Name: three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)
Uploaded: 2016-12-01T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

YW ve PK minnetle PK (NRF-NRFF-2011-04 ve NRF2012NRF-CRP001-084) verilir Singapur Ulusal Araştırma Vakfı, finansman desteği kabul. Biz de altyapı desteği için MTE açık laboratuvar ve mikroskopi çekirdek imkanları teşekkür ederim.

1. Görüntüleme Numune Hazırlama <ol><li> Arka plan floresan sinyalleri florofor etiketlerden floresan ile etkileştikleri ilk (GKD 2 O) deiyonize suda durulama ve daha sonra basınçlı hava kullanarak hava-kurutma ile coverglasses temizleyin. Daha sonra, 15 saniye için bir plazma temizleme plazma aşındırma yerine veya gerekirse daha fazla. </li><li> sürüklenme düzeltme ve iPALM kalibrasyon etkinleştirmek için, # 1.5 yuvarlak (22 mm çap) güvenilir kalibrasyon ve sürüklenme düzeltilmesi iç...

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Şekil 1A &#39;de gösterildiği gibi iPALM optik sistemi, 4-π çift karşıt amaçlı tasarım dayanmaktadır. Tablo 1&#39;de önceki 23 anlatıldığı ve listelenen kurulum, özel işlenmiş ve ticari opto-mekanik parçalar kullanılarak inşa edilmiştir. Bizim kurulumuna ek olarak, Howard Hughes Tıp Enstitüsü (HHMI) Janelia Araştırma Kampüsü&#39;nde İleri Görüntüleme Merkezi bilim dünyası için erişilebilir bir sistem barındırır. Tam me...

kompleks hücresel yapıların görselleştirme uzun biyolojik anlayışlar ve keşif ayrılmaz olmuştur. floresan mikroskopi, onun çözme gücü ~ 200 resim düzleminde nm (x, y, veya lateral boyut) ve&gt; 500 nm optik eksen boyunca (z, ya da eksenel boyut) kırınım yüksek moleküler özgüllük ile görüntü hücreleri sınırlı olsa da 1,2. Bu nedenle, ultrastrüktürel özellikleri gözlem tarihsel elektron mikroskobu (EM) ile sınırlı kalmıştır. 100 nm aralığında 1-6 - Neyse, süper çözünürlük mikroskopisi son gelişme 10 uzaysal çözünürlüğü sağlayan bu sınırı hile vardır. Özellikle, süper çözünürlük gibi PALM (photoactivated Yerelleştirme Mikroskobu) 4, FPALM (Floresan photoactivated Yerelleştirme Mikroskobu) 5 (d) STORM (doğrudan Stokastik Optik İmar Mikroskopi) 6,7, BOYA (as kısaltmaları tarafından bilinen tek bir molekül lokalizasyonu dayalı yaklaşımlar poGörüntüleme Nano Topografya) 8 için int Birikim, GSDIM (Ground Devlet tükenmesi Mikroskopi 9) bireysel moleküler dönüşü takiben, ya da SMACM (Tek Molekül Aktif Kontrol Mikroskopi) 10, yanı sıra bunların 3 boyutlu (3D) uygulamaları, interferometrik PALM (iPALM) 11 veya 3D-STORM 12, nöronal aksonlar, çok sayıda biyolojik yapıların nano örgüte yeni bakış açıları ortaya dahil olmak üzere değerli olmuştur ve fokal yapışıklıklar 14,15, hücre-hücre kavşak 16, nükleer 17 gözeneklerin, 13 sinapsların ve sentrozomlar 18-20, birkaç isim. süper çözünürlüklü mikroskopi potansiyel olarak yararlı olduğu hücrelerde başka ultrastrüktürel özelliği aktin hücre iskeleti olduğunu. Hücre kortekste filamentli (f) aktin için kompleks ağ örgüsü hücre şekli ve mekanik özellikleri 21 kontrolünde önemli bir rol oynar. organizasyon of f-aktin aktif ve dinamik güçlü polimerizasyonu, çapraz bağlanmayı, ciro, istikrar ve ağ topolojisini 22 etkileyen çok sayıda düzenleyici proteinler olsa düzenlenir. Ancak, f-aktin ağ örgüsü mimarisinin karakterizasyonu hücresel süreçlerin çeşitli bir yelpazede, geleneksel kırınım sınırlı ışık mikroskobu ile kendi gözlemini engelleyen f-aktin filamentler küçük boyutlu (~ 8 nm) içine mekanistik anlayışlar için önemli olmasına rağmen; böylece, aktin ince yapısı görselleştirme, şimdiye kadar sadece EM ile yapılmaktadır. Burada, biz, yapışık memeli hücrelerinde f-aktin hücre iskeleti görselleştirmek 3D 11,23 yılında çok yüksek hassasiyetli yeteneği yararlanmak için iPALM süper çözünürlük mikroskopi tekniği kullanarak protokolleri açıklar. IPALM enstrüman son derece uzmanlaşmış olmasına rağmen, böyle bir araç kurma hakkında talimat, son zamanlarda 23 tarif edilmiştir Ho tarafından barındırılan iPALM mikroskop erişim sırasındakoğuş Hughes Tıp Enstitüsü de minimum maliyetle araştırma topluluğuna kullanılabilir hale getirilmiştir. Buna ek olarak, burada açıklanan numune hazırlama yöntemleri daha geniş mevcut gibi nokta dağılım fonksiyonu (PSF) ve astigmat odaktan uzaklaşma dayalı olanlar gibi alternatif 3D süper çözünürlük yaklaşımları, 12 veya bi-düzlem algılama 24, doğrudan uygulanabilir. Biz genel olarak tek-molekül yerelleştirme tabanlı süper çözünürlük mikroskopi için gerekli bir maddedir yerine getirilmesi tek-molekül yerelleştirme tabanlı süper çözünürlük mikroskopi için üç kritik gereksinimleri izin veren photoswitchable fluorofor 25 olduğuna dikkat: i) yüksek tek-molekül arka plan sinyallerine parlaklık ve kontrast akraba; ii) Belirli bir resim çerçevesi içinde tek moleküllerin seyrek dağılımı; Ayrıca Nyquist-Sha olarak bilinen altta yatan yapının (profilini yakalamak için yeterli etiketleme ve iii) yüksek uzaysal yoğunluknnon örnekleme kriteri) 26. Bu nedenle, tatmin edici sonuçlar elde etmek için ağırlık florofor photoswitching optimize etmek ve altta yatan ultrastrüktürel korumak hem de deney cihazı ve alıcı yönleri için numunelerin uygun hazırlanması hem de eşit yerleştirilmelidir.

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			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
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			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
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			<td>laser-642</td>
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			<td>laser-561</td>
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			<td>Newport, CA&#160;</td>
			<td>1137970</td>
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			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
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			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
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			<td>dichroic beamsplitter&#160;</td>
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			<td>LM01-427-25</td>
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			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
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			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</td>
			<td>05LP-VIS-B</td>
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			<td>baseplate</td>
			<td>local workshop</td>
			<td>customized</td>
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			<td>turning mirror (22.5&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
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			<td>motorized optic mounts&#160;</td>
			<td>New Focus, CA</td>
			<td>8816</td>
			<td></td>
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			<td>motorized XYZ translation stage</td>
			<td>Thorlabs, NJ</td>
			<td>MT3/M-Z6</td>
			<td>sample holder</td>
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			<td>T-Cube DC servo motor controller</td>
			<td>Thorlabs, NJ</td>
			<td>TDC001</td>
			<td></td>
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			<td>Piezo Phase Shifter</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-303.CD</td>
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			<td>objective lens</td>
			<td>Nikon, Japan</td>
			<td>MRD01691</td>
			<td>objective. Apo TIRF 60X/1.49oil</td>
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			<td>translation stage</td>
			<td>New Focus, CA</td>
			<td>9062-COM-M</td>
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			<td>Pico Motor Actuator</td>
			<td>New Focus, CA</td>
			<td>8301</td>
			<td></td>
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			<td>rotary Solenoid/Shutter</td>
			<td>DACO Instruments, CT</td>
			<td>5423-458</td>
			<td></td>
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			<td>3-way beam splitter</td>
			<td>Rocky Mountain Instruments, CO</td>
			<td>customized</td>
			<td>beamsplitter</td>
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			<td>Piezo Z/Tip/Tilt scanner</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-316.10</td>
			<td></td>
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			<td>motorized five-axis tilt aligner&#160;</td>
			<td>New Focus, CA</td>
			<td>8081</td>
			<td></td>
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			<td>Picmotor ethernet controller</td>
			<td>New Focus, CA</td>
			<td>8752</td>
			<td></td>
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			<td>Piezo controllers/amplifier/digital operation module</td>
			<td>Physik Instrumente, Germany</td>
			<td>E-509/E-503/E-517</td>
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			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-523/20</td>
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			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-588/21</td>
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			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-607/30</td>
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			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-676/37</td>
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			<td>notch filter</td>
			<td>Semrock, NY</td>
			<td>NF01-405/488/561/635</td>
			<td></td>
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			<td>motorized filter wheel with controllter</td>
			<td>Thorlabs, NJ</td>
			<td>FW103H</td>
			<td></td>
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			<td>EMCCD</td>
			<td>Andor, UK&#160;</td>
			<td>DU-897U-CSO-#BV</td>
			<td>3 sets</td>
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			<td>Desktop computers for controlling cameras and synchronization</td>
			<td>Dell</td>
			<td>Precision T3500</td>
			<td>PC, 4 sets</td>
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		<tr>
			<td>coverslips with fiducial</td>
			<td>Hestzig, VA</td>
			<td>600-100AuF</td>
			<td>sample preparation. fiducial marks with various density and spectra&#160; available</td>
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			<td>fibronectin&#160;</td>
			<td>Millipore, MT</td>
			<td>FC010</td>
			<td></td>
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		<tr>
			<td>paraformaldehyde</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>15710</td>
			<td>fixation. 16%</td>
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		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16220</td>
			<td>25%</td>
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			<td>triton X-100</td>
			<td>Sigma aldrich, MO</td>
			<td>T8787</td>
			<td></td>
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			<td>HUVEC cells</td>
			<td>Life Technologies, CA</td>
			<td>C-015-10C</td>
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			<td>Medium 200</td>
			<td>Life Technologies, CA</td>
			<td>M-200-500</td>
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			<td>Large Vessel Endothelial Factors</td>
			<td>Life Technologies, CA</td>
			<td>A14608-01</td>
			<td></td>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
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			<td>Pennicillin/Streptomycin</td>
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			<td>15140122</td>
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			<td>Trypsin/EDTA</td>
			<td>Life Technologies, CA</td>
			<td>25200056</td>
			<td></td>
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			<td>PIPES</td>
			<td>Sigma aldrich, MO</td>
			<td>P1851</td>
			<td>PHEM</td>
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		<tr>
			<td>HEPES</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1825</td>
			<td></td>
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		<tr>
			<td>EGTA</td>
			<td>Sigma aldrich, MO</td>
			<td>E3889</td>
			<td></td>
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		<tr>
			<td>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
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			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
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			<td>sodium borohydride (NaBH4)&#160;</td>
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			<td>480886</td>
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			<td>1st base, Malaysia</td>
			<td>BIO-1101</td>
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			<td>glucose oxidase</td>
			<td>Sigma aldrich, MO</td>
			<td>G2133</td>
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			<td>catalase</td>
			<td>Sigma aldrich, MO</td>
			<td>C9322</td>
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			<td>Sigma aldrich, MO</td>
			<td>30070</td>
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			<td>Epoxy</td>
			<td>Thorlabs, NJ</td>
			<td>G14250</td>
			<td></td>
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			<td>vaseline</td>
			<td>Sigma aldrich, MO</td>
			<td>16415</td>
			<td>sample sealing</td>
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		<tr>
			<td>lanolin</td>
			<td>Sigma aldrich, MO</td>
			<td>L7387</td>
			<td></td>
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		<tr>
			<td>parafin wax</td>
			<td>Sigma aldrich, MO</td>
			<td>327204</td>
			<td></td>
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			<td></td>
			<td></td>
			<td></td>
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			<td>Immersion oil&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16915-04</td>
			<td>imaging. Cargille Type HF</td>
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three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

Floresan mikroskopi hücreleri içinde belirli biyomoleküllerin doğrudan görüntülenmesine olanak tanıyan. Bununla birlikte, geleneksel floresan mikroskobu için, uzamsal çözünürlük optik eksen boyunca görüntü düzleminde ve&gt; 500 nm olan ~ 200 nm ila kırınımı ile sınırlıdır. Bunun bir sonucu olarak, floresan mikroskobu uzun ciddi hücre içinde ultrastrüktürel özellikler gözlem sınırlıdır. süper çözünürlüklü mikroskopi yöntemleri son gelişmeler bu sınırlamanın üstesinden gelmiştir. Özellikle, photoswitchable fluorophores gelişi moleküler uzunlukta ölçeği yaklaşan çözme gücü sağlar yerelleştirme tabanlı süper çözünürlük mikroskopi, sağlar. Burada, interferometrik photoactivated Yerelleştirme Mikroskobu (iPALM) olarak adlandırılan tek-molekül yerelleştirme mikroskopi ve çok fazlı interferometriye dayalı üç boyutlu süper çözünürlük mikroskopi yöntemi uygulanmasını açıklar. Bu yöntem neredeyse izotropik çözünürlük sağlarher üç boyutta da 20 nm sırası. iPALM aletin numune hazırlama ve işlemler dahil, ipliksi aktin hücre iskeleti görselleştirmek için protokoller, burada açıklanmıştır. Bu protokoller de hücrelerde diğer ultrastrüktürel özellikleri çalışma için kolaylıkla adapte ve öğreticidir.

iPALM için kritik şartlar optik sistemlerin hizalama, kayıt ve kalibrasyon vardır. çıkarma z koordinatı için bu 3 yollu ışın ayırıcı koşul içinde uygun girişim sağlamak için gereklidir. sürekli izleme etkinleştirmek için, floresan sabit nokta kaynağı gereklidir. Bu flüoresan Au veya olan Fotolüminesans lokalize yüzey plazmon rezonansı (lspr) ortaya bimetalik nano-tanecikleri 23 kullanılarak elde edilebilir. Onlar aydınlatma üzerine kararlı bir t...

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Biz yapışık memeli hücrelerinde aktin hücre iskeletinin görüntüleme, interferometrik photoactivated Yerelleştirme Mikroskopi (iPALM), 3-boyutlu tek-molekül yerelleştirme süper çözünürlük mikroskopi yöntemi uygulaması için bir protokol mevcut. Bu yaklaşım, aksi takdirde geleneksel kırınım sınırlı optik mikroskopi ile çözülmemiş kalacağını nano yapısal özellikleri ışık tabanlı görselleştirme sağlar.

Üç boyutlu Süper İnterferometrik photoactivated Yerelleştirme Mikroskopi F-aktin Filament Çözünürlük Mikroskobu (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Research

JoVE Journal

Biology

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

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

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

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

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

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Nano-fem: Foto-aktif Yerelleştirme Mikroskobu ve Elektron Mikroskop Kullanarak Protein Yerelleştirme

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

Optimize STORM Süper Çözünürlük Mikroskopi Testi Örnekleri

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Tek Virion Atomik Kuvvet Mikroskopisi ve Süper Çözünürlüklü Floresan Görüntüleme için Numune Hazırlama

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Hızlı 3D-Yapılandırılmış Aydınlatma mikroskopi ile Canlı Bakteri Cytokinetic Z Yüzük Süper çözünürlük Görüntüleme (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

VirusMapper Süper çözünürlüklü Mikroskopi Açık kaynak Tek parçacık Analizi

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

Yüksek hızlı süper çözünürlük hızı mikroskobu canlı birincil Cilium içinde uygulanması

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Yüksek basınç dondurucu, mikrodalga destekli kontrast geliştirme ve en az reçine birim görüntüleme için katıştırma tarafından biyolojik örnek hazırlama

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...