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 og PK ønsker å takke for finansiell støtte fra Singapore National Research Foundation, tildelt PK (NRF-NRFF-2011-04 og NRF2012NRF-CRP001-084). Vi takker også MBI åpne lab og mikroskjernefasiliteter for infrastruktur støtte.

1. Imaging Specimen Forberedelse <ol><li> Siden bakgrunn fluorescens signaler forstyrrer fluorescens fra fluoroforen etiketter, rengjøre dekkglass ved først å skylle dem i de-ionisert vann (DDH 2 O) og deretter luft-tørke dem ved hjelp av trykkluft. Deretter utfører plasmaetsing i en plasmarenser i 15 sekunder, eller lenger om nødvendig. </li><li> For å aktivere korreksjon av drifting og iPALM kalibrering, bruk # 1,5 runde (22 mm diameter) pre-renset dekkglass innebygd med fluorescerende nanopartikle...

<ol>
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Det optiske system av iPALM er basert på en 4-π to motstående objektiv utforming, som vist i figur 1A. Oppsettet er bygget ved hjelp både custom-maskinerte og kommersielle opto-mekanisk deler, som beskrevet tidligere 23 og oppført i tabell 1. I tillegg til vårt oppsett, Howard Hughes Medical Institute (HHMI) er vert for et system som er tilgjengelig for det vitenskapelige miljøet ved Advanced Imaging Center ved Janelia Forskning Campus. For den fulle m...

Visualisering av komplekse cellulære strukturer har lenge vært en integrert del av biologiske innsikter og funn. Selv om fluorescens mikroskopi kan bildet celler med høy molekyl spesifisitet, dets oppløsningsevne er begrenset av diffraksjon til ~ 200 nm i bildeplanet (x, y, eller tverrdimensjon) og&gt; 500 nm langs den optiske aksen (z, eller aksial dimensjon) 1,2. Derfor har observasjon av ultrastrukturelle egenskaper historisk sett vært begrenset til elektronmikroskopi (EM). Heldigvis har den siste utviklingen av super oppløsning mikros omgått denne grensen, slik romlig oppløsning på 10-100 nm range 1-6. Spesielt tilnærminger super oppløsning basert på enkelt molekyl lokalisering, kjent av akronymer som PALM (PhotoActivated Lokalisering Mikros) 4, FPALM (Fluorescence PhotoActivated Lokalisering Mikros) 5 (d) STORM (direkte Stochastic Optical Rekonstruksjon mikroskopi) 6,7, maling ( Point Opphopning for Imaging nanoskala Topografi) 8, GSDIM (Ground State Nedbryting Mikrosetterfulgt av individuell molekylær retur) 9, eller SMACM (Single-Molecule Active-Control Mikroskopi) 10, samt deres 3-dimensjonale (3D) implementeringer, for eksempel interferometrisk PALM (iPALM) 11 eller 3D-STORM 12, har vært verdifulle i å avsløre ny innsikt i nanoskala organiseringen av mange biologiske strukturer, inkludert nevronale aksoner og synapser 13, fokale sammenvoksninger 14,15, celle-celle veikryss 16, kjernekraft porer 17 og centrosomes 18-20, for å nevne noen. En annen ultrastructural funksjon i celler som super oppløsning mikroskopi er potensielt nyttig er aktin cytoskjelettet. Komplekset nettvaren av filamentøse (f) p-aktin i cellen cortex spiller en vesentlig rolle i kontrollen av celle form og mekaniske egenskaper 21. Organisasjonen of f-aktin er aktivt og dynamisk regulert om mange regulatoriske proteiner som sterkt påvirker polymerisering, kryssbinding, omsetning, stabilitet og nettverkstopologi 22. Men selv om karakterisering av f-aktin nettvaren arkitekturen er viktig for mekanistiske innsikt i et variert utvalg av cellulære prosesser, den lille størrelse (~ 8 nm) av F-aktin filamenter vanskeliggjør deres observasjon ved konvensjonell diffraksjon begrensede lysmikroskopi; således, visualiseringen av aktin fin struktur har hittil vært utelukkende utført ved EM. Her beskriver vi protokollene for å visualisere f-aktin cytoskjelettet i heftpattedyrceller, bruker iPALM super oppløsning mikroskopi teknikk for å dra nytte av sin svært høy presisjon evne i 3D 11,23. Selv om iPALM instrumentet er svært spesialiserte, har instruksjon om å sette opp et slikt instrument er beskrevet nylig 23, mens tilgang til iPALM mikroskop arrangert av Hosengepost Hughes Medical Institute har også blitt gjort tilgjengelig for forskersamfunnet med minimal kostnad. I tillegg er prøvefremstillingsmetoder som er beskrevet her, er direkte anvendelige for alternative 3D superoppløsningsfremgangsmåter, slik som de basert på astigmatiske defokusering av punktspredefunksjonen (PSF) 12 eller bi-plan deteksjons 24, som er mer bredt tilgjengelig. Vi merker oss at en nødvendig ingrediens for enkelt-molekyl lokalisering basert super oppløsning mikroskopi generelt er photoswitchable fluorophore 25, noe som gjør at de tre kritiske krav til enkelt-molekyl lokalisering basert super oppløsning mikros være oppfylt: i) høy enkelt-molekyl lysstyrke og kontrast i forhold til bakgrunnssignaler; ii) sparsom distribusjon av enkle molekyler i et gitt bilde ramme; og iii) med høy romlig tetthet av merking tilstrekkelig til å fange profilen til den underliggende strukturen (også kalt Nyquist-ShanIkke sampling kriterium) 26. Således kan tilfredsstillende resultater, bør det legges vekt likt på både den riktige fremstillingen av prøver for å optimalisere fluoroforen photoswitching og for å bevare de underliggende ultrastruktur, så vel som på de instrumentering og anskaffelses aspekter av eksperimentene.

<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 muliggjør direkte visualisering av spesifikke biomolekyler i cellene. Men for konvensjonelle fluorescens mikroskopi, er den romlige oppløsningen begrenset av diffraksjon til ~ 200 nm i bildeplanet og&gt; 500 nm langs den optiske aksen. Som et resultat av fluorescens mikroskopi har lenge vært sterkt begrenset i observasjon av ultrastrukturelle egenskaper i cellene. Den siste utviklingen av super oppløsning mikroskopi metoder har overvunnet denne begrensningen. Særlig bruk av photoswitchable fluoroforer muliggjør lokaliseringsbasert super oppløsning mikroskopi, noe som gir oppløsningsevne som nærmer seg den molekylære lengdeskala. Her beskriver vi anvendelse av en tre-dimensjonal super oppløsning mikroskopi metode basert på enkelt-molekyl lokalisering mikroskopi og flerfase interferometri, kalt interferometrisk PhotoActivated Localization Mikroskopi (iPALM). Denne metoden gir nesten isotrop oppløsning påorden 20 nm i alle tre dimensjoner. Protokoller for å visualisere det trådformede aktin cytoskjelettet, inkludert prøven forberedelse og drift av iPALM instrumentet, blir beskrevet her. Disse protokollene er også lett tilpasses og rikt for studiet av andre ultra funksjoner i celler.

Kritiske krav til iPALM er justering, registrering og kalibrering av optiske systemer. Dette er nødvendig for å sikre riktig inngrep i tre-veis strålesplitter nødvendig for z-koordinaten ekstraksjon. For å aktivere kontinuerlig overvåking, konstant punktkilder for fluorescens er nødvendig. Dette kan oppnås ved hjelp av fluorescerende Au eller bi-metallholdige nanopartikler 23 som har photoluminescence oppstår fra lokal overflate-plasmonresonans (LSPR). De fungerer som...

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 presenterer en protokoll for anvendelse av interferometriske PhotoActivated Localization Mikroskopi (iPALM), en tre-dimensjonal enkelt-molekyl lokalisering super oppløsning mikroskopi fremgangsmåte til avbildning av aktin cytoskjelettet i adherente pattedyrceller. Denne fremgangsmåten gjør det mulig for lys-basert visualisering av nanoskala strukturelle trekk som ellers ville forbli uløst ved konvensjonell diffraksjon begrenset optisk mikroskopi.

Tredimensjonal Super Resolution Mikroskopi av F-aktin filamenter av Interferometric PhotoActivated Lokalisering Mikroskopi (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

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: Protein Lokalisering Bruke Photo-aktivert Lokalisering Mikroskopi og 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 Prøver for Optimalisere STORM Super-oppløsning Mikros

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

Prøvepreparering for mikroskopi av enkeltvirionakroskopi og superoppløselig 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-Resolution Imaging av Cytokinetic Z Ring i levende bakterier Bruke Fast 3D-Strukturert Illumination Mikros (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 Klonal Sporing av Hematopoetiske Stem og stamceller Preget av fem Fluorescent Proteiner bruker Confocal og multiphoton Mikros

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 Sporing av Hematopoietiske Stem og stamceller i Adult Mouse Calvarial Bone Marrow

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 Single-partikkel Analyse for Super-oppløsning Mikroskopi 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

Anvendelse av høyhastighets super-oppløsning hastighet mikroskopi i Live primære Flimmerhår

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

Biologiske utvalg forberedelse av høytrykks fryser, mikrobølgeovn-assistert kontrast ekstrautstyr og Minimal harpiks innebygging volum Imaging

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