyutta olabilir, örneğin kısıtlayıcıları kırınım daha küçük ya da daha büyük, kendi zar üzerinde dağıtılmış çok sayıda floresan proteinleri ile bir organel (~ 1 mm) teşekkül edebilir. Fiona hala ortalama merkezden-of-kütlesinin çok doğru (nanometre) ortalama verim kullanma. FIONA tarafından yerelleştirme hassasiyetle büyük bir gelişme nanome çözme sağlarzaman içinde tert-ölçekli hareketleri. Bu moleküler uzunluk ölçüsü 4 8 içine mikroskopi itti. Onun icadından bu yana, Fiona çeşidi geliştirilmiştir. Örneğin, parlak-alan bir-nanometre doğruluk (bFIONA) 9, FIONA hafif bir varyantı, görüntüleri ile görüntüleme ve iletilen ışık ile böyle Melanozomlar in vivo (pigment melanin içeren koyu nesneler) gibi yoğun nesneleri lokalize. Buna ek olarak, birden fazla boyayı Fiona gidermek için kullanılmıştır. Örneğin, ışıkla ağartma (karides) ile birlikte tek bir molekül yüksek çözünürlüklü bir 10,11 veya tek molekül yüksek çözünürlüklü ko (SHREC) 12 yaklaşık 10 nm olan, iki boya çözmek için geliştirilmiştir. (Bu kararın bu bir arayla aynı boyaları söyleyebilirim ne kadar doğru, yani dikkat edin.) Daha yakın zamanlarda, Fiona analizi bazı süper çözünürlük mikroskopi yerelleştirme sürecine katkıda böyle Stokastik optik reco gibinstruction mikroskobu (STORM) 13-15 ve geçici koyu flüoroforlar heyecan ve floresan lokalize edildiği fotoaktif localisation mikroskobu (PALM) 16,. Defalarca heyecanlı oldukça düşük boyaların yoğunluğu (az bir başına kırılma sınırlı nokta), ve daha sonra FIONA her bunların analiz, floresan toplayarak, tek bir yüksek çözünürlüklü haritayı kurabilirsiniz. Çözünürlüğü sonra sadece her bir boya dışarı koyar fotonların sayısı, yanı sıra satın alma sırasında (örneğin dahil, mikroskop sahne) numune sabit tutmak gibi şeyler sınırlıdır. Bu makalede, bir Fiona tekniğin bir özeti ve kısaca Fiona bildirilmektedir kullanılarak yapılmıştır araştırma örneklerini tarif eder. İlk olarak, nasıl optik hizalama hakkında ayrıntılar yani Fiona deneylerde, toplam iç yansıma floresan mikroskobu (TIRFM) için gerekli ekipman kurmak, tarif edilir. Sonra nasıluygun protokolleri kullanarak hareketsiz Cy3-DNA tek moleküllerini lokalize basit Fiona deneyi yürütmek, gösterilmektedir. Bundan sonra, Fiona&#39;yla kullanımı kuantum nokta ile işaretlenmiş tek bir tepe bölümü kesik miyozin Va motorun 36 nm aşaması boyutunu ölçmek için sunulmuştur. Miyozin Va aktin filamentler boyunca translocating Hücresel kargo taşıyan bir temel süreçsel motoru proteindir. Burada Va kesik oluşturmak miyozin adım boyutuyla ilgisiz etki ayrılması için kullanılan ve bir FLAG etiketi C-terminaline ilave Anti-Flag antikorları ile işlevselleştirilmiş kuantum noktaları ile etiketleme kolaylığı sağlamak için. Bu deney miyozini yavaşlatmak ve her karede iyi bir foton sayısı almak için yeterince uzun pozlama süreleri kullanımına izin düşük ATP altında yapılır. Herhangi yeterince parlak floresan etiket aşağıdaki protokolde ikame edilebilir. Son olarak, kalın örnekleri FIONA uygulamasını uzanan son çaba bildirdi. Proof-of-prensip olarak, kuantum noktalar batırılmışSol-jel ve tavşan gözü kornealar ve sonra yansıması ve FIONA&#39;sı kullanılarak lokalize. Görüntüleme için, NA ile 60X su daldırma objektif bu hedefi daha önceden kullanılan 100X immersiyon yağı hedefi daha uzun çalışma mesafesi vardır, çünkü 1.2 kullanıldı =. Objektifine büyütmede kaybını telafi etmek için fazladan bir büyütme merceği (3.3X veya 4.0x) emisyon yoluna yerleştirilen edilmiştir. Buna ek olarak, epi-floresans (değil TIR) mikroskopisi kalın örneklerde derin bölgeleri ulaşmak için kullanılması gerekir. Bu sol-jel ve 2-3 nm hassasiyetle lokalize edilebilir tavşan gözü kornealar (Z&gt; 200 mikron) derin batırılmış kuantum noktalar gösterilmiştir.

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			<td style="width:211px;">Company</td>
			<td style="width:211px;">Catalog Number</td>
			<td style="width:443px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:220px;">Double-sided tape</td>
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			<td style="width:211px;">--</td>
			<td>~ 75 um thick</td>
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			<td height="21" style="height:21px;width:220px;">Fluorescence filter set</td>
			<td style="width:211px;">Chroma</td>
			<td style="width:211px;">49016</td>
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			<td height="21" style="height:21px;width:220px;">Actin polymerization buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA02</td>
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			<td height="21" style="height:21px;width:220px;">Biotin G-actin</td>
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			<td style="width:211px;">AB07</td>
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			<td height="21" style="height:21px;width:220px;">G-actin&#160;</td>
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			<td style="width:211px;">AKL95</td>
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			<td height="21" style="height:21px;width:220px;">General actin buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA01</td>
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			<td height="21" style="height:21px;width:220px;">Laser shutter (with driver)</td>
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			<td style="width:211px;">SH-10-MP</td>
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			<td style="width:211px;">--</td>
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			<td style="width:211px;">PI-31000</td>
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			<td height="21" style="height:21px;width:220px;">Coverslip</td>
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			<td style="width:211px;">22X30-1.5</td>
			<td>0.16-0.19 mm thick</td>
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			<td height="21" style="height:21px;width:220px;">Microscope slide</td>
			<td style="width:211px;">Gold Seal Microslides&#160;</td>
			<td style="width:211px;">30103X1</td>
			<td>0.93-1.05 mm thick</td>
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			<td height="21" style="height:21px;width:220px;">Glass bottom dish&#160;</td>
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			<td style="width:211px;">D35-20-1.5-N</td>
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			<td height="21" style="height:21px;width:220px;">Cy3-DNA oligos</td>
			<td style="width:211px;">Integrated DNA Technologies</td>
			<td style="width:211px;">--</td>
			<td>5&#39;-Cy3/GCCTCGCTGCCGTCGCCA-3&#39;Bio</td>
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			<td height="21" style="height:21px;width:220px;">Fluorescent beads&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">T-7280</td>
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			<td height="21" style="height:21px;width:220px;">Qdot 605-streptavidin&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q10101MP</td>
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			<td height="21" style="height:21px;width:220px;">Qdot605&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q21301MP</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;">Qdot705</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22021MP&#160;</td>
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			<td height="42" style="height:42px;width:220px;">Qdot705 Antibody Conjugation Kit</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22061MP</td>
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			<td height="21" style="height:21px;width:220px;">Matlab</td>
			<td style="width:211px;">MathWorks</td>
			<td style="width:211px;">--</td>
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			<td height="21" style="height:21px;width:220px;">Optical table</td>
			<td style="width:211px;">Newport Corp</td>
			<td style="width:211px;">--</td>
			<td>RS4000 Series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:211px;">Plan Apo VC 60x WI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">100X Objective</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">PlanApo 100X/1.45 Oil &#8734;/0.17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">UPlanApo 60X/1.20W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Inverted microscope</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">IX71/IX70/IX81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Origin</td>
			<td style="width:211px;">OriginLab</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Anti-FLAG antibody</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">F7425-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ATP</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A7699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BME&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">63689-25ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BSA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BSA-biotin</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A8549-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">CK</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">C3755</td>
			<td>Creatine Phosphokinase from rabbit muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">CP</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P1937</td>
			<td>Phosphocreatine di(tris) salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">DTT</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">43815</td>
			<td>DL-Dithiothreitol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">EGTA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">E3889</td>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">HCl&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">93363-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">HEPES&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">KCl</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MgCl2</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">NaCl</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">PCA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">03930590</td>
			<td>Protocatechuic acid&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">PCD</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P8279</td>
			<td>Protocatechuate-3,4-dioxygenase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">TMOS</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">341436-25G</td>
			<td>Tetramethyl orthosilicate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Tris-HCl&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">93363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Trolox</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">238813</td>
			<td>6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">1&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB1-E02, KM100</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">&#189;&#8221; diameter posts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">TR4</td>
			<td>Quantity &#8805; 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">10x beam expander</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BE10M-A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB2-E02, KM200</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter f = 300 mm lens with mount</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LA1256-A, LMR2</td>
			<td>TIR lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescent alignment target&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">VRC2SM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser safety goggles</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LG3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ND filter(s)</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">FW1AND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Optical beam profiler</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BP209-VIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Post-mounted iris diaphragm</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">ID25</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Shearing interferometer</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">SI100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">XYZ translation stage, &#189;&#8221; travel&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">T12XYZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser</td>
			<td style="width:211px;">World Star Technologies&#160;</td>
			<td style="width:211px;">TECGL-30</td>
			<td>532 nm, 30 mW</td>
		</tr>
	</tbody>
</table>

fluorescence imaging with one-nanometer accuracy (fiona)

Bir nanometre doğruluk (Fiona) ile floresan görüntüleme xy düzleminde nanometre hassasiyetle tek florosforlar lokalize için basit ama kullanışlı bir tekniktir. Burada FIONA tekniğin bir özeti ve rapor edilir FIONA&#39;sı kullanılarak yapılmıştır araştırma örnekleri kısaca tarif edilmektedir. İlk olarak, nasıl optik hizalama hakkında ayrıntılar yani Fiona deneylerde, toplam iç yansıma floresan mikroskobu (TIRFM) için gerekli ekipman kurmak, tarif edilir. O kadar bir kuantum nokta ile işaretlenmiş tek bir tepe bölümü kesik miyozin Va motorun 36 nm Adım boyutunu ölçmek için FIONA kullanılması, ardından uygun olan protokolü kullanılarak immobilize Cy3 DNA tek molekülleri lokalize basit Fiona deneyi yürütmek için görüntülenmiştir. Son olarak, kalın örnekleri FIONA uygulama genişletmek için son çaba bildirilmiştir. &gt; (Bu bir suya daldırma objektif kullanılarak, olduğu gösterilmiştir ve kuantum noktaları, sol-jel ve tavşan gözü kornealar derin batırılmış200 um), 2,3 nm yeri hassas elde edilebilir.

Tipik amaç tipi TIRFM kurulumu, Şekil 3 &#39;de gösterilmiştir. İlk olarak, yüzey hareketsizleştirilmiş Cy3 DNA örneği görüntülenmiştir. Tipik bir görüntü Şekil 4a&#39;da gösterilmektedir. Görüntü EM kazanç = 50 ve CCD hassasiyet = kamera için 12,13 ile, pozlama süresi ile 0.5 sn alınmıştır. Tek bir Cy3 ile DNA molekülünün bir nokta-yayılmış bir fonksiyonu (PSF) Şekil 4b&#39;de gösterilmiştir renk çubuğu piksel yoğunluğunun öl?...

Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

Fluorescence Imaging with One-nanometer Accuracy FIONA

Tek flüoroforlar FIONA&#39;sı kullanarak nanometre hassasiyetle lokalize edilebilir. Burada FIONA tekniğin bir özet bilgi ve nasıl Fiona deneyleri tarif edilir.

Bir nanometre Hassasiyet Floresans Görüntüleme (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

fluorescence-imaging-with-one-nanometer-accuracy-fiona

Research

JoVE Journal

Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Tüm Kalp Görüntüleme Floresan Optik Projeksiyon Tomografi tarihi Normalizasyon

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Biyoloji

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Floresan Mikroskop ile görselleştirme Tek molekül DNA replikasyonu

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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 ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Hücre Elektrofüzyon Floresan Mikroskop ile görüntülendi

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Hızlı Analiz ve Floresans Mikroskopi Görüntüler Exploration

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Floresan Mikroskopi ile Stromule Frekansı görselleştirme

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Enteroendokrin Hücre Gut Hormon salgısını Çalışması ve Canlı Hücre Görüntüleme için Amaçlanan Kemirgen Küçük Bağırsak Karışık İlköğretim Kültürler

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Photobleaching, sarı floresan Protein etiketli p62 indüklenen Aggresome benzeri yapılarda sonra Floresans kurtarma

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...