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 takker Mickael Lelek, Orestis Faklaris og Nicolas Bourg nemlig givtig diskussion, Andrey Aristov og Elena Rensen til hjælp med super-opløsning teknik og Shailaja Seetharaman for grundig gennemlæsning af manuskriptet. Vi parlamentsarbejdet UtechS fotoniske BioImaging (Imagopole) Citech af Institut Pasteur (Paris, Frankrig) samt Frankrig-BioImaging infrastruktur-netværk understøttes af det franske nationale forskning agentur (ANR-10-INSB-04; Investeringer i fremtiden), og Région Ile-de-France (program Domaine d'Intérêt Majeursøen-Malinf) for brug af Elyra mikroskop. Dette arbejde blev støttet af den franske National forskning agentur (ANR-16-CE13-0019) og Ligue Contre le Cancer.

1. Coverslips forberedelse (dag 1, 30 min)
<ol><li>Placer 18-mm nº1.5H (170 µm +/-5µm) glas coverslips på en plast rist.</li>
	<li>Sætte rack i et 100 mL bægerglas fyldt med acetone og blød i 5 min.</li>
	<li>Overføre rack til et 100 mL bægerglas fyldt med absolut ethanol og blød i 5 min.</li>
	<li>Overføre rack til en ny 100 mL bægerglas fyldt med absolut ethanol og Bægerglasset anbringes i en ultralyds renere enhed (se tabel over materialer) ved stuetemperatur. Tryk på "on" og vente på 10 min.</li>
	<l...

<ol>
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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>

Kritiske trin i protokollen
Vi præsenterer her en protokol, som minimerer artefakter i dSTORM billeder af mikrotubuli og IFs i gliaceller. Artefakter kan oprettes på hvert trin af prøveforberedelsen og billedbehandling: fiksering, blokering, immunolabeling, glider under erhvervelse, ikke-optimale blinkende betingelser23. Vi liste nedenfor de mest kritiske trin.
Rengøring af coverslips er et vigtigt skridt til at begrænse...

Cytoplasmatisk mellemliggende filamenter (IFs) er 10 nm-diameter homo- eller heteropolymers af en celle type bestemt undersæt af hvis proteiner. IFs deltage i en lang række cellulære funktioner såsom celle motilitet, spredning og stress reaktioner. Deres vigtigste rolle understreges af det faktum, at mere end 90 menneskelige sygdomme er direkte forårsaget af mutationer i hvis proteiner; for eksempel, ændringer i hvis sammensætning ledsager tumor vækst og spredning af1,2,3. Der stigende bevis for, at de tre cytoskeleton systemer fungerer i samarbejde at kontrol cellulære funktioner såsom celle polarisering, division og migration2,3. Da der er en tæt kobling mellem rumlige arkitektur og funktioner, er det afgørende at få indsigt i den strukturelle organisering af IF med de andre filamenter og bedre forstå den cytoskeletal cross-talk. denne artikel indeholder en protokol for at udføre super-resolution teknik tofarvet dSTORM (direkte stokastiske optisk genopbygning mikroskopi)4 og hvordan det bruges til at undersøge samspillet mellem hvis og mikrotubuler i faste, kulturperler celler i 2 dimensioner (2D).
Flere super-resolution teknikker er blevet udviklet i løbet af sidste årti, og der opdagelser var på oprindelsen af Nobelprisen i kemi i 20145. Blandt alle disse teknikker ligger enkelt molekyle lokalisering-baserede metoder som PALM6 (Photo-Activated lokalisering mikroskopi) som bruger photoswitchable fluorescerende proteiner, STORM7 med par fluorophores eller dSTORM4 med konventionel fluorophores. Alle disse metoder er baseret på samme princip, som består af (i) de fleste af de fluorescerende reportere skiftet ind i en "off" tilstand"(ikke-fluorescerende), (ii) den stokastiske aktivering af en delmængde af dem ind i et fluorescerende stat for at lokalisere deres geografiske position med nanometer nøjagtighed, og (iii) en gentagelse af denne proces for at aktivere så mange delmængder af fluorophores som muligt. En endelige billede er rekonstrueret ved hjælp af alle lokaliseringer af de aktiverede molekyler, giver en lateral opløsning ned til ~ 20-40 nm. Flere kommercialiseret optiske systemer så PALM/STORM er nu tilgængelige for biologer for rutinemæssig eksperimenter. Her, blev et sådant system brugt til at studere de strukturelle sammenslutning af mikrotubuli og IFs. Blandt alle de enkelt-molekyle lokalisering-baserede metoder, tofarvet dSTORM teknik blev valgt, fordi det er velegnet til at observere meget tynd gråt regioner (< 1 µm) af vedhængende celler og kan give betydelig forbedring af billedopløsning med minimal investering i tid og penge. DSTORM er faktisk en meget praktisk og alsidigt teknik, der er kompatibel med standard organiske farvestoffer rutinemæssigt anvendes til cellulære farvning og immunfluorescens.
Mens én farve dSTORM billeder er relativt enkle at opnå ved hjælp af fluorophore Alexa6478, kræver dobbelt farve dSTORM for at optimere de eksperimentelle betingelser, så to farvestoffer kan blinke korrekt i den valgte buffer, især når der er begrænset Laser power. Fremragende papirer er allerede tilgængelige på metoder til at oprette flerfarvede imaging med dSTORM9,10,11,12,13, og disse papirer forklare i detaljer de mulige kilder til artefakter og forringet billedopløsningen samt hvordan man kan overvinde dem. I denne artikel, de optimale eksperimentelle betingelser med hensyn til celler fiksering, immunfarvning, prøveforberedelse er imaging erhvervelse og billede genopbygning beskrevet for at erhverve billeder af tætte cytoplasmatisk hvis netværk og mikrotubuli i glial celler med dual-farve dSTORM. Kort efter ekstraktion/fiksering metoden i Chazeau mfl12 blev tilpasset til glial celler og bruges sammen med en efter fiksering skridt, optimeret antistoffer koncentration, en STORM buffer med 10 mM MEA beskrevet i Dempsey9 et al., som var fundet for at være optimal for eksperimenterende sæt op og prøvetypen.
Gliaceller express primært tre typer af hvis proteiner: vimentin, nestin og GFAP (glial sure fibrillære proteiner). Disse tre proteiner blev vist til co polymerisere i astrocytter14. Vi viste tidligere ved hjælp af super-opløsning struktureret belysning mikroskopi (SR SIM), at disse tre hvis proteiner kan findes i samme enkelt hvis glødetråden og at de viser lignende fordeling og dynamik i gliaceller 15. På grund af ligheder mellem de tre hvis proteiner, blev vimentin farvning brugt som en reporter for hele hvis netværket. Ved hjælp af dSTORM, vi har kunnet løse, hvordan hvis form bundter langs mikrotubuli, hvilket ikke var muligt med diffraktion begrænset mikroskopi teknikker15. Disse bemærkninger kan hjælpe til at forstå, hvordan vimentin IFs kan skabelon mikrotubuli og regulere deres vækst bane, fremme bevarelsen af en polaritet akse i celle migration16. Super-opløsning billeder givet vigtige oplysninger om den gensidige interaktion mellem de to cytoskeletal delsystemer og bragt indsigt i sammenhængen mellem fysisk association og lokale funktion, som kunne være celletype specifikke17. I almindelighed, kan tofarvet dSTORM bruges til at studere krydstale mellem cytoskeletal elementer eller andre typer af organeller, forudsat at god immunfarvning betingelser er nået med hensyn til tæthed og specificitet. Denne teknik vil være nyttigt til at bedre karakterisere cytoskeleton ændringer observeret under astrogliosis og i glioblastom, den mest almindelige og mest maligne tumor i centralnervesystemet, hvor udtryk for hvis proteiner er ændret 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

Cytoskeleton, der består af aktin microfilaments, mikrotubuli og mellemliggende filamenter (IF), spiller en central rolle i kontrollen af celle form, polaritet og motilitet. Tilrettelæggelsen af aktin og mikrotubulus net har været grundigt undersøgt, men som af IFs er ikke endnu fuldt karakteriseret. IFs har en gennemsnitlig diameter på 10 nm og form et netværk strækker sig i hele cellen cytoplasma. De er fysisk forbundet med aktin og mikrotubuli gennem molekylære motorer og cytoskeletal linkers. Dette stramme sammenslutning er kernen i de reguleringsmekanismer, der sikrer den koordinerede regulering af de tre cytoskeletal netværk kræves for de fleste cellefunktioner. Det er derfor afgørende at visualisere IFs alene og også sammen med hver af de andre cytoskeletal netværk. Men hvis netværk er meget tætte i de fleste celletyper, navnlig glial celler, hvilket gør sin beslutning meget vanskeligt at opnå med standard Fluorescens mikroskopi (lateral opløsning på ~ 250 nm). Direkte stokastiske optisk genopbygning mikroskopi (dSTORM) er en teknik, der tillader en gevinst i laterale opløsning af en størrelsesorden. Vi viser her, at lateral dSTORM beslutning er tilstrækkelig til at løse den tætte organisation Hvis netværk og navnlig, hvis bundter omkring mikrotubuli. Sådan stram association er tilbøjelige til at deltage i den koordinerede regulering af disse to netværk og maj, forklare hvordan vimentin IFs skabelon og stabilisere mikrotubulus organisation samt kunne påvirke mikrotubulus afhængige vesikulære handel. Mere generelt viser vi hvordan observation af to cytoskeletal komponenter med tofarvet dSTORM teknik bringer ny indsigt i deres gensidigt samspil.

Et mikroskop udstyret med 50 mW 405 nm og 100 mW 488, 561 og 642 nm solid-state lasere, en EMCCD 512 x 512 kamera, en alfa planlægger Apo 100 X / 1.46 mål og Band Pass 570-650 / lang Pass 655 emission filtre blev anvendt til de repræsentative resultater præsenteres nedenfor.
Figur 1A giver et eksempel molekyle tæthed og signal-støj-forhold, der skal bruges under raw-billede erhvervelse. Billed...

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 mellemliggende filamenter og mikrotubuli med 2-dimensionelle direkte stokastiske optisk genopbygning mikroskopi

Det overordnede mål med denne metode er at give de optimale forsøgsbetingelser fra prøveforberedelse til billede erhvervelse og genopbygning for at udføre 2D dobbelt farve dSTORM billeder af mikrotubuli og mellemliggende filamenter i faste 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.

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