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 tackar Xufeng Wu för åtkomst till STORM-mikroskopet. Denna forskning stöddes av Intramural Research Programmet från National Cancer Institute (NCI) Center for Cancer Research och National Heart Lung and Blood Institute (NHLBI).

Varning: Vänligen se alla relevanta säkerhetsdatablad (MSDS) före användning. Flera av de kemikalier som används i detta protokoll är giftiga och cancerframkallande. Använd alla lämpliga säkerhetsmetoder vid genomförandet av protokollet, inklusive användning av tekniska kontroller (spishäll, handskfack) och personlig skyddsutrustning (skyddsglasögon, handskar, labbrock, fulla längdbyxor, stängda tånskor). 1. Multiplexerad bildning av aktiverade T-celler <ol><li> Direktkonju...

<ol>
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De sekventiella multiplexerings-, driftkorrigerings- och anpassningsförfarandena i madSTORM möjliggör exakt, högmultiplexerad visualisering av heterogena strukturer i celler. 10 Dessutom undviker madSTORM gränserna för multi-color STORM, såsom kromatisk aberration och suboptimal photowitching / emissionsegenskaper för icke-farfärgade färgämnen 9 , 12 . Eftersom elueringssteget signifikant minskar sterisk interferens från sekve...

En mängd mikrosupplösningsmikroskopitekniker har utvecklats för att övervinna diffraktionsgränsen för ljusmikroskopi (~ 200 nm). Bland dessa är en kategori av tekniker som kallas single molecule localization microscopy (SMLM) som inkluderar fotoaktivering lokalisering mikroskopi (PALM) och stokastisk optisk rekonstruktion mikroskopi (STORM). SMLM-tekniker delar i användningen av fluoroforer som kan växlas mellan på (fluorescerande) och av (mörka / fotoomkopplade) tillstånd, vilket möjliggör sekventiell lokalisering av fluorescens från enskilda molekyler 1 , 2 , 3 . På grund av dess kompatibilitet med kommersiellt tillgängliga färgämnen och mikroskop har direkt STORM (dSTORM) blivit en allmänt vedertagen SMLM-teknik 4 . DSTORM kan rutinmässigt uppnå ~ 10 nm lokaliserings precision, definierad som osäkerheten vid beräkning av centrum för en diffraktionJon-begränsad punktspridningsfunktion (PSF). Trots den höga precisionen som uppskattats med användning av lokaliseringsalgoritmerna 5 , 6 , 7 har emellertid en bestämd bestämning av den faktiska placeringen av enskilda molekyler hindrats av ett antal problem. För det första lägger mekanisk rörelse av mikroskopsteget under bildförvärv stor osäkerhet på lokaliserings precision. När SMLM-bilder erhålls över tusentals tidsramar kan nanoskala-rörelser i mikroskopsteget på ett väsentligt sätt kompromissa med precisionen hos den slutliga superupplösningsbilden 8 . För att kompensera för scenrörelse under bildförvärv beräknas stegdrift vanligtvis från regressionsbaserad montering av inåtpositioneringar från själva bilden (korskorrelation) eller sekventiella lokaliseringar från fiducialmarkörer (fiducial-korrektion) 1 , 9 . howevDet innebär att dessa metoder kräver optimering av flera parametrar för varje bildstapel och kan inte redovisa scenrörelser vid korta tidsskalor som mekanisk vibration. Guldnanopartiklar och flerfärgade fluorescerande pärlor har använts som fiducialmarkörer i SMLM, men de är inte fotostabila och resulterar i signifikant lägre precision efter driftskorrigering än de fluorescerande nanodiamanterna (FND) som används för kvävgascentraler. I madSTORM 10 . Förutom diffraktionsgränsen är ljusmikroskopi ytterligare begränsad av spektralgränser. Samtidig visualisering av flera mål kräver fluorescerande sonder med icke-överlappande spektralprofiler, vilket i allmänhet begränsar fluorescensbaserad ljusmikroskopi till 6 färger och SMLM till 2-3 färger 4 , 11 , 12 . Dessutom orsakar icke-linjär kromatisk avvikelse felinriktning av mångfärgade bilder, wSom kräver omfattande anpassningsförfaranden med hjälp av flerfärgade fiducialmarkörer 8 , 13 . För att övervinna dessa gränser har tidigare studier avbildat flera mål med användning av repetitiv fotobildning eller kemisk släckning av sekventiellt bundna fluoroforer 14 , 15 , 16 , 17 , 18 , 19 . Medan dessa metoder kan övervinna de spektrala gränserna för mikroskopi, är fluorescensblekning känt för att vara en toxisk process 20 , och långvarig blekning eller släckning kan orsaka oönskade bieffekter såsom förlust av tvärbindning. Vidare kan ackumuleringen av fluorescerande prober leda till sterisk blockering av bindningsställen i provet, förhindrande av storskalig multiplexering och robust målning av epitoper. För att undvika sådan sterisk störning, en nyligen sTudy uppnådd multiplexering med användning av stokastisk utbyte av fritt diffunderande proteinfragment 21 . Medan denna metod tillåter tät märkning av cellulära strukturer krävs det omfattande biokemisk beredning för att isolera peptidfragment, kan inte lokalisera molekylpositioner och lättar inte lätt för storskalig multiplexering med användning av kommersiellt tillgängliga prober. Vi presenterar ett detaljerat videoprotokoll som beskriver sekventiell bindning och eluering av fluorescensantikroppar för multiplexerad, antikroppstorleksbegränsad dSTORM (madSTORM) avbildning och användning av fluorescerande nanodiamanter för att uppnå exakt driftskorrigering och anpassning.

<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 heterogena cellulära strukturer med användning av molekyl-lokaliseringsmikroskopi har hindrats av otillräcklig lokaliserings precision och multiplexeringsförmåga. Med hjälp av fluorescerande nano-diamantfiducialmarkörer beskriver vi driftkorrigerings- och anpassningsförfarandena som erfordras för att erhålla hög precision i enkelmolekyl lokaliseringsmikroskopi. Dessutom beskrivs en ny multiplexstrategi, madSTORM, i vilken multipla molekyler är riktade i samma cell med användning av sekventiell bindning och eluering av fluorescerande antikroppar. MadSTORM demonstreras på en aktiverad T-cell för att visualisera läget för olika komponenter i en membranbunden, multi-proteinstruktur kallad T-cellreceptormikroklusten. Dessutom diskuteras applicering av madSTORM som ett allmänt verktyg för visualisering av multi-proteinstrukturer.

Den sekventiella eluerings- och färgningsmetoden användes för framställning av den multiplexerade madSTORM-bilden av mikrokluster och andra strukturer i en aktiverad Jurkat T-cell ( Figur 1 , kontrollfigurinställningar). Varje pseudo-färgad bild representerar en runda av madSTORM-bildbehandling som erhållits i steg 1.1.1 till 1.3.13. Som beskrivits tidigare 10 bör synfältet övervakas för restsignalen från tidigare, icke-el...

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 demonstrerar en metod för att bilda flera molekyler inom heterogena nanostrukturer vid enkel molekyls noggrannhet med användning av sekventiell bindning och eluering av fluorescensmärkta antikroppar.

Mycket multiplexerad, superupplösning av T-celler med hjälp av 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

Retroviral Transduction av T-cell receptorer i Mus T-celler

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 Imaging av T-celler i murina Slices lymfkörtel med Widefield och Confocal Mikroskop

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

Utbyggnad av Human Peripheral Blood γδ T-celler med hjälp zoledronate

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

Generering av Inducerade regulatoriska T-celler från primära humana naiva och minne T-celler

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

Mycket Lösta Intravital Randig-belysning Mikroskopi germinalcenter

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

Medelvärdes av Viral höljesglykoproteinet Spikes från Electron Cryotomography rekonstruktioner använder 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-upplösning avbildning av Natural Killer Cell Immunologisk Synapse på en Glass-stött Planar lipidbiskiktet

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

Analys av<em&gt; Yersinia enterocolitica</em&gt; Effector translokation i värdceller Använda Beta-laktamas 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

Kortikal Actin Flow i T-celler kvantifieras genom Spatio-temporal bild korrelationsspektroskopi av Structured belysning mikroskopi Data

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-differentiering av humana CD4 + Foxp3 + Induced regulatoriska T-celler (iTregs) från naiva CD4 + -T-celler med ett TGF-β-innehållande protokoll