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 et PK remercient le soutien financier de la Fondation de recherche national de Singapour, décerné à PK (NRF-NRFF-2011-04 et NRF2012NRF-CRP001-084). Nous remercions également les laboratoires et de base de microscopie ouvertes installations MBI pour le soutien de l&#39;infrastructure.

1. Préparation des échantillons d&#39;imagerie <ol><li> Comme les signaux de fluorescence de fond interfèrent avec la fluorescence des étiquettes fluorophores, nettoyer les couvre - objets d&#39;abord les rincer dans de l&#39;eau déminéralisée (ddH 2 O), puis les séchage à l&#39; air à l&#39;air comprimé. Par la suite, effectuer une gravure par plasma dans un nettoyeur à plasma pendant 15 secondes, ou plus si nécessaire. </li><li> Pour activer la correction de la dérive et de l&#39;étalonnage...

<ol>
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Le système optique de iPALM est basé sur une conception à double objectif opposé 4-π, comme illustré sur la figure 1A. La configuration est construit en utilisant des pièces opto-mécaniques à la fois sur mesure usinées et commerciales, comme décrit plus haut 23 et énumérés dans le tableau 1. En plus de notre configuration, le Howard Hughes Medical Institute (HHMI) héberge un système qui est accessible à la communauté scientifique au Centre d...

La visualisation des structures cellulaires complexes depuis longtemps partie intégrante de connaissances biologiques et de découverte. Bien que la microscopie de fluorescence peut imager des cellules ayant une spécificité moléculaire élevée, son pouvoir de résolution est limitée par la diffraction à ~ 200 nm dans le plan d&#39;image (x, y ou dimension latérale), et&gt; 500 nm le long de l&#39;axe optique (z, ou dimension axiale) 1,2. Par conséquent, l&#39;observation des caractéristiques ultrastructurales ont toujours été limitées à la microscopie électronique (EM). Heureusement, le développement récent de nanoscope a contourné cette limite, ce qui permet une résolution spatiale dans le 10 - gamme de 100 nm 1-6. En particulier, super résolution approches basées sur la localisation unique molécule, connue par des acronymes tels que PALM (photoactive Localisation Microscopy) 4, FPALM (Fluorescence photoactive Localisation Microscopy) 5 (d) STORM (direct Stochastic Optical Reconstruction Microscopy) 6,7, PAINT ( Point Accumulation for Imaging Nanoscale Topographie) 8, GSDIM (État sol Depletion Microscopie suivi par retour moléculaire individuel) 9, ou SMACM (Single-Molecule Active-contrôle Microscopy) 10, ainsi que leurs (3D) implémentations 3 dimensions, telles que PALM interférométrique (iPALM) 11 ou 3D-STORM 12, ont été précieux pour révéler de nouvelles perspectives dans l&#39;organisation à l&#39; échelle nanométrique de nombreuses structures biologiques, y compris les axones neuronaux et synapses 13, adhérences focales 14,15, jonctions cellule-cellule 16, les pores nucléaires 17 et centrosomes 18-20, pour ne citer que quelques - uns. Une autre caractéristique ultrastructural dans les cellules pour lesquelles nanoscope est potentiellement utile est le cytosquelette d&#39;actine. Le maillage complexe de filamenteuse (f) actine dans le cortex cellulaire joue un rôle essentiel dans le contrôle de la forme cellulaire et les propriétés mécaniques 21. L&#39;organisation of f-actine est active et dynamique régulée si de nombreuses protéines régulatrices qui influencent fortement la polymérisation, la réticulation, le chiffre d&#39; affaires, la stabilité et la topologie du réseau 22. Cependant, bien que la caractérisation de l&#39;architecture meshwork f-actine est important pour des idées mécanistes dans un large éventail de processus cellulaires, la petite taille (~ 8 nm) des filaments f-actine entrave leur observation par microscopie optique conventionnelle limitée par la diffraction; ainsi, la visualisation de structure fine actine a jusqu&#39;à présent été exclusivement réalisé par EM. Ici, nous décrivons les protocoles pour visualiser le cytosquelette d&#39;actine F dans les cellules de mammifères adhérentes, en utilisant la technique de microscopie super résolution iPALM pour tirer parti de sa capacité de très haute précision en 3D 11,23. Bien que l&#39;instrument iPALM est hautement spécialisée, des instructions sur la mise en place d&#39; un tel instrument a été récemment décrit 23, alors que l&#39; accès au microscope iPALM hébergé par le Hopupille Hughes Medical Institute a également été mis à la disposition de la communauté scientifique à un coût minime. En outre, les méthodes de préparation des échantillons décrits ici sont directement applicables à d&#39; autres approches 3D super - résolution, tels que ceux basés sur défocalisation astigmate de la fonction d&#39;étalement de point (PSF) 12 ou bi-plan de détection 24, qui sont plus largement disponibles. Nous notons qu&#39;un ingrédient nécessaire pour la microscopie de super - résolution basée sur la localisation unique de molécule en général est le fluorophore photoswitchable 25, qui permet aux trois exigences critiques pour la microscopie super - résolution basée sur la localisation unique de molécule à être remplies: i) élevé seule molécule luminosité et le contraste par rapport aux signaux d&#39;arrière-plan; ii) la distribution clairsemée de molécules simples dans une trame d&#39;image donnée; et iii) de haute densité spatiale d&#39;étiquetage suffisant pour capturer le profil de la structure sous-jacente (également connue sous le nom de Nyquist-Shacritère d&#39;échantillonnage nLes) 26. Ainsi, pour obtenir des résultats satisfaisants, l&#39;accent doit être mis aussi à la fois sur la bonne préparation des échantillons pour optimiser fluorophore photocommutation et de préserver l&#39;ultrastructure sous-jacent, ainsi que sur les instruments et d&#39;acquisition aspects des expériences.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>optical table</td>
			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
			<td>iPALM, installed on 4 Newport Stabilizer vibration isolators&#160;</td>
		</tr>
		<tr>
			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>laser-642</td>
			<td>Newport, CA&#160;</td>
			<td>1185055</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>laser-561</td>
			<td>Newport, CA&#160;</td>
			<td>1168931</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-488</td>
			<td>Newport, CA&#160;</td>
			<td>1137970</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
		</tr>
		<tr>
			<td>dichroic beamsplitter&#160;</td>
			<td>Semrock, NY</td>
			<td>LM01-427-25</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic tunable filter&#160;</td>
			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</td>
			<td>05LP-VIS-B</td>
			<td></td>
		</tr>
		<tr>
			<td>baseplate</td>
			<td>local workshop</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr>
			<td>turning mirror (22.5&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
		</tr>
		<tr>
			<td>motorized optic mounts&#160;</td>
			<td>New Focus, CA</td>
			<td>8816</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized XYZ translation stage</td>
			<td>Thorlabs, NJ</td>
			<td>MT3/M-Z6</td>
			<td>sample holder</td>
		</tr>
		<tr>
			<td>T-Cube DC servo motor controller</td>
			<td>Thorlabs, NJ</td>
			<td>TDC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo Phase Shifter</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-303.CD</td>
			<td></td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Nikon, Japan</td>
			<td>MRD01691</td>
			<td>objective. Apo TIRF 60X/1.49oil</td>
		</tr>
		<tr>
			<td>translation stage</td>
			<td>New Focus, CA</td>
			<td>9062-COM-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pico Motor Actuator</td>
			<td>New Focus, CA</td>
			<td>8301</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary Solenoid/Shutter</td>
			<td>DACO Instruments, CT</td>
			<td>5423-458</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way beam splitter</td>
			<td>Rocky Mountain Instruments, CO</td>
			<td>customized</td>
			<td>beamsplitter</td>
		</tr>
		<tr>
			<td>Piezo Z/Tip/Tilt scanner</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-316.10</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized five-axis tilt aligner&#160;</td>
			<td>New Focus, CA</td>
			<td>8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Picmotor ethernet controller</td>
			<td>New Focus, CA</td>
			<td>8752</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo controllers/amplifier/digital operation module</td>
			<td>Physik Instrumente, Germany</td>
			<td>E-509/E-503/E-517</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-523/20</td>
			<td>filters</td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-588/21</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-607/30</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-676/37</td>
			<td></td>
		</tr>
		<tr>
			<td>notch filter</td>
			<td>Semrock, NY</td>
			<td>NF01-405/488/561/635</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized filter wheel with controllter</td>
			<td>Thorlabs, NJ</td>
			<td>FW103H</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD</td>
			<td>Andor, UK&#160;</td>
			<td>DU-897U-CSO-#BV</td>
			<td>3 sets</td>
		</tr>
		<tr>
			<td>Desktop computers for controlling cameras and synchronization</td>
			<td>Dell</td>
			<td>Precision T3500</td>
			<td>PC, 4 sets</td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>fibronectin&#160;</td>
			<td>Millipore, MT</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>15710</td>
			<td>fixation. 16%</td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16220</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>triton X-100</td>
			<td>Sigma aldrich, MO</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC cells</td>
			<td>Life Technologies, CA</td>
			<td>C-015-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 200</td>
			<td>Life Technologies, CA</td>
			<td>M-200-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Vessel Endothelial Factors</td>
			<td>Life Technologies, CA</td>
			<td>A14608-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td></td>
			<td>14190367</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin/Streptomycin</td>
			<td></td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Life Technologies, CA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma aldrich, MO</td>
			<td>P1851</td>
			<td>PHEM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1825</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma aldrich, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>480886</td>
			<td>quenching</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1101</td>
			<td>imaging buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase</td>
			<td>Sigma aldrich, MO</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>catalase</td>
			<td>Sigma aldrich, MO</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteamine&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Thorlabs, NJ</td>
			<td>G14250</td>
			<td></td>
		</tr>
		<tr>
			<td>vaseline</td>
			<td>Sigma aldrich, MO</td>
			<td>16415</td>
			<td>sample sealing</td>
		</tr>
		<tr>
			<td>lanolin</td>
			<td>Sigma aldrich, MO</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr>
			<td>parafin wax</td>
			<td>Sigma aldrich, MO</td>
			<td>327204</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16915-04</td>
			<td>imaging. Cargille Type HF</td>
		</tr>
	</tbody>
</table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

La microscopie à fluorescence permet la visualisation directe de biomolécules spécifiques dans les cellules. Cependant, pour la microscopie par fluorescence classique, la résolution spatiale est limitée par la diffraction à ~ 200 nm dans le plan d&#39;image et&gt; 500 nm le long de l&#39;axe optique. Par conséquent, la microscopie par fluorescence a longtemps été sévèrement limité à l&#39;observation des caractéristiques ultrastructurales dans les cellules. Le développement récent des méthodes de microscopie super-résolution a surmonté cette limitation. En particulier, l&#39;avènement de fluorophores photoswitchable permet de super résolution microscopie à base de localisation, ce qui fournit un pouvoir de résolution approcher l&#39;échelle moléculaire longueur. Ici, nous décrivons l&#39;application d&#39;une méthode ultra tridimensionnelle résolution microscopie basée sur une seule molécule microscopie localisation et interférométrie polyphasique, appelé interférométrique photoactive Localisation Microscopie (iPALM). Cette méthode offre une résolution quasi isotrope sur laordre de 20 nm dans les trois dimensions. Des protocoles pour la visualisation du cytosquelette d&#39;actine filamenteuse, y compris la préparation des échantillons et le fonctionnement de l&#39;instrument iPALM, sont décrits ici. Ces protocoles sont également facilement adaptable et instructif pour l&#39;étude d&#39;autres caractéristiques ultrastructurales dans les cellules.

exigences critiques pour iPALM sont l&#39;alignement, l&#39;enregistrement, et le calibrage des systèmes optiques. Ceux-ci sont nécessaires pour assurer une bonne interférence dans le 3-way diviseur de faisceau requis pour l&#39;extraction coordonnée z. Pour permettre une surveillance continue, de sources ponctuelles constantes de fluorescence sont nécessaires. Ceci peut être réalisé en utilisant des nanoparticules fluorescentes Au ou bimétalliques 23 dont la photolum...

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

Nous présentons un protocole pour l&#39;application de localisation interférométrique photoactive Microscopie (iPALM), une méthode nanoscope trois dimensions molécule unique de localisation, à l&#39;imagerie du cytosquelette d&#39;actine dans les cellules de mammifères adhérentes. Cette approche permet la visualisation à base de lumière des caractéristiques structurelles nanométriques qui autrement resteraient en suspens par microscopie optique à diffraction limitée classique.

Trois dimensions Super Resolution Microscopie de Filaments F-actine par interférométrique photoactive Localisation Microscopy (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

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (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 ...

Recherche

Biologie

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

Nano-FEM: la localisation des protéines en utilisant la microscopie Localisation Photo-actif et microscopie électronique

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

Les échantillons de test pour optimiser STORM Super-Résolution Microscopie

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

Préparation d’échantillons pour la microscopie à force atomique à virion unique et l’imagerie par fluorescence à super-résolution

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

Super-résolution d&#39;imagerie de l&#39;Anneau Z cytokinétique en utilisant des bactéries vivantes 3D structuré rapide Illumination Microscopy (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

In vivo suivi clonale de cellules souches hématopoïétiques et progénitrices Marqué par cinq protéines fluorescentes confocale et multiphotonique Microscopie

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

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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

Open Source Analyse unique pour particules super-résolution Microscopie avec VirusMapper

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

Application de la microscopie vitesse haut débit Super-résolution en direct cil primaire

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

Préparation de l’échantillon biologique par congélation à haute pression, amélioration du contraste assistée par micro-ondes et minime résine enrobage pour l’imagerie de Volume

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