Name: imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy
Uploaded: 2018-03-06T13:30:00
Description: Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

Vi tackar Mickael Lelek, Orestis Faklaris och Nicolas Bourg för givande diskussion, Andrey Aristov och Elena Rensen för hjälp med den super resolution-tekniken och Shailaja Seetharaman för noggrann läsning av manuskriptet. Vi erkänna tacksamt UtechS fotoniska BioImaging (Imagopole) Citech av Institut Pasteur (Paris, Frankrike) samt Frankrike-BioImaging infrastrukturnätverket stöds av franska National Research Agency (ANR-10-INSB-04; Investeringar för framtiden), och de Région Ile-de-France (programmera Domaine d'Intérêt majeure-Malinf) för användning av mikroskopet Elyra. Detta arbete stöds av den Ligue Contre le Cancer och den franska National Research Agency (ANR-16-CE13-0019).

1. Coverslips förberedelse (dag 1, 30 min)
<ol><li>Placera 18-mm nº1.5H (170 µm +/-5µm) glas coverslips på en plast rack.</li>
	<li>Lägg racket i en 100 mL-bägare fylld med aceton och blöt i 5 min.</li>
	<li>Överföra racket till en 100 mL-bägare fylld med absolut etanol och blöt i 5 min.</li>
	<li>Överföra racket till en ny 100 mL-bägare fylld med absolut etanol och placera bägaren i ett ultraljud renare enhet (se tabell material) vid rumstemperatur. Tryck på ”on” och vänta i 10 min.</li>
	<li>Rac...

<ol>
	<li>Herrmann, H., Aebi, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+Filaments:+Structure+and+Assembly.">Intermediate Filaments: Structure and Assembly.</a> Cold Spring Harb Perspect Biol. 8 (11), (2016).</li><li>Huber, F., Boire, A., Lopez, M. P., Koenderink, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+crosstalk:+when+three+different+personalities+team+up.">Cytoskeletal crosstalk: when three different personalities team up.</a> Curr Opin Cell Biol. 32, 39-47 (2015).</li><li>Leduc, C., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+filaments+in+cell+migration+and+invasion:+the+unusual+suspects.">Intermediate filaments in cell migration and invasion: the unusual suspects.</a> Curr Opin Cell Biol. 32, 102-112 (2015).</li><li>Heilemann, M., 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=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angew Chem Int Ed Engl. 47 (33), 6172-6176 (2008).</li><li>Gevaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nobel+Prize+in+Chemistry:+seeing+the+nanoscale.">Nobel Prize in Chemistry: seeing the nanoscale.</a> Nat Nanotechnol. 9 (11), 878 (2014).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3 (10), 793-795 (2006).</li><li>Metcalf, D. J., Edwards, R., Kumarswami, N., Knight, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+samples+for+optimizing+STORM+super-resolution+microscopy.">Test samples for optimizing STORM super-resolution microscopy.</a> J Vis Exp. (79), (2013).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nat Methods. 8 (12), 1027-1036 (2011).</li><li>Dempsey, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+localization-based+super-resolution+fluorescence+imaging.">A user's guide to localization-based super-resolution fluorescence imaging.</a> Methods Cell Biol. 114, 561-592 (2013).</li><li>van de Linde, 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+stochastic+optical+reconstruction+microscopy+with+standard+fluorescent+probes.">Direct stochastic optical reconstruction microscopy with standard fluorescent probes.</a> Nat Protoc. 6 (7), 991-1009 (2011).</li><li>Chazeau, A., Katrukha, E. A., Hoogenraad, C. C., Kapitein, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+neuronal+microtubule+organization+and+microtubule-associated+proteins+using+single+molecule+localization+microscopy.">Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy.</a> Methods Cell Biol. 131, 127-149 (2016).</li><li>Olivier, N., Keller, D., Rajan, V. S., Gonczy, P., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+buffers+for+3D+STORM+microscopy.">Simple buffers for 3D STORM microscopy.</a> Biomed Opt Express. 4 (6), 885-899 (2013).</li><li>Eliasson, C., 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=Intermediate+filament+protein+partnership+in+astrocytes.">Intermediate filament protein partnership in astrocytes.</a> J Biol Chem. 274 (34), 23996-24006 (1999).</li><li>Leduc, C., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+microtubule-associated+motors+drives+intermediate+filament+network+polarization.">Regulation of microtubule-associated motors drives intermediate filament network polarization.</a> J Cell Biol. 216 (6), 1689-1703 (2017).</li><li>Gan, Z., 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=Vimentin+Intermediate+Filaments+Template+Microtubule+Networks+to+Enhance+Persistence+in+Cell+Polarity+and+Directed+Migration.">Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration.</a> Cell Syst. 3 (5), 500-501 (2016).</li><li>Nieuwenhuizen, R. 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=Co-Orientation:+Quantifying+Simultaneous+Co-Localization+and+Orientational+Alignment+of+Filaments+in+Light+Microscopy.">Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.</a> PLoS One. 10 (7), e0131756 (2015).</li><li>Yang, Z., Wang, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein:+from+intermediate+filament+assembly+and+gliosis+to+neurobiomarker.">Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker.</a> Trends Neurosci. 38 (6), 364-374 (2015).</li><li>Maslehaty, H., Cordovi, S., Hefti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symptomatic+spinal+metastases+of+intracranial+glioblastoma:+clinical+characteristics+and+pathomechanism+relating+to+GFAP+expression.">Symptomatic spinal metastases of intracranial glioblastoma: clinical characteristics and pathomechanism relating to GFAP expression.</a> J Neurooncol. 101 (2), 329-333 (2011).</li><li>Hol, E. M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein+(GFAP)+and+the+astrocyte+intermediate+filament+system+in+diseases+of+the+central+nervous+system.">Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system.</a> Curr Opin Cell Biol. 32, 121-130 (2015).</li><li>Skalli, O., 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=Astrocytoma+grade+IV+(glioblastoma+multiforme)+displays+3+subtypes+with+unique+expression+profiles+of+intermediate+filament+proteins.">Astrocytoma grade IV (glioblastoma multiforme) displays 3 subtypes with unique expression profiles of intermediate filament proteins.</a> Hum Pathol. 44 (10), 2081-2088 (2013).</li><li>Endesfelder, U., Malkusch, S., Fricke, F., Heilemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+estimate+the+average+localization+precision+of+a+single-molecule+localization+microscopy+experiment.">A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment.</a> Histochem Cell Biol. 141 (6), 629-638 (2014).</li><li>Whelan, D. R., Bell, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+artifacts+in+single+molecule+localization+microscopy:+why+optimization+of+sample+preparation+protocols+matters.">Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.</a> Sci Rep. 5, 7924 (2015).</li><li>Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z., Hagen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThunderSTORM:+a+comprehensive+ImageJ+plug-in+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging.">ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li><li>Olivier, N., Keller, D., Gonczy, P., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+doubling+in+3D-STORM+imaging+through+improved+buffers.">Resolution doubling in 3D-STORM imaging through improved buffers.</a> PLoS One. 8 (7), e69004 (2013).</li><li>Nahidiazar, L., Agronskaia, A. V., Broertjes, J., van den Broek, B., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Imaging+Conditions+for+Demanding+Multi-Color+Super+Resolution+Localization+Microscopy.">Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.</a> PLoS One. 11 (7), e0158884 (2016).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Mikhaylova, M., 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=Resolving+bundled+microtubules+using+anti-tubulin+nanobodies.">Resolving bundled microtubules using anti-tubulin nanobodies.</a> Nat Commun. 6, 7933 (2015).</li><li>Nieuwenhuizen, R. 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=Measuring+image+resolution+in+optical+nanoscopy.">Measuring image resolution in optical nanoscopy.</a> Nat Methods. 10 (6), 557-562 (2013).</li><li>Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., Jungmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+with+DNA-PAINT.">Super-resolution microscopy with DNA-PAINT.</a> Nat Protoc. 12 (6), 1198-1228 (2017).</li></ol>

Kritiska steg i protokollet
Vi presenterar här ett protokoll som minimerar artefakter i dSTORM bilder av mikrotubuli och IFs i gliaceller. Artefakter kan skapas vid varje steg av provberedning och imaging: fixering, blockering, immunolabeling, glida under förvärv, icke-optimala blinkande villkor23. Vi listar nedan de viktigaste stegen.
Rengöring av coverslips är ett viktigt steg för att begränsa den icke-specifik bindn...

Cytoplasmiska mellanliggande glödtrådar (IFs) är 10 nm-diameter homo- eller heteropolymers av en cell typ specifika delmängd av om proteiner. IFs delta i ett stort utbud av cellulära funktioner såsom cell motilitet, spridning och stress svaren. Deras viktiga roll understryks av det faktum att mer än 90 mänskliga sjukdomar orsakas direkt av mutationer i om proteiner; exempelvis förändringar om sammansättning medföljer tumörens tillväxt och spridning1,2,3. Det finns växande bevis för att de tre cytoskelettsystemen fungerar i samarbete till kontroll cellulära funktioner såsom cell polarisering, division och migration2,3. Eftersom det finns en tät koppling mellan rumsliga arkitektur och funktioner, är det viktigt att få insikter i den strukturella organisationen om den med andra filament och bättre förstå den cytoskeletal överhörning. den här artikeln innehåller ett protokoll för att utföra den super-upplösning teknik tvåfärgad dSTORM (direkt stokastiska optiska återuppbyggnad Microscopy)4 och hur den används att undersöka samspelet mellan om och mikrotubuli i fast, odlade celler i 2 dimensioner (2D).
Flera super-resolution tekniker har utvecklats under senaste decenniet, och där upptäckter var på beskärningen av Nobelpriset i kemi i 20145. Bland alla dessa tekniker ligger de enda molekyl lokalisering-baserade metoderna som PALM6 (Photo-Activated lokalisering Microscopy) som använder photoswitchable fluorescerande proteiner, STORM7 med par fluorophores eller dSTORM4 med konventionell fluorophores. Alla dessa metoder bygger på samma princip som består av a växeln av mest av de fluorescerande reportrarna till en ”off” stat ”(icke-fluorescerande), (ii) den stokastiska aktiveringen av en delmängd av dem in i en fluorescerande staten för att lokalisera sin rumsliga position med nanometer precision, och (iii) upprepning av proceduren för att aktivera så många grupper av fluorophores som möjligt. En slutliga bilden rekonstrueras med hjälp av alla lokaliseringar av aktiverade molekylerna, som tillhandahåller en lateral upplösning ner till ~ 20-40 nm. Flera kommersialiserade optiska system tillåter PALM/STORM är nu tillgängliga för biologer för rutinmässig experiment. Här, användes ett sådant system för att studera strukturella associering av mikrotubuli och IFs. Bland alla singel-molekyl lokalisering-baserade metoder, tvåfärgad dSTORM tekniken valdes, eftersom det är väl lämpad att observera mycket tunn lamellär regioner (< 1 µm) av vidhäftande celler och kan ge betydande förbättring av bildupplösning med minimal investering av tid och pengar. DSTORM är faktiskt en mycket bekväm och mångsidig teknik som är kompatibel med de vanliga organiska färgämnen som rutinmässigt används för cellulär färgning och immunofluorescens.
Medan en färg dSTORM bilder är relativt enkel att få med fluorophore Alexa6478, kräver tvåfärgad dSTORM för att optimera de experimentella förhållandena så att två färgämnen kan blinka ordentligt i valda bufferten, särskilt när det finns begränsad lasereffekt. Utmärkt papper finns redan på metoderna för att ställa in flerfärgade imaging med dSTORM9,10,11,12,13, och dessa papper förklara i detalj de möjliga källorna till artefakter och försämrad upplösning samt hur man övervinna dem. I denna artikel, de optimala experimentella förhållandena när det gäller celler fixering, immunfärgning, provberedning som imaging förvärv och bildrekonstruktion beskrivs för att förvärva bilder av tät cytoplasmiska om nätverk och mikrotubuli i gliaceller med tvåfärgad dSTORM. Kort, utvinning/fixering metoden beskrivs i Chazeau et al12 anpassades till gliaceller och används med ett efter fixering steg, optimerad antikroppar koncentration, en STORM buffert med 10 mM MEA beskrivs i Dempsey9 et al. som var befunnits vara optimal för experimentell uppsättning upp och Provtyp.
Gliaceller express huvudsakligen tre typer av om proteiner: vimentin, nestin och Fredsgenomförande (glial fibrillary sura protein). Dessa tre proteiner visades att samtidig polymerisera i astrocyterna14. Vi visade tidigare använda super-resolution strukturerade belysningen mikroskopi (SR SIM) att dessa tre om proteiner kan hittas i samma enda om glödtråden och att de visar liknande fördelning och dynamik i gliaceller 15. På grund av likheterna mellan de tre om proteinerna användes vimentin färgning som en reporter för hela om nätverket. Med hjälp av dSTORM, lyckades vi lösa hur om formuläret buntar längs mikrotubuli, vilket inte var möjligt med diffraktion begränsad mikroskopi teknik15. Dessa observationer kan hjälpa till att förstå hur vimentin IFs kan mallen mikrotubuli och reglera deras tillväxt bollbana, främja upprätthållandet av en polaritet axel under cell migration16. Super-upplösning bilder nyckelinformation om ömsesidig samverkan mellan de två cytoskeletal delsystem samt förde inblick i sambandet mellan rumsliga association och lokala funktion som kunde vara celltyp specifika17. I allmänhet, kan tvåfärgad dSTORM användas för att studera överhörning mellan cytoskeletal element eller andra typer av organeller, förutsatt att bra immunfärgning villkor uppnås vad gäller täthet och specificitet. Denna teknik kommer att vara användbart för att bättre karaktärisera de cytoskelettet förändringar som observerats under astrogliosis och glioblastom, den vanligaste och mest maligna tumören i centrala nervsystemet, där uttrycket av om proteiner är ändras 18 ,19,20,21.

<table>
	<tbody>
		<tr>
			<td>Minimum Essential Media (MEM)</td>
			<td>Gibco</td>
			<td>41090-028</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>non essential amino acid</td>
			<td>Gibco</td>
			<td>1140-050</td>
			<td>cell culture 1/100e</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>cell culture 1/100e</td>
		</tr>
		<tr>
			<td>F&#339;tal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>cell culture 1/10e</td>
		</tr>
		<tr>
			<td>0,05 % Trypsin-EDTA (1X)</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>fischer scientific</td>
			<td>11540486</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>coverslip 18 mm n&#176;1,5H</td>
			<td>Marienfield/Dominic dutsher</td>
			<td>900556</td>
			<td>coverslips</td>
		</tr>
		<tr>
			<td>paraformaldehyde (PFA) solution</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>triton X100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>non ionic surfactant. Used for cell fixation</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)</td>
			<td>Sigma</td>
			<td>452904-25ML</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>sample passivation 3% in PBS</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Vimentin (V9) antibody produced in mouse</td>
			<td>Sigma</td>
			<td>V6630</td>
			<td>primary antibodies</td>
		</tr>
		<tr>
			<td>Rat anti alpha tubulin</td>
			<td>Biorad</td>
			<td>MCA77G</td>
			<td>primary antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 555</td>
			<td>Fisher scientific</td>
			<td>10635923</td>
			<td>secondary antibodies</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647</td>
			<td>Fisher scientific</td>
			<td>10226162</td>
			<td>secondary antibodies</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td>Fisher scientific</td>
			<td>T7279</td>
			<td>fluorescent beads</td>
		</tr>
		<tr>
			<td>Chamlide magnetic chamber</td>
			<td>Live Cell Instrument</td>
			<td>CM-B18-1</td>
			<td>magnetic sample holder, maximum volume 1,2 mL</td>
		</tr>
		<tr>
			<td>Cysteamine (MEA)</td>
			<td>Sigma</td>
			<td>30070</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase from Aspergillus niger</td>
			<td>Sigma</td>
			<td>G0543</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>sigma</td>
			<td></td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>catalase from bovine liver</td>
			<td>Sigma</td>
			<td>C9322</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>Tris (trizma)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>Wash-N-Dry&#160;coverslip rack</td>
			<td>Sigma</td>
			<td>Z688568</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>ultrasonic cleaner</td>
			<td>Fisher scientific</td>
			<td>15-335-6</td>
			<td></td>
		</tr>
		<tr>
			<td>plasma cleaner</td>
			<td>Harrick plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy

Cytoskelettet, sammansatt av aktin microfilaments, mikrotubuli och mellanliggande glödtrådar (om), spelar en viktig roll i kontrollen av cell form, polaritet och motilitet. Organisationen av aktin- och mikrotubuli har studerats men som av IFs inte ännu fullt kännetecknas. IFs har en genomsnittlig diameter av 10 nm och bildar ett nätverk som sträcker sig över hela cellens cytoplasma. De är fysiskt associerade med aktin och mikrotubuli genom molekylära motorer och cytoskeletal linkers. Denna snäva association är kärnan i de reglerande mekanismer som säkerställer samordnad reglering av de tre cytoskeletal nätverk som krävs för de flesta cellfunktioner. Det är därför avgörande att visualisera IFs ensam och även tillsammans med alla andra cytoskeletal nätverk. Dock om nätverk är extremt tät i de flesta celltyper, särskilt i gliaceller, som gör sin resolution mycket svårt att uppnå med standard fluorescensmikroskopi (lateral resolution av ~ 250 nm). Direkta stokastiska optisk återuppbyggnad mikroskopi (dSTORM) är en teknik som ger en vinst i lateral resolution en storleksordning. Här visar vi att laterala dSTORM resolution är tillräckliga för att lösa tät organisationen om nätverk och, i synnerhet om buntar kring mikrotubuli. Sådana snäva association är sannolikt att delta i samordnad reglering av dessa två nät och maj, förklara hur vimentin IFs mall och stabiliserar mikrotubuli organisation som skulle kunna påverka mikrotubulära beroende vesikulär människohandel. Generellt visar vi hur observationen av två cytoskeletal komponenter med tvåfärgad dSTORM teknik ger nya insikter om deras växelverkan.

Ett Mikroskop utrustade med 50 mW 405 nm och 100 mW 488, 561 och 642 nm solid-state-lasrar, en elektrisk 512 x 512 kamera, en alfa planerar Apo 100 X / 1,46 mål och Band passera 570-650 / långa Pass 655 utsläpp filter användes för representativa resultat presenteras nedan.
Figur 1A ger ett exempel på molekylen densitet och signal-brus-förhållande som ska användas under raw-bild förvärv. B...

Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Imaging intermediär filament och mikrotubuli med 2-dimensionell direkt stokastiska optiska återuppbyggnad mikroskopi

Det övergripande målet med denna metod är att ge de optimala experimentella förhållandena från provberedning till bild förvärv och återuppbyggnad för att utföra 2D tvåfärgad dSTORM bilder av mikrotubuli och mellanliggande glödtrådar i fasta celler

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

The overall goal of this methodology is to create the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual-colored D-STORM imaging of microtubules and intermediate filaments in fixed cells. This method can help characterize the physical interactions between dense and complex cytoskeletal networks such as intermediate filaments and microtubules. The main advantage of this technique is that it allows the observation of cellular structures, which are not resolved with conventional fluorescence microscopy.Visual demonstration of this method is critical as the control of sample preparation and acquisition of robe-linking data is crucial in order to limit the generation of artifacts. After growing U373 cells according to the text protocol, fix the cells by first using PBS to wash them once. Then, remove the PBS and add one milliliter per well of preheated extraction fixation solution.Incubate the cells at room temperature for 60 seconds. Then remove the solution and add one milliliter per well of freshly prepared and preheated 4%PFA diluted in cytoskeleton buffer. Place the 12-well plate back into the incubator at 37 degrees Celsius for 10 minutes.Then remove the PFA and add three milliliters of PBS per well. Use PBS to wash the cells twice. Then remove the PBS and add freshly prepared 10 millimolars sodium borohydride diluted in PBS in order to quench the autofluorescence from the glutaraldehyde.Incubate the cells at room temperature for seven minutes and transfer the recycled sodium borohydride into a specific bin. Use PBS to wash the wells three times for five minutes each. Then remove the PSB and add 5%BSA in PBS as a blocking solution.After immunostaining samples and preparing solutions in buffers according to the text protocol, mount the coverslip containing the labeled cells on a magnetic sample holder. And add the imaging buffer to completely fill the chamber. Apply the lid and make sure that there are no air bubbles between the imaging buffer and the lid.Using absolute ethanol, clean the bottom of the sample and verify that there are no leaks. Then if necessary, use grease to properly seal the sample hold. To carry out imaging, switch on the microscope and the acquisition software.Set up the acquisition parameters according to the text protocol, using the 100-fold NA 1.46 objective, the 1.6-fold Optivar lens, and the TIRF UHP mode. Create a 647 track to image the vimentin filaments labeled with Alexa Fluor 647. Select the 405 and 642 nanometer laser lines and the LP 655 emission filter.Then, create a 555 track to image the microtubules labeled with Alexa Fluor 555. Select the 561, 488, and 405 nanometer laser lines to image the fluorophores and the BP 570 to 650 emission filter. Next, add oil to the objective and place the sample on the lens.Then, select the locate tab and open the shutter of the fluorescence lamp. Find the focus and use the eyepiece to look for the cells to image. Then use the joystick to position the cells in the center of the field.Click on the acquisition tab to return to acquisition mode. Then select the 647 track and set the 642 laser power to 0.2%and the 405 laser power to its minimal value. Set the exposure time to 50 milliseconds and the gain to 50.Then choose continuous acquisition mode to observe the cell on the screen. Make sure that there is at least one fluorescent bead in the field of view. Use an autoscale mode for the display and adjust the focus.Next, take a wide field epifluorescence image of the vimentin network by clicking on snap and save the image. To check TIRF illumination, click on TIRF, press continuous, adjust the acquisition parameters in order to have a homogenous field of illumination. Then press snap and save the image.Check the 555 track. Uncheck the 647 track, and select the 555 track. Take an epifluorescence image of the microtubule network and save it.Then take a TIRF image and save it. Now, check the 647 track. Uncheck the 555 track, and select the 647 track.Set the gain to zero and the exposure time to 10 milliseconds. Then press continuous. Set the 642 nanometer laser power to 100%in order to pump most of the Alexa 647 dyes to the dark state.Once the fluorophores start blinking, increase the exposure to 15 milliseconds and the gain to 300. Use the range indicator mode to verify that single molecule signals do not saturate the camera as indicated by the red color. In that case, decrease the gain until the saturation disappears.The beads will remain saturated at the beginning of the acquisition. Press stop to discontinue observation of the cell. Expand the online processing tool and click online processing PALM to visualize the STORM image during acquisition.Use nine for the peak mask size and six for the peak intensity to noise. Now, press start acquisition. During acquisition, refocus on the cell if necessary.Adjust the exposure time and eventually switch on the 405 nanometer laser line starting at the 0.001%to keep a medium density of fluorophores with a minimum distance of one micrometer between single molecules. Make sure that the peak of localization precision on the histogram below the reconstructed image is centered around or below 10 nanometers. Press stop to end the movie when more than one million localizations have been reached.Save the raw data from the STORM acquisition with the CZI format. Then in the channels tool, check track 555, uncheck track 647, and select track 555. Next, press the continuous button.Set the exposure time to 30 milliseconds and the gain to zero. Then set both 488 and 561 lasers to 100%to pump Alexa 555 dyes to the dark state. Use the HILO mode of illumination.Once the fluorophores are depleted, press stop. Set the gain to 250 and decrease the 488 laser to 50%Use the range indicator to check that single molecules do not saturate the camera. If that is the case, decrease the gain.Now, press start acquisition. During acquisition, increase the gain gradually to reach the limit of saturation. Increase step-by-step the 405 nanometer laser power to activate the molecules and reach a medium density in the field of view.Decrease the 488 nanometer laser power to 30%when the 405 laser is about 15%Then decrease gradually the 488 while increasing the 405 laser power in order to keep the blinking of the molecules as fast as possible and with a medium density. The 488 laser line coupled with the 561 excitation laser at maximal intensity increases both the on and off rates of the fluorophores, whereas the 405 laser line only increases the on rate. Stop the acquisition when 500, 000 localizations or more have been reached or when no more molecules are blinking.Then save the raw data from the STORM acquisition with the CZI format. To carry out image reconstruction using the PAL-Drift tool, choose the model-based correction type with automatic segments with a maximum size of eight. Press apply multiple times in a row until the drift is corrected.This model-based correction applies an algorithm based on cross-correlation analysis, which corrects the drift. Using the PAL filter tools, improve the appearance of the STORM image by selecting only the molecules which were best localized. For example, limit the localization precision to 30 nanometers and the PSF to 120 to 180 nanometers.With the PAL-Render tool, use a pixel resolution of five or 10 nanometers and parameters according to the text protocol. Select render auto dynamic range HR with a 95%value and render auto dynamic range SWF with a 90%value. Then select render best quality.In the PALM menu of the processing tab, select PALM convert. Press select and then apply. Finally, save the created image with the format LSM and not CZI.This figure shows a typical example of a D-STORM image of vimentin filaments immuno-labeled with Alexa 647. The increase in resolution can clearly be observed upon comparison with the image acquired with a standard, wide-field microscope. The fluorescence intensity profiles presented in this graph show that super resolution microscopy imaging allows the resolution of vimentin bundles.The D-STORM resolution is sufficient to count the number of filaments present in the bundles. Shown here is a typical example of the dual-colored D-STORM imaging of vimentin and microtubule networks immuno-labeled with Alexa 647 and Alexa 555, respectively. Good vimentin images should have at least 5, 000 localizations per micrometer squared and 2, 500 per micrometer squared for microtubules.Once mastered, this technique can be done in three days if it performed properly. While attempting this procedure, it is important to adapt the methods of fixation and parametrization to the protein that would be imaged. For example, fixation with only glutaraldehyde gives poor results for intermediate filaments, but is best for microtubule fixation.It is important to remember to use freshly prepared samples and solutions, especially MEA, whose pH has to be carefully adjusted. After its development, this technique paved the way for researchers in the field of cell biology to explore the morphology of nanoscopic cellular structures. After watching this video, you should have a good understanding of how to perform sample preparation, image acquisition, and reconstruction of 2D dual-colored D-STORM images of intermediate filaments and microtubules.Don't forget that working with some solutions, especially PFA, NABH4 can be extremely hazardous and precautions such as wearing gloves, working under the chemical hood, and using specific bins should always been taken while performing this procedure.

Research

JoVE Journal

Biology

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Märkning stamceller med fluorescerande färger för icke-invasiv Detection med optisk avbildning

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Korrekt vård och rengöring av mikroskopet

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Viktigaste komponenterna i ljusmikroskop

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Imaging exocytos i näthinnan bipolär Celler med TIRF Mikroskopi

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Imaging och 3D rekonstruktion av Cerebrovascular Structures i embryonala Zebrafish

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Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Tandem Högtrycks Frysning och Quick Freeze Substitution av växtvävnad för transmissionselektronmikroskopi

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