Name: highly multiplexed, super-resolution imaging of t cells using madstorm
Uploaded: 2017-06-24T12:30:00
Description: Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Vi takker Xufeng Wu for adgang til STORM-mikroskopet. Denne forskning blev støttet af Intramural Research Programmet fra National Cancer Institute (NCI) Center for Cancer Research og National Heart Lung and Blood Institute (NHLBI).

Forsigtig: Se venligst alle relevante sikkerhedsdatablade (MSDS) inden brug. Flere af de kemikalier, der anvendes i denne protokol, er toksiske og kræftfremkaldende. Brug venligst alle relevante sikkerhedsprincipper, når du udfører protokollen, herunder brug af tekniske kontroller (dampkåbe, handskerum) og personlige værnemidler (sikkerhedsbriller, handsker, lab coat, fuld længde bukser, lukkede sko). 1. Multiplexet billeddannelse af aktiverede T-celler <ol><li> Direkte konjugerede a...

<ol>
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Den sekventielle multiplexering, driftskorrektion og justeringsprocedurer i madSTORM tillader præcis, højmultiplexeret visualisering af heterogene strukturer i celler. 10 Derudover undgår madSTORM begrænsningerne i multi-farve STORM, såsom kromatisk aberration og suboptimale fotoswitching / emissionsegenskaber af ikke-farfarfarvestoffer 9 , 12 . Da elueringstrinnet signifikant reducerer sterisk interferens fra sekventielt bundne anti...

En række superopløsningsmikroskopiteknikker er blevet udviklet til at overvinde diffraktionsgrænsen for lysmikroskopi (~ 200 nm). Blandt disse er en kategori af teknikker kaldet single molecule localization microscopy (SMLM), som omfatter fotoaktivering lokalisering mikroskopi (PALM) og stokastisk optisk rekonstruktion mikroskopi (STORM). SMLM teknikker deler i brugen af ​​fluoroforer, der kan skiftes mellem på (fluorescerende) og off (mørke / foto-switchede) tilstande, hvilket muliggør sekventiel lokalisering af fluorescens fra enkeltmolekyler 1 , 2 , 3 . På grund af dets kompatibilitet med kommercielt tilgængelige farvestoffer og mikroskoper er direkte STORM (dSTORM) blevet en almindeligt vedtaget SMLM-teknik 4 . DSTORM kan rutinemæssigt opnå ~ 10 nm lokaliseringspræcision, defineret som usikkerheden ved beregning af centrum for en diffraktionIonbegrænset punktspredningsfunktion (PSF). På trods af den høje præcision, der anslås ved hjælp af lokaliseringsalgoritmerne 5 , 6 , 7 , er nøjagtig bestemmelse af den faktiske placering af enkeltmolekyler blevet hæmmet af en række problemer. For det første tilføjer mekanisk bevægelse af mikroskopfasen under billedopsamling betydelig usikkerhed omkring lokaliseringspræcision. Da SMLM-billeder opnås over tusindvis af tidsrammer, kan nano-skala bevægelser i mikroskopetrinnet betydeligt kompromittere præcisionen af ​​det endelige superopløsningsbillede 8 . For at kompensere for trinbevægelse under billedkøb, estimeres scenydrift almindeligvis fra regressionsbaseret montering af in-situ lokaliseringer fra selve billedet (krydskorrelation) eller sekventielle lokaliseringer fra fiducial-markører (fiducial-korrektion) 1 , 9 . HowevEr, disse metoder kræver optimering af flere parametre for hver billedstabel og kan ikke tage højde for scenebevægelser på kort tidskalaer som mekanisk vibration. Guldnano-partikler og flerfarvede fluorescerende perler er blevet anvendt som fiducial markører i SMLM, men de er ikke fotostabile og resulterer i signifikant lavere præcision efter driftskorrektion end nitrogen fluorescerende nanodiamanter (FND&#39;er) I madSTORM 10 . Ud over diffraktionsgrænsen er lysmikroskopi yderligere begrænset af spektrale grænser. Samtidig visualisering af flere mål kræver fluorescerende sonder med ikke-overlappende spektrale profiler, der generelt begrænser fluorescensbaseret lysmikroskopi til 6 farver og SMLM til 2-3 farver 4 , 11 , 12 . Desuden forårsager ikke-lineær kromatisk afvigelse forskydning af flerfarvede billeder, wDer kræver omfattende justeringsprocedurer ved brug af flerfarvede fiducial markører 8 , 13 . For at overvinde disse grænser har tidligere undersøgelser afbildet flere mål ved anvendelse af gentagen fotoblegning eller kemisk quenching af sekventielt bundne fluoroforer 14 , 15 , 16 , 17 , 18 , 19 . Selvom disse fremgangsmåder kan overvinde mikroskopiens spektrale grænser, er fluorescensblegning kendt for at være en toksisk proces 20 , og langvarig blegning eller slukning kan forårsage uønskede bivirkninger såsom tab af tværbinding. Desuden kan akkumuleringen af ​​fluorescerende prober føre til sterisk blokering af bindingssteder i prøven, hvilket forhindrer storskala multiplexering og robust målretning af epitoper. For at undgå sådan sterisk indblanding er en nylig sTudy opnået multipleksering ved anvendelse af stokastisk udveksling af frit diffunderende proteinfragmenter 21 . Mens denne metode tillader tæt mærkning af cellulære strukturer, kræver det omfattende biokemisk præparation til isolering af peptidfragmenter, kan ikke lokalisere enkeltmolekylpositioner og letter ikke let ved hjælp af storskala multiplexering ved anvendelse af kommercielt tilgængelige prober. Vi præsenterer en detaljeret videoprotokol, der beskriver sekventiel binding og eluering af fluorescensantistoffer til multiplexeret, antistofstørrelsesbegrænset dSTORM (madSTORM) billedbehandling og anvendelse af fluorescerende nanodiamanter for at opnå præcis driftskorrektion og justering.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">8 well coverslip chamber</td>
			<td style="width:117px;">Lab-tek</td>
			<td style="width:124px;">155409</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% Poly-L-Lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NV-100nm Fluorescent Nano-diamond</td>
			<td>Adamas</td>
			<td>Red FND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD3&#949; antibody</td>
			<td>BD Biosciences</td>
			<td>555329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, Fraction V</td>
			<td>KSE Scientific</td>
			<td>98-100P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Paraformaldehyde</td>
			<td>EMS</td>
			<td>15712</td>
			<td>Carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from fresh water fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647 antibody labeling kit</td>
			<td>Thermo Fisher</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7154</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>30070</td>
			<td>Highly toxic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclooctatetraene 98%</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>KD Medical</td>
			<td>RGF-3210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x TBS</td>
			<td>KD Medical</td>
			<td>RGF-3385</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly multiplexed, super-resolution imaging of t cells using madstorm

Imaging heterogene cellulære strukturer ved anvendelse af enkeltmolekyl lokalisering mikroskopi er blevet forhindret af utilstrækkelig lokalisering præcision og multiplexing evne. Ved hjælp af fluorescerende nano-diamant fiducial markører beskriver vi driftskorrektions- og justeringsprocedurerne, der kræves for at opnå høj præcision i enkeltmolekyle lokaliseringsmikroskopi. Derudover er der beskrevet en ny multiplexeringsstrategi, madSTORM, hvori flere molekyler er målrettet i den samme celle ved anvendelse af sekventiel binding og eluering af fluorescerende antistoffer. MadSTORM er demonstreret på en aktiveret T-celle for at visualisere placeringen af ​​forskellige komponenter i en membranbundet, multi-proteinstruktur kaldet T-cellereceptor-mikroklassen. Derudover er anvendelsen af ​​madSTORM som et generelt værktøj til visualisering af multi-proteinstrukturer diskuteret.

Den sekventielle eluerings- og farvningsfremgangsmåde blev anvendt til at fremstille det multipleksede madSTORM-billede af mikroklyngere og andre strukturer i en aktiveret Jurkat T-celle ( Figur 1 , kontrolfigurjusteringer). Hvert pseudo-farvet billede repræsenterer en runde madSTORM-billeddannelse erhvervet i trin 1.1.1 til 1.3.13. Som beskrevet tidligere 10 skal synsfeltet overvåges for restsignal fra tidligere, ikke-elueret anti...

Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Vi demonstrerer en metode til at vise flere molekyler inden for heterogene nanostrukturer ved nøjagtighed af enkeltmolekyler ved hjælp af sekventiel binding og eluering af fluorescensmærkede antistoffer.

Meget multiplexeret, superopløselig billeddannelse af T-celler ved hjælp af madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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