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 och PK tack erkänna finansieringsstöd från Singapore National Research Foundation, tilldelas PK (NRF-NRFF-2011-04 och NRF2012NRF-CRP001-084). Vi tackar också MBI Open Lab och mikroskopi kärnanläggningar för infrastrukturstöd.

1. Imaging Provberedning <ol><li> Eftersom bakgrundsfluorescens signaler störa fluorescens från fluoroforen etiketter, rengöra täckglas genom att först skölja dem i avjoniserat vatten (DDH 2 O) och sedan lufttorkning dem med tryckluft. Därefter utför plasmaetsning i en plasma renare under 15 sekunder, eller längre om det behövs. </li><li> För att aktivera driftkorrigering och iPALM kalibrering, använda # 1,5 runda (22 mm diameter) i förväg rengjorda täckglas inbäddade med fluorescerande nano...

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Det optiska systemet i iPALM är baserad på en 4-π dubbla motsats objektiv konstruktion, såsom visas i figur 1A. Installationen är konstruerad med användning av både specialanpassade maskinbearbetade och kommersiella optomekaniska delar, såsom beskrivits tidigare 23 och räknas upp i tabell 1. Förutom vår inställning, Howard Hughes Medical Institute (HHMI) är värd ett system som är tillgängligt för det vetenskapliga samfundet vid Advanced Imagi...

Visualisering av komplexa cellulära strukturer har länge varit en integrerad del av biologiska insikter och upptäckter. Även fluorescensmikroskopi kan bildceller med hög molekylspecificitet, dess upplösningsförmåga är begränsad av diffraktion till ~ 200 nm i bildplanet (x, y, eller lateral dimension) och&gt; 500 nm längs den optiska axeln (z, eller axiell dimension) 1,2. Därför har observationen av ultra funktioner historiskt varit begränsade till elektronmikroskopi (EM). Lyckligtvis har den senaste utvecklingen av superupplösning mikroskopi kringgås denna gräns, vilket möjliggör spatial upplösning i 10-100 nm 1-6. I synnerhet, strategier superupplösning baserad på samma molekyl lokalisering, känd av akronymer såsom PALM (photoactivated Lokalisering Microscopy) 4, FPALM (Fluorescence photoactivated Lokalisering Microscopy) 5 (d) STORM (direkt Stochastic Optical återuppbyggnad Microscopy) 6,7, färg ( point Ackumulering för Imaging Nanoscale Topography) 8, GSDIM (grundtillståndet Depletion Mikroskopi följt av enskilda molekyl avkastning) 9, eller SMACM (Single-Molecule Active-Control Mikroskopi) 10, såväl som deras 3-dimensionella (3D) implementeringar, såsom interferometrisk PALM (iPALM) 11 eller 3D-STORM 12, har varit värdefulla i att avslöja nya insikter i nano organisation av ett flertal biologiska strukturer, inklusive neuronala axoner och synapser 13, fokala sammanväxningar 14,15, cellcellkontakter 16, kärnpor 17 och centrosom 18-20, för att nämna några. En annan ultra funktion i celler som superupplösning mikroskopi är potentiellt användbart är aktin cytoskelettet. Den komplexa nätverket av fintrådiga (f) aktin i cellen cortex spelar en viktig roll i kontrollen av cell form och mekaniska egenskaper 21. Organisationen of f-aktin aktivt och dynamiskt regleras även många regulatoriska proteiner som starkt påverkar polymerisation, tvärbindning, omsättning, stabilitet och nätverkstopologin 22. Men även om karakterisering av F-aktin nätverket arkitektur är viktigt för mekanistiska insikter i en rad olika cellulära processer, den lilla storleken (~ 8 nm) av F-aktin filament försvårar deras observation genom konventionell diffraktionsbegränsad ljusmikroskop; därmed visualisering av aktin fin struktur har hittills uteslutande utförs av EM. Här beskriver vi protokoll för att visualisera f-aktin cytoskelettet i vidhäftande däggdjursceller, med hjälp av iPALM superupplösning mikroskopi teknik för att dra fördel av sin mycket hög precision kapacitet i 3D 11,23. Även om iPALM instrumentet högt specialiserad, har instruktioner om att inrätta ett sådant instrument beskrivits nyligen 23, medan tillgång till iPALM mikroskop värd HoWard Hughes Medical Institute har också gjorts tillgängliga för forskarsamhället med minimal kostnad. Dessutom är provframställningsmetoder som beskrivs häri är direkt tillämpliga på alternativa 3D superupplösning metoder, såsom de som är baserade på astigmatisk oskärpa på punktspridningsfunktion (PSF) 12 eller bi-planet upptäckt 24, som är mer allmänt tillgängliga. Vi noterar att en nödvändig ingrediens för enda molekyl lokalisering baserade superupplösning mikroskopi i allmänhet är photoswitchable fluoroforen 25, vilket gör att de tre kritiska kraven på enda molekyl lokalisering baserade superupplösning mikroskopi vara uppfyllda: i) hög enda molekyl ljusstyrka och kontrast i förhållande till bakgrundssignaler; ii) gles fördelning av enskilda molekyler i en given bildram; och iii) hög spatial densitet av märkning är tillräcklig för att fånga profilen hos den underliggande strukturen (även känd som Nyquist-ShanIcke provtagning kriterium) 26. Således, för tillfredsställande resultat, bör betonas lika på både korrekt beredning av prover för att optimera fluoroforen photoswitching och bevara den underliggande ultra, liksom på instrumentering och förvärvs aspekter av experimenten.

<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)

Fluorescensmikroskopi möjliggör direkt visualisering av specifika biomolekyler inom celler. Men för konventionell fluorescensmikroskopi, är den spatiala upplösningen begränsad av diffraktion till ~ 200 nm inom bildplanet och&gt; 500 nm längs den optiska axeln. Som ett resultat, fluorescensmikroskopi har länge varit starkt begränsad i observationen av ultrastrukturella egenskaper inom celler. Den senaste tidens utveckling av superupplösning mikroskopimetoder har övervinna denna begränsning. Särskilt tillkomsten av photoswitchable fluoroforer möjliggör lokalisering baserade superupplösning mikroskopi, som ger upplösningsförmågan närmar molekyl-längdskala. Här beskriver vi tillämpningen av en tredimensionell superupplösning mikroskopi metod som bygger på en enda molekyl lokalisering mikroskopi och flerfasig interferometri, kallas interferometriska photoactivated Lokalisering Microscopy (iPALM). Denna metod ger nästan isotropiska upplösning påstorleksordningen 20 nm i alla tre dimensioner. Protokoll för att visualisera den filamentösa aktincytoskelettet, inklusive extraktion och drift av iPALM instrumentet, beskrivs här. Dessa protokoll är också lätt anpassningsbar och lärorikt för studier av andra ultra funktioner i celler.

Kritiska krav iPALM är inriktningen, registrering och kalibrering av de optiska systemen. Dessa är nödvändiga för att säkerställa korrekt störningar inom 3-vägs stråldelare förutsättning för z-koordinat extraktion. För att möjliggöra kontinuerlig övervakning, fasta punktkällor för fluorescens är nödvändiga. Detta kan åstadkommas med hjälp av fluorescerande Au eller bi-metalliska nanopartiklar 23, vars fotoluminescens uppstår lokaliserad ytplasmonresona...

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

Vi presenterar ett protokoll för tillämpning av interferometriska photoactivated Lokalisering Microscopy (iPALM), en 3-dimensionell enda molekyl lokalisering superupplösning mikroskopi metoden, avbildning av aktin cytoskelettet i vidhäftande däggdjursceller. Detta tillvägagångssätt gör ljusbaserad visualisering av nanoskala strukturella egenskaper som annars skulle förbli olösta genom konventionell diffraktionsbegränsad optisk mikroskopi.

Tredimensionell Super Resolution mikroskopi av F-aktinfilament av interferometriska photoactivated Lokalisering 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

Levande cell imaging av F-aktin Dynamics via Fluorescent 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: proteinlokalisering Använda Photo-aktiverad lokalisering Mikroskopi och elektronmikroskopi

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

Test Prover för Optimera STORM Super-resolution Mikroskopi

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

Provberedning för single virion atomic force mikroskopi och superupplösning fluorescensavbildning

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-upplösning avbildning av Cytokinetic Z Ring i levande bakterier Använda Snabb 3D-Structured Illumination Mikroskopi (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 Klon Spårning av hematopoetiska stam och progenitorceller märkta med fem fluorescerande proteiner med hjälp Konfokal och multifoton mikroskopi

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 4-Dimensional Spårning av Hematopoietic Stem och stamceller i vuxen mus calvarial Benmärg

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

Öppen källkod Single-partikelanalys för Super högupplösande mikroskopering med 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

Tillämpningen av höghastighetståg super-resolution hastighet mikroskopi i levande primära cilier

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

Biologiska provberedning genom högtrycks frysning, mikrovågsugn-assisted kontrastförbättring, och Minimal harts inbäddning för volym Imaging

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