Name: correlative microscopy for 3d structural analysis of dynamic interactions
Uploaded: 2013-06-24T13:00:00
Description: Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

Forfatterne vil gerne takke Travis Wheeler og maskinværksted på Institut for Cellebiologi og Fysiologi, University of Pittsburgh for opførelsen af ​​cryo-fluorescens prøve scenen, Changlu Tao og Cheng Xu ved University of Science and Technology i Kina til teknisk assistance, og Dr. Teresa Brosenitsch for kritisk læsning af manuskriptet. Dette arbejde blev støttet af National Institutes of Health (GM082251 &amp; GM085043).

1.. HeLa Cell Culture på Carbon-belagt, guld EM Finder Grids <ol><li> Glimudladning carbon-siden af ​​en 200-mesh R2 / 2 Quantifoil guld EM finderen gitter under 25 mA for 25 sek. </li><li> Coat EM nettet med fibronectin, ved at flyde det carbon-side ned på en 40 ul dråbe af 50 ug / ml fibronektin løsning, så desinficere det under UV-lys i 2 timer i en vævskultur hætte. </li><li> Plate HeLa-celler på elnettet med en tæthed på 2 x 10 4 celler / ml (i alt 2 ml kultur) i et glas-bund dyrkningssk?...

<ol>
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D., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+biogenesis+of+individual+HIV-1+virions+in+live+cells.">Imaging the biogenesis of individual HIV-1 virions in live cells.</a> Nature. 454, 236-240 (2008).</li><li>Koch, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+fusion+of+pseudotyped+HIV-1+particles+in+real+time+by+live+cell+microscopy.">Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy.</a> Retrovirology. 6, 84 (2009).</li><li>Zhang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+receptor+arrays+in+frozen-hydrated+sections+and+plunge-frozen+specimens+of+E.+coli+engineered+to+overproduce+the+chemotaxis+receptor+Tsr.">Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr.</a> Journal of Microscopy. 216, 76-83 (2004).</li><li>Wang, K., Strunk, K., Zhao, G., Gray, J. L., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+structure+determination+of+native+mammalian+cells+using+cryo-FIB+and+cryo-electron+tomography.">3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.</a> Journal of Structural Biology. 180, 318-326 (2012).</li><li>Sartori, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+microscopy:+bridging+the+gap+between+fluorescence+light+microscopy+and+cryo-electron+tomography.">Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.</a> Journal of Structural Biology. 160, 135-145 (2007).</li><li>Schwartz, C. L., Sarbash, V. I., Ataullakhanov, F. I., McIntosh, J. R., Nicastro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-fluorescence+microscopy+facilitates+correlations+between+light+and+cryo-electron+microscopy+and+reduces+the+rate+of+photobleaching.">Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching.</a> Journal of Microscopy. 227, 98-109 (2007).</li><li>van Driel, L. F., Valentijn, J. A., Valentijn, K. M., Koning, R. I., Koster, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+correlative+cryo-fluorescence+microscopy+and+cryo-electron+tomography+applied+to+whole+mitochondria+in+human+endothelial+cells.">Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells.</a> European Journal of Cell Biology. 88, 669-684 (2009).</li><li>Rigort, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromachining+tools+and+correlative+approaches+for+cellular+cryo-electron+tomography.">Micromachining tools and correlative approaches for cellular cryo-electron tomography.</a> Journal of Structural Biology. 172, 169-179 (2010).</li><li>Gruska, M., Medalia, O., Baumeister, W., Leis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+tomography+of+vitreous+sections+from+cultured+mammalian+cells.">Electron tomography of vitreous sections from cultured mammalian cells.</a> Journal of Structural Biology. 161, 384-392 (2008).</li><li>Jun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+HIV-1+with+correlative+live-cell+microscopy+and+cryo-electron+tomography.">Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography.</a> Structure. 19, 1573-1581 (2011).</li><li>Dubochet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+vitrified+specimens.">Cryo-electron microscopy of vitrified specimens.</a> Quarterly Reviews of Biophysics. 21, 129-228 (1988).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> Journal of Structural Biology. 116, 71-76 (1996).</li><li>Whitfield, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+periodically+expressed+in+the+human+cell+cycle+and+their+expression+in+tumors.">Identification of genes periodically expressed in the human cell cycle and their expression in tumors.</a> Molecular Biology of the Cell. 13, 1977-2000 (2002).</li><li>Agronskaia, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+fluorescence+and+transmission+electron+microscopy.">Integrated fluorescence and transmission electron microscopy.</a> Journal of Structural Biology. 164, 183-189 (2008).</li><li>Marko, M., Hsieh, C., Schalek, R., Frank, J., Mannella, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused-ion-beam+thinning+of+frozen-hydrated+biological+specimens+for+cryo-electron+microscopy.">Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy.</a> Nature Methods. 4, 215-217 (2007).</li></ol>

Vi har fremlagt en enkel sæt af protokoller til at levere en avanceret korrelativ tilgang til at analysere dynamiske cellulære begivenheder ved hjælp af time-lapse konfokal, live-cell fluorescensimagografi efterfulgt af cryoET. Vores metodeudvikling at korrelere 3D live-cell imaging med høj opløsning cryoET er kritisk at undersøge mange udfordrende biologiske problemer, såsom visualisering sjældne, dynamiske (ikke statisk, som tidligere rapporteret) og diffraktion begrænsede virale partikler og deres vekselvirk...

Cryo-elektron tomografi (cryoET) er en kraftfuld imaging teknik, der giver tre-dimensionelle (3D) visualisering af celler og væv og giver indsigt i tilrettelæggelsen af indfødte organeller og cellulære strukturer på molekylært opløsning i et tæt-på fysiologiske tilstand 1. Men den iboende lav kontrast af ufarvet frosne hydreret prøve, kombineret med deres stråling følsomhed, gør det vanskeligt at lokalisere områder af interesse inde i en celle og derefter udføre tilt serien succes uden at beskadige målområdet. For at overvinde disse problemer, en modsvarende tilgang, der kombinerer lys og elektronmikroskopi er nødvendig. Særlige forhold fremhæves af fluorescerende mærkning identificeres og lokaliseres ved fluorescens lysmikroskopi, og så deres koordinater overføres til elektronmikroskop for erhvervelse af høj opløsning 3D strukturelle data. Denne korrelativ metode hjælper lokalisere målet enreas af interesse at blive behandlet. På grund af den begrænsning prøvetykkelse med cryoEM (&lt;300 nm), som i øjeblikket kun de perifere områder af cellen er egnet til 3D strukturel analyse af CryoET. Yderligere at reducere tykkelsen af frosset-hydratiserede prøver ved glasagtige sektionering 8 eller ved kryo-fokuseret ionstråle (FIB) fræsning 9 ville udvide evnen til korrelationstabet billeddannelse. Tidligere blev korrelationsmaalinger metoder primært brugt til at lette cryoET dataopsamling til store og statiske konstruktioner 10-13. I disse undersøgelser har cryo faser er gennemført for at acceptere cryoEM net og passe på enten en opretstående mikroskop eller et inverteret mikroskop 10,11,14. Selvom skifte net forekommer temmelig ligetil i deres designs, der er yderligere overførsel trin involveret i EM nettet, øger chancen for, at nettet kan blive deformeret, beskadiget og forurenet. Vi har for nylig demonstreret et teknisk fremskridt ikorrelativ mikroskopi, der giver os mulighed for direkte at visualisere dynamiske begivenheder, som af natur svært at fange, såsom HIV-1 og vært celle interaktioner på tidlige stadier af infektionen 15.. Vi udført dette ved at designe og gennemføre en cryo-lysmikroskopi prøve fase, tilpasser en patron system til at minimere modellen skader som følge af grid håndtering, hvilket letter korrelation. Vores design omfatter en integreret eksemplar patron holder, så både cryo-lys og cryo-elektronmikroskopi, der skal udføres, sekventielt på samme prøveholder uden prøve overførsel, og dermed effektivisere korrelativ proces. Derudover har vi implementeret en præcis og pålidelig korrelation procedure ved hjælp fluorescerende latexperler som Referencemærkernes markører.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>DMEM 4.5 g/L Glucose w/ L-Glutamine</td>
			<td>Atlanta Biologicals, Inc. Lawrenceville, GA</td>
			<td>12-604F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma, St. Louis, MO</td>
			<td>F1141-1MG</td>
			<td>HeLa cell culture on EM grids</td>
		</tr>
		<tr>
			<td>Cell tracker</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>C34552</td>
			<td>Red CMTPX</td>
		</tr>
		<tr>
			<td>Protein A gold conjugates</td>
			<td>Ted Pella, Inc., Redding, CA</td>
			<td>15822-1</td>
			<td>15 nm diameter</td>
		</tr>
		<tr>
			<td>0.2 &mu;m fluorescent microspheres</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>F8811</td>
			<td>Yellow-green fluorescent</td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal calf bovine serum</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>10082-139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass-bottom culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gold quantifoil finder EM grids</td>
			<td>Quantifoil Micro Tools, Jena, Germany</td>
			<td>R2/2 Au NH2 200 mesh</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>70011-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Glow-discharge device 100X</td>
			<td>EMS, Hatfield, PA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tecnai Polara G2 electron microscope with a Field Emission Gun</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>300 keV</td>
		</tr>
		<tr>
			<td>Vitrobot Mark III</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus IX 71 microscope</td>
			<td>Olympus America Inc., Center Valley, PA</td>
			<td>&nbsp;</td>
			<td>LUCPlanFLN 40X/0.6 NA (2.7-4 mm working distance) objective lens</td>
		</tr>
		<tr>
			<td>Nikon TiE microscope</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>&nbsp;</td>
			<td>using a 60X/1.35 NA oil immersion objective lens</td>
		</tr>
		<tr>
			<td>Sweptfield confocal microscope</td>
			<td>Prairie Technologies, Middleton, WI</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tokai Hit live cell chamber</td>
			<td>Tokyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryo-fluorescence sample stage</td>
			<td>Home-made</td>
			<td>&nbsp;</td>
			<td>Homebuilt by machine shop, see reference 15 for the design.</td>
		</tr>
	</tbody>
</table>

correlative microscopy for 3d structural analysis of dynamic interactions

Cryo-elektron tomografi (cryoET) giver mulighed for 3D-visualisering af cellulære strukturer på molekylært opløsning i et tæt-på-fysiologiske tilstand 1. Imidlertid er direkte visualisering af individuelle virale komplekser i deres vært cellulære miljø med cryoET udfordrende 2, på grund af den sjældne og dynamiske karakter af viral indgang, især i tilfælde af HIV-1. Mens time-lapse live-cell imaging har givet en hel del oplysninger om mange aspekter af livscyklus af HIV-1 3-7 opløsningen ydes af live-cell mikroskopi er begrænset (~ 200 nm). Vores arbejde var rettet mod at udvikle en korrelation metode, der tillader direkte visualisering af tidlige begivenheder i HIV-1 infektion ved at kombinere live-cell fluorescerende lysmikroskopi, cryo-fluorescerende mikroskopi og cryoET. På denne måde, kan levende celle og cryo-fluorescerende signaler anvendes til præcist at styre prøvetagning i cryoET. Desuden strukturel information opnået fra cryoET kan be suppleret med de dynamiske funktionelle data opnået gennem live-cell imaging af fluorescerende mærkede mål. I denne video artiklen, giver vi detaljerede metoder og protokoller for strukturel undersøgelse af HIV-1 og host-celle interaktioner ved hjælp af 3D-correlative high-speed live-cell imaging og høj opløsning cryoET strukturel analyse. HeLa-celler inficeret med HIV-1-partikler blev karakteriseret først ved konfokal live-cell mikroskopi, og regionen indeholdende det samme virale partikel blev derefter analyseret ved cryo-elektron tomografi til 3D strukturelle detaljer. Sammenhængen mellem to sæt af billeddata, optisk billeddannelse og elektronmikroskopi billeddannelse, blev opnået ved hjælp af en hjemme-bygget cryo-fluorescens lysmikroskopi scenen. Den fremgangsmåde beskrevet her, vil være værdifuld, ikke kun for undersøgelse af virus-vært-celle interaktioner, men også for bredere anvendelser inden for cellebiologi, såsom celle signalering, membran-receptor handel, og mange andre dynamiske cellulære processer. &lt;/ P&gt;

At karakterisere den dynamiske opførsel af viruspartikler blev HeLa-celler inficeret med HIV-1 afbildet af high-speed konfokal live-cell mikroskopi og partikel bevægelser blev analyseret ved automatiseret 3D partikel sporing (Figur 1). For at undgå tidsforløb på flere minutter, der kan opstå mellem indsamling af den sidste konfokal live-cell billede og styrte-frysning (som kan være lang nok til at tabe korreleret HIV-1-partikler), en cryo-fluorescens lysmikroskopi fase (Figur 3 )...

Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Vi beskriver en modsvarende mikroskopi metode, der kombinerer high-speed 3D live-cell lysstofrør mikroskopi og høj opløsning cryo-elektron tomografi. Vi demonstrerer evne til korrelativ metode imaging dynamisk, små HIV-1 partikler vekselvirker med værts HeLa-celler.

Korrelativ mikroskopi til 3D Strukturel analyse af dynamiske vekselvirkning

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

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Korrelationsmaalinger lys Elektron Mikroskopi (CLEM) til sporing og Imaging Viral Protein associeret strukturer i Cryo-immobiliserede celler

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

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