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

Agradecemos ao Xufeng Wu o acesso ao microscópio STORM. Esta pesquisa foi apoiada pelo Programa de Pesquisa Intramural do Instituto Nacional do Câncer (NCI) Center for Cancer Research e do National Heart Lung and Blood Institute (NHLBI).

Cuidado: consulte todas as folhas relevantes de dados de segurança de material (MSDS) antes de usar. Vários dos produtos químicos utilizados neste protocolo são tóxicos e cancerígenos. Use todas as práticas de segurança adequadas ao executar o protocolo, incluindo o uso de controles de engenharia (exaustor, porta-luvas) e equipamento de proteção pessoal (óculos de segurança, luvas, bata de laboratório, calças completas, sapatos fechados). 1. Imagem multiplexada de células T ativ...

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Os procedimentos de multiplexagem seqüencial, correção de deriva e alinhamento em madSTORM permitem uma visualização precisa e altamente multiplexada de estruturas heterogêneas nas células. 10 Além disso, madSTORM evita as limitações de STORM multicolor, como a aberração cromática e as propriedades de troca / troca de fotos sub-ótimas de corantes vermelhos não distantes 9 , 12 . À medida que o passo de eluição reduz sign...

Uma variedade de técnicas de microscopia de super resolução foi desenvolvida para superar o limite de difracção do microscópio de luz (~ 200 nm). Entre estas, uma categoria de técnicas denominada microscopia de localização de moléculas únicas (SMLM), que inclui microscopia de localização foto-ativação (PALM) e microscopia estocástica de reconstrução óptica (STORM). As técnicas SMLM compartilham o uso de fluoróforos que podem ser alternados entre os estados fluorescentes (offs) e off (dark / photo-switched), permitindo a localização sequencial da fluorescência a partir de moléculas únicas 1 , 2 , 3 . Devido à sua compatibilidade com corantes e microscópios comercialmente disponíveis, o STORM direto (dSTORM) tornou-se uma técnica SMLM amplamente adotada 4 . DSTORM pode normalmente atingir uma precisão de localização de ~ 10 nm, definida como a incerteza no cálculo do centro de uma difraçãoFunção de espalhamento de ponto limitado a iões (PSF). No entanto, apesar da alta precisão estimada usando os algoritmos de localização 5 , 6 , 7 , a determinação precisa da localização real de moléculas únicas foi prejudicada por uma série de problemas. Primeiro, o movimento mecânico do estágio do microscópio durante a aquisição da imagem aumenta a incerteza significativa na precisão da localização. À medida que as imagens SMLM são obtidas através de milhares de quadros de lapso de tempo, os movimentos de nano-escala do estágio do microscópio podem comprometer significativamente a precisão da imagem super-resolução final 8 . Para compensar o movimento do palco durante a aquisição da imagem, a derivação do estágio é geralmente estimada a partir de ajustes baseados em regressão de localizações binadas da própria imagem (correlação cruzada) ou localizações seqüenciais de marcadores fiduciais (correção fiducial) 1 , 9 . HowevEsses métodos requerem a otimização de vários parâmetros para cada pilha de imagens e não podem ser considerados movimentos de estágio em escalas de curta duração, como a vibração mecânica. As nanopartículas de ouro e as esferas fluorescentes multicoloridas foram usadas como marcadores fiduciários no SMLM, mas não são estáveis ​​em fotos e resultam em precisão significativamente menor após a correção da deriva do que os nano-diamantes fluorescentes de centro de vacância de nitrogênio (FNDs) utilizados Em madSTORM 10 . Além do limite de difracção, o microscópio de luz é ainda restringido por limites espectrais. A visualização simultânea de múltiplos alvos requer sondas fluorescentes com perfis espectrais não sobrepostos, geralmente restringindo o microscópio de luz à base de fluorescência a 6 cores e SMLM a 2-3 cores 4 , 11 , 12 . Além disso, a aberração cromática não linear provoca desalinhamento de imagens multicoloridas, wQue exigem extensos procedimentos de alinhamento usando marcadores fiduciários multicoloridos 8 , 13 . Para superar esses limites, estudos anteriores criaram imagens de múltiplos alvos usando fotobloco repetitivo ou extinção química de fluoróforos 14 , 15 , 16 , 17 , 18 , 19 . Embora estes métodos possam superar os limites espectrais da microscopia, o branqueamento com fluorescência é conhecido por ser um processo tóxico 20 , e o branqueamento prolongado ou a extinção pode causar efeitos colaterais indesejáveis, como perda de reticulação. Além disso, o acúmulo de sondas fluorescentes poderia levar ao bloqueio estérico de locais de ligação na amostra, evitando a multiplexação em grande escala e a segmentação robusta de epítopos. Para evitar tais interferências estéticas, uma recente sTudy conseguiu a multiplexação usando a troca estocástica de fragmentos de proteínas que difundem livremente 21 . Considerando que este método permite rotulagem densa de estruturas celulares, requer uma preparação bioquímica extensa para isolar fragmentos de péptidos, não pode localizar posições de moléculas únicas e não prontamente facilita a multiplexação em larga escala usando sondas comercialmente disponíveis. Apresentamos um protocolo de vídeo detalhado que descreve a ligação seqüencial e a eluição de anticorpos fluorescentes para imagens multiplexadas, tamanho limitado de anticorpos dSTORM (madSTORM) e uso de nano-diamantes fluorescentes para obter correção e alinhamento preciso da deriva.

<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

A imagem de estruturas celulares heterogêneas usando microscopia de localização de molécula única foi dificultada por precisão de localização inadequada e capacidade de multiplexação. Usando marcadores fiduciários de nano-diamante fluorescentes, descrevemos os procedimentos de correção e alinhamento da deriva necessários para obter alta precisão em microscopia de localização única de moléculas. Além disso, uma nova estratégia de multiplexação, madSTORM, é descrita em que moléculas múltiplas são direcionadas na mesma célula usando ligação seqüencial e eluição de anticorpos fluorescentes. MadSTORM é demonstrado em uma célula T ativada para visualizar as localizações de diferentes componentes dentro de uma estrutura de múltiplas proteínas ligada à membrana chamada de microcluster do receptor de células T. Além disso, a aplicação de madSTORM como uma ferramenta geral para a visualização de estruturas multi-proteínas é discutida.

O método de eluição e coloração seqüencial foi utilizado para produzir a imagem madSTORM multiplexada de microclusters e outras estruturas em uma célula T Jurkat ativada ( Figura 1 , verifique os alinhamentos das figuras). Cada imagem pseudo-colorida representa uma rodada de imagens madSTORM adquiridas nas etapas 1.1.1 a 1.3.13. Conforme descrito anteriormente 10 , o campo de visão deve ser monitorado quanto ao sinal residual ...

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

Demonstamos um método para imagem de múltiplas moléculas dentro de nanoestruturas heterogêneas com precisão de molécula única usando ligação sequencial e eluição de anticorpos marcados fluorescentemente.

Imagem altamente multiplexada e de super resolução de células T usando 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

Transdução retroviral de receptor de células T no rato células T

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 ...

Imunologia e Infecção

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 de imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

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

Expansão do sangue periférico humano γδ células T usando zoledronato

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

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

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

Altamente Resolvido Microscopia intravital Striped-iluminação dos centros germinativos

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

Calculando a média de Viral glicoproteína do envelope Spikes de Electron Cryotomography reconstruções com 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-Resolution Imaging da Célula Natural Killer imunológica Synapse em um vidro com suporte Planar Lipid Bilayer

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

Análises de<em&gt; Yersinia enterocolitica</em&gt; Effector translocação em células hospedeiras Usando 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

Cortical actina Fluxo nas células T quantificada pela correlação espacial e temporal Imagem Espectroscopia de iluminação estruturada Microscopia Dados

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

Na diferenciação in vitro de células T CD4 + humano FOXP3 + Induzida T reguladoras (iTregs) a partir de células naive T CD4 + usando um protocolo de TGF-β contendo