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

We bedanken Xufeng Wu voor toegang tot de STORM-microscoop. Dit onderzoek werd ondersteund door het Intramuraal Onderzoeksprogramma van het NCI-Centrum voor Kankeronderzoek en het National Heart Lung and Blood Institute (NHLBI).

Voorzichtigheid: Raadpleeg al de relevante veiligheidsinformatiebladen (MSDS) voor gebruik. Verscheidene van de in dit protocol gebruikte chemicaliën zijn giftig en kankerverwekkend. Gebruik alstublieft alle passende veiligheidspraktijken bij het uitvoeren van het protocol, inclusief het gebruik van technische controles (dampkoker, handschoenkoffer) en persoonlijke beschermingsmiddelen (veiligheidsbril, handschoenen, laboratoriumjas, broek met volledige lengte, gesloten schoenen). 1. Multiple...

<ol>
	<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>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys J. 91 (11), 4258-4272 (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>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>Mortensen, K. I., Churchman, L. S., Spudich, J. A., Flyvbjerg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+localization+analysis+for+single-molecule+tracking+and+super-resolution+microscopy.">Optimized localization analysis for single-molecule tracking and super-resolution microscopy.</a> Nat Methods. 7 (5), 377-381 (2010).</li><li>Thompson, R. E., Larson, D. R., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+nanometer+localization+analysis+for+individual+fluorescent+probes.">Precise nanometer localization analysis for individual fluorescent probes.</a> Biophys J. 82 (5), 2775-2783 (2002).</li><li>Rieger, B., Stallinga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lateral+and+axial+localization+uncertainty+in+super-resolution+light+microscopy.">The lateral and axial localization uncertainty in super-resolution light microscopy.</a> Chemphyschem. 15 (4), 664-670 (2014).</li><li>Pertsinidis, A., Zhang, Y., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnanometre+single-molecule+localization,+registration+and+distance+measurements.">Subnanometre single-molecule localization, registration and distance measurements.</a> Nature. 466 (7306), 647-651 (2010).</li><li>Bates, M., Jones, S. A., 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=Stochastic+optical+reconstruction+microscopy+(STORM):+a+method+for+superresolution+fluorescence+imaging.">Stochastic optical reconstruction microscopy (STORM): a method for superresolution fluorescence imaging.</a> Cold Spring Harb Protoc. (6), 498-520 (2013).</li><li>Yi, 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=madSTORM:+a+superresolution+technique+for+large-scale+multiplexing+at+single-molecule+accuracy.">madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy.</a> Mol Biol Cell. 27 (22), 3591-3600 (2016).</li><li>Bates, M., Huang, B., Dempsey, G. T., 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=Multicolor+super-resolution+imaging+with+photo-switchable+fluorescent+probes.">Multicolor super-resolution imaging with photo-switchable fluorescent probes.</a> Science. 317 (5845), 1749-1753 (2007).</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>Erdelyi, 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=Correcting+chromatic+offset+in+multicolor+super-resolution+localization+microscopy.">Correcting chromatic offset in multicolor super-resolution localization microscopy.</a> Opt Express. 21 (9), 10978-10988 (2013).</li><li>Gerdes, M. 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=Highly+multiplexed+single-cell+analysis+of+formalin-fixed,+paraffin-embedded+cancer+tissue.">Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.</a> Proc Natl Acad Sci U S A. 110 (29), 11982-11987 (2013).</li><li>Jungmann, R., 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=Multiplexed+3D+cellular+super-resolution+imaging+with+DNA-PAINT+and+Exchange-PAINT.">Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT.</a> Nat Methods. 11 (3), 313-318 (2014).</li><li>Nanguneri, S., Flottmann, B., Horstmann, H., Heilemann, M., Kuner, T. <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+tomographic+super-resolution+fluorescence+imaging+of+serially+sectioned+thick+samples.">Three-dimensional tomographic super-resolution fluorescence imaging of serially sectioned thick samples.</a> PLoS One. 7 (5), e38098 (2012).</li><li>Schubert, W., 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=Analyzing+proteome+topology+and+function+by+automated+multidimensional+fluorescence+microscopy.">Analyzing proteome topology and function by automated multidimensional fluorescence microscopy.</a> Nat Biotechnol. 24 (10), 1270-1278 (2006).</li><li>Tam, J., Cordier, G. A., Borbely, J. S., Sandoval Alvarez, A., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk-free+multi-color+STORM+imaging+using+a+single+fluorophore.">Cross-talk-free multi-color STORM imaging using a single fluorophore.</a> PLoS One. 9 (7), e101772 (2014).</li><li>Valley, C. C., Liu, S., Lidke, D. S., Lidke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+superresolution+imaging+of+multiple+targets+using+a+single+fluorophore.">Sequential superresolution imaging of multiple targets using a single fluorophore.</a> PLoS One. 10 (4), e0123941 (2015).</li><li>Hoebe, R. 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=Controlled+light-exposure+microscopy+reduces+photobleaching+and+phototoxicity+in+fluorescence+live-cell+imaging.">Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.</a> Nat Biotechnol. 25 (2), 249-253 (2007).</li><li>Kiuchi, T., Higuchi, M., Takamura, A., Maruoka, M., Watanabe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multitarget+super-resolution+microscopy+with+high-density+labeling+by+exchangeable+probes.">Multitarget super-resolution microscopy with high-density labeling by exchangeable probes.</a> Nat Methods. 12 (8), 743-746 (2015).</li><li>Campi, G., Varma, R., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+agonist+MHC-peptide+complex-dependent+T+cell+receptor+microclusters+as+scaffolds+for+signaling.">Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.</a> J Exp Med. 202 (8), 1031-1036 (2005).</li><li>Sarkar, S. K., 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=Wide-field+in+vivo+background+free+imaging+by+selective+magnetic+modulation+of+nanodiamond+fluorescence.">Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence.</a> Biomed Opt Express. 5 (4), 1190-1202 (2014).</li></ol>

De sequentiële multiplexing, driftcorrectie, en uitlijningsprocedures in madSTORM maken het mogelijk om nauwkeurige, hoogmultiplexeerde visualisatie van heterogene structuren in cellen toe te staan. 10 Bovendien vermijdt madSTORM de beperkingen van multi-color STORM, zoals chromatische aberratie en suboptimale fotowisseling / emissie eigenschappen van niet-ver rode kleurstoffen 9 , 12 . Aangezien de elutiestap sterische interferentie sig...

Een verscheidenheid aan superresolutie microscopie technieken zijn ontwikkeld om de diffractie grens van lichtmicroscopie (~ 200 nm) te overwinnen. Onder deze is een categorie technieken genaamd single molecule localization microscopy (SMLM), waaronder foto-activering lokalisatie microscopie (PALM) en stochastische optische reconstructie microscopie (STORM). SMLM technieken delen in het gebruik van fluorophoren die kunnen worden omgeschakeld tussen aan (fluorescerende) en off (donker / foto-geschakeld) staten, waardoor sequentiële lokalisatie van fluorescentie uit enkele moleculen 1 , 2 , 3 . Door zijn compatibiliteit met commercieel verkrijgbare kleurstoffen en microscopen is direct STORM (dSTORM) uitgegroeid tot een algemeen aanvaarde SMLM techniek 4 . DSTORM kan routinematig ~ 10 nm lokalisatie precisie bereiken, gedefinieerd als de onzekerheid bij het berekenen van het middelpunt van een diffractieIon-beperkte puntverdeling functie (PSF). Ondanks de hoge nauwkeurigheid die is geschat met behulp van lokalisatiealgoritmen 5 , 6 , 7 , is de nauwkeurige bepaling van de werkelijke locatie van enkele moleculen gehinderd door een aantal problemen. Ten eerste, mechanische beweging van de microscoopfase tijdens beeldverwerving voegt aanzienlijke onzekerheid toe aan de nauwkeurigheid van lokalisatie. Aangezien SMLM beelden worden verkregen over duizenden time-lapse frames, kunnen nano-schaalbewegingen van de microscoopfase de precisie van de uiteindelijke superresolutiebeeld 8 aanzienlijk in gevaar brengen. Voor het compenseren van de stadiumbeweging tijdens de beeldverzameling wordt de stadiumdrift algemeen geraamd uit regressiegebaseerde montage van binnenste lokalisaties van de afbeelding zelf (kruiscorrelatie) of sequentiële lokalisaties van fiduciale markeringen (fiduciale correctie) 1 , 9 . HowevEr zijn deze methoden nodig voor het optimaliseren van meerdere parameters voor elke beeldstapel en kunnen niet rekening houden met stadiumbewegingen op korte tijdschalen zoals mechanische trillingen. Gouden nanodeeltjes en multicolor fluorescerende kralen zijn gebruikt als fiduciale markers in SMLM, maar ze zijn niet fotostabiel en resulteren in een aanzienlijk lagere precisie na drijfcorrectie dan de stikstofcentrale fluorescerende nano-diamanten (FND&#39;s) gebruikt In madSTORM 10 . Naast de diffractiegrens wordt de lichtmicroscopie verder beperkt door spectrale grenzen. Gelijktijdige visualisatie van meerdere doelen vereist fluorescerende probes met niet-overlappende spectrale profielen, waarbij de fluorescentie gebaseerde lichtmicroscopie in het algemeen beperkt wordt tot 6 kleuren en SMLM tot 2-3 kleuren 4 , 11 , 12 . Bovendien veroorzaakt niet-lineaire chromatische aberratie verkeerde aanpassing van veelkleurige beelden, wHiervoor zijn uitgebreide uitlijningsprocedures nodig met behulp van multi-colored fiducial markers 8 , 13 . Om deze grenzen te overwinnen hebben eerdere studies meerdere doelen getoond door gebruik te maken van herhalende photobleaching of chemische quenching van achtereenvolgens gebonden fluorophoren 14 , 15 , 16 , 17 , 18 , 19 . Hoewel deze methoden de spectrale grenzen van microscopie kunnen overwinnen, is fluorescentiebleken bekend dat het een toxisch proces 20 is , en langdurig bleken of blussen kunnen ongewenste neveneffecten veroorzaken, zoals verlies van verknoping. Bovendien kan de accumulatie van fluorescerende probes leiden tot sterische blokkering van bindingsplaatsen in het monster, het voorkomen van grootschalige multiplexing en robuuste targeting van epitopen. Om dergelijke sterische interferentie te vermijden, is een recente sTudy bereikt multiplexing met behulp van stochastische uitwisseling van vrij diffuus eiwitfragmenten 21 . Terwijl deze methode een dichte etikettering van cellulaire structuren mogelijk maakt, vereist het uitgebreide biochemische bereiding om peptidefragmenten te isoleren, geen enkele molecuulposities kan isoleren en niet gemakkelijk grootschalige multiplexing vergemakkelijken met gebruik van commercieel verkrijgbare probes. We presenteren een gedetailleerd videoprotocol dat de sequentiële binding en elutie van fluorescente antilichamen voor multiplexed, antilichaamgrootte beperkte dSTORM (madSTORM) imaging, en het gebruik van fluorescerende nano-diamanten om nauwkeurige drijfcorrectie en uitlijning te bereiken, beschrijft.

<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 cellulaire structuren met behulp van single molecule lokalisatie microscopie is gehinderd door onvoldoende lokalisatie precisie en multiplexing vermogen. Met behulp van fluorescerende nano-diamant fiduciale markers beschrijven we de drijfcorrectie- en uitlijningsprocedures die nodig zijn om hoge precisie te verkrijgen in single-molecule lokalisatiemicroscopie. Daarnaast wordt een nieuwe multiplexingstrategie, madSTORM, beschreven waarin meerdere molecules in dezelfde cel worden geteeld met behulp van sequentiële binding en elutie van fluorescerende antilichamen. MadSTORM wordt aangetoond op een geactiveerde T-cel om de locaties van verschillende componenten te visualiseren in een membraan gebonden, multi-eiwitstructuur genaamd de T-celreceptor microcluster. Daarnaast wordt de toepassing van madSTORM als een algemeen hulpmiddel voor het visualiseren van multi-eiwitstructuren besproken.

De sequentiële elutie- en kleurmethode werd gebruikt voor het produceren van het multiplexed madSTORM beeld van microclusters en andere structuren in een geactiveerde Jurkat T-cel ( Figuur 1 , controle-uitlijnen). Elk pseudo-gekleurd beeld vertegenwoordigt één ronde van madSTORM-beeldvorming, verkregen in stappen 1.1.1 tot 1.3.13. Zoals eerder beschreven 10 , moet het zichtveld worden gecontroleerd voor residueel signaal van eerder...

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

We tonen een methode aan om meerdere molecuelen te identificeren binnen heterogene nano-structuren bij nauwkeurige molecuul nauwkeurigheid, met behulp van sequentiële binding en elutie van fluorescent gelabelde antilichamen.

Zeer Multiplexed, Superresolutie Imaging van T-cellen Met behulp van madSTORM

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

The overall goal of this imaging protocol is to visualize multiple proteins in an activated T cell, using single molecule localisation microscopy. This method can help answer key questions in the T cell activation field such as, the single molecule distribution of TC or microcluster components. The main advantage of this technique is that, it allows greater multiplexing than achieved by current supervised solution microscopy methods.To begin, directly conjugate antibodies with Alexa 647 dye, using the Alexa 647 antibody labeling kit, following the labeling protocol provided by the manufacturer. Centrifuge the labeled antibodies for five minutes at 20, 800 times G.Following centrifugation, collect the supernatant to remove the aggregated antibodies. Next, coat A12 cover slip chambers with 250 microliters of 01%poly-l-lyzine for 15 minutes.Then, aspirate the solution without rinsing and dry the chambers at 65 degree celsius for 30 minutes. Prepare dilution of 100 nanometer fluorescent nanodiamonds or FNDs, in one PBS, test various dilutions to ensure that enough FNDs will be visible in each field. Vortex the diluted FNDs for one minute.Next, sonicate the FND supernatant for 30 seconds at a high power setting. Incubate the sonicated FND supernatant in a poly-l-lyzine coated A12 cover slip chamber for 30 minutes at room temperature. After washing the chamber five times, with 1x PBS, visualize the FND coated chamber using 647 nanometer laser excitation on the turf microscope.Use a 100x objective tealed a 61 by 61 micron field of view. Ideally, four to 10 individual FNDs should be visible in the field of view, given the appropriate dilution of FNDs, with at least one FND present in each quadrant of the imaging field. Once the proper density of FNDs has been achieved, add 250 microliters of anti-CD3 antibody to each well.Incubate the chamber for one hour at 37 degree celsius or overnight at four degree celsius. Following incubation, remove the solution from the wells and add 1x PBS for 30 seconds. Repeat this wash step five times.Spin one milliliter of Jurkat T cells at 800 times G for six minutes. Jurkat T cells should ideally be at a concentration of 0.5 to one million cells per milliliter concentration before spinning. Re-suspend the cells in 300 microliters of 1x HBS solution.Meanwhile, add 150 microliters of 1x HBS solution to each well of the chamber and incubate at 37 degree celsius for 15 minutes. Then, add 50 microliters of the re-suspended Jurkat T cells to each well and incubate for three minutes at 37 degree celsius. Next, add 300 microliters of 4%paraformaldehyde to each well and incubate for 30 minutes at 37 degree celsius.After washing three times with 1x PBS, permeabilise the cells, by adding 250 microliters of 01%Triton x solution for five minutes at room temperature. Following three additional washes with 1x PBS, add 250 microliters of 1%fish gelatin solution to each well for 30 minutes at room temperature. Finally, wash the cells three more times with 1x PBS.Add 200 microliters of labeled antibody at 0.1 to 0.5 micrograms per milliliter to the fixed cells for one hour at room temperature. After washing the cells five times with 1x PBS, add one milliliter of STORM buffer and cover the chamber with a glass cover slip to limit exposure. Use clamps on the microscope stage to hold the cover slip chamber in position.This step is critical for large scale multiplexing, since the same field of view must be present after several rounds of washing and incubation. Using a low 647 nanometer laser power in turf mode, locate a stained cell with at least three FNDs in the field of view. Increase the 647 nanometer laser power and acquire the images.Typical parameters can be found in the text protocol. Wash the cells five times with 1x tris-buffered saline or 1x TBS. Then, add one milliliter of elution buffer and incubate at room temperature for one minute.Repeat the elution three times. Use the exact elution conditions and a number of elution rinses needed to remove the signal as specified for each antibody. Then, wash the cells three times with 1x TBS and add one milliliter of 1x PBS.Proper execution of this elution step, will result in removal of previously bound antibody and allow higher efficiency for subsequent antibody labeling. It is important to use TBS for washes to minimize precipitation between elution steps. Photo bleach the cells using a 647 nanometer laser at high power, with a 405 nanometer laser at two to five milliwatts to photo activate Alexa 647 dyes in the dark state.Wait until all remaining signal from non-eluted antibody is photo bleached. Typically, this takes two to five seconds of laser exposure. Acquire sample images, using the STORM settings after illusion and photo bleaching, to confirm removal of the signal.Next, add 250 microliters of 4%paraformaldehyde for 15 minutes. This prevents reverse cross-linking of fixed molecules in the cell. Wash the cells three times with 1x PBS.Repeat these steps for sequential labeling of multiple targets. Finally, perform drift correction and alignment of multiplex image stacks at described in the text protocol. The sequential illusion and standing method was used to produce the multiplex MET STORM image of microclusters and other structures in an activated Jurkat T cell.The final MET STORM image has been corrected for a drift and alignment, using average fiducial correction and FND fiducial markers. Shown here, are single molecule localisation microscopy images of a single FND localized in 30, 000 image frames before and after drift correction with average fiducial correction. Similarly, here are single molecule localisation microscopy images of an activated Jurkat T cell stained with anti-phosphorelated SLP76 antibody and FND futusial markers, before and after drift correction with average futusial correction.After watching this video, you should have a good understanding of how to perform multiplexed super resolution imaging of proteins in an activated T cell, using MET STORM. Once mastered, this technique can be done in three hours for each multiplexing round. While attempting this procedure, it's important to remember to keep the specimen in the same position on the microscope stage.This can be done using stage clamps. Don't forget that working with our modified STORM buffer, can be extremely hazardous and precautions such as, respirators and goggles should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Retrovirale Transductie van T-cel-receptoren in Mouse T-cellen

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo beeldvorming van T-cellen in muizen lymfkliertest Plakjes met Widefield en confocale microscopen

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Uitbreiding van de humane perifere bloed γδ T-cellen met behulp van zoledronaat

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

Genereren van Induced regulatoire T-cellen uit primaire menselijke naïef en geheugen T-cellen

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Hoogst vastbesloten Intravitale Striped-verlichting Microscopie van Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Een gemiddelde van virale envelop glycoproteïne Spikes uit Electron Cryotomography Reconstructies met Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Super-resolutie beeldvorming van de Natural Killer Cell Immunological Synapse op een glas-ondersteunde Planar lipidendubbellaag

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analyse van<em&gt; Yersinia enterocolitica</em&gt; Effector Translocatie in gastheercellen met behulp van beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Corticale actine Flow in T-cellen gekwantificeerd door ruimte en tijd Afbeelding Correlatie Spectroscopie van Structured Verlichting Microscopie Gegevens

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In vitro differentiatie van humane CD4 + FOXP3 + geïnduceerde regulatoire T-cellen (iTregs) van naïeve CD4 + T cellen met behulp van een TGF-β-bevattende Protocol