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

Damos las gracias a Xufeng Wu por el acceso al microscopio STORM. Esta investigación fue apoyada por el Programa de Investigación Intramural del Instituto Nacional del Cáncer (NCI) Centro de Investigación del Cáncer y el Instituto Nacional de Pulmón del Corazón y la Sangre (NHLBI).

Precaución: Antes de usar, consulte todas las hojas de datos de seguridad del material (MSDS) pertinentes. Varios de los productos químicos utilizados en este protocolo son tóxicos y carcinógenos. Utilice todas las prácticas de seguridad apropiadas cuando realice el protocolo, incluyendo el uso de controles de ingeniería (campana extractora de humos, guantera) y equipo de protección personal (gafas de seguridad, guantes, bata de laboratorio, pantalones largos, zapatos cerrados). 1. Imá...

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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. 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Los procedimientos de multiplexación secuencial, corrección de deriva y alineación en madSTORM permiten una visualización precisa y altamente multiplexada de estructuras heterogéneas en células. 10 Además, madSTORM evita las limitaciones de la TORMENTA multicolor como la aberración cromática y las propiedades de fotosensibilización / emisión subóptimas de colorantes rojos no lejanos 9 , 12 . A medida que la etapa de elución r...

Se han desarrollado una variedad de técnicas de microscopía de super resolución para superar el límite de difracción de la microscopía óptica (~ 200 nm). Entre éstas se encuentra una categoría de técnicas denominadas microscopía de localización de moléculas individuales (SMLM) que incluye microscopía de localización por fotoactivación (PALM) y microscopía óptica de reconstrucción estocástica (STORM). Las técnicas SMLM comparten el uso de fluoróforos que pueden ser conmutados entre los estados (fluorescente) y apagado (oscuro / foto-conmutado), permitiendo la localización secuencial de la fluorescencia de las moléculas individuales 1 , 2 , 3 . Debido a su compatibilidad con colorantes y microscopios comercialmente disponibles, STORM directa (dSTORM) se ha convertido en una técnica SMLM ampliamente adoptada 4 . DSTORM puede alcanzar rutinariamente una precisión de localización de ~ 10 nm, definida como la incertidumbre en el cálculo del centro de un difractoFunción de propagación de puntos limitados por iones (PSF). Sin embargo, a pesar de la alta precisión estimada utilizando los algoritmos de localización 5 , 6 , 7 , la determinación exacta de la ubicación real de las moléculas individuales se ha visto obstaculizada por una serie de cuestiones. En primer lugar, el movimiento mecánico de la etapa del microscopio durante la adquisición de imágenes añade una incertidumbre significativa a la precisión de localización. Como las imágenes SMLM se obtienen a lo largo de miles de marcos de lapso de tiempo, los movimientos a escala nanométrica de la etapa del microscopio pueden comprometer significativamente la precisión de la imagen de superresolución final 8 . Para compensar el movimiento de la etapa durante la adquisición de la imagen, la deriva de la etapa es comúnmente estimada a partir de la adaptación basada en regresión de localizaciones binned de la propia imagen (correlación cruzada) o localizaciones secuenciales de los marcadores fiduciales (corrección fiducial) 1 , 9 . Sin embargoEstos métodos requieren la optimización de múltiples parámetros para cada pila de imágenes y no pueden dar cuenta de movimientos de escenario a escalas de tiempo cortas tales como vibración mecánica. Las nanopartículas de oro y las bolas fluorescentes multicolores se han utilizado como marcadores fiduciales en SMLM, pero no son foto-estables y resultan en una precisión significativamente menor después de la corrección de la desviación que los nano-diamantes fluorescentes (FND) En madSTORM 10 . Además del límite de difracción, la microscopía óptica está restringida además por límites espectrales. La visualización simultánea de objetivos múltiples requiere sondas fluorescentes con perfiles espectrales que no se superponen, restringiendo generalmente la microscopía de luz basada en fluorescencia a 6 colores y SMLM a 2-3 colores 4 , 11 , 12 . Además, la aberración cromática no lineal causa desalineación de las imágenes multicolor, wQue requieren amplios procedimientos de alineación utilizando marcadores fiduciales multicolor 8 , 13 . Para superar estos límites, los estudios previos han imaginado múltiples objetivos utilizando fotoblanqueo repetitivo o quimioterapia química de fluoróforos unidos secuencialmente 14 , 15 , 16 , 17 , 18 , 19 . Aunque estos métodos pueden superar los límites espectrales de la microscopia, el blanqueo por fluorescencia es conocido por ser un proceso tóxico 20 , y el blanqueo o apagado prolongado puede causar efectos secundarios no deseados tales como pérdida de reticulación. Además, la acumulación de sondas fluorescentes podría conducir al bloqueo estérico de sitios de unión en la muestra, impidiendo la multiplexación a gran escala y la focalización robusta de epítopos. Para evitar tal interferencia estérica, una recienteTudy logrado multiplexing utilizando intercambio estocástico de libremente difundir fragmentos de proteínas [ 21] . Mientras que este método permite el etiquetado denso de las estructuras celulares, requiere una preparación bioquímica extensa para aislar fragmentos peptídicos, no puede localizar posiciones de una sola molécula y no facilita fácilmente el multiplexado a gran escala usando sondas comercialmente disponibles. Presentamos un protocolo detallado de video que describe la unión secuencial y la elución de anticuerpos fluorescentes para la obtención de imágenes dSTORM (madSTORM) de tamaño limitado de anticuerpos, y el uso de nano-diamantes fluorescentes para lograr corrección y alineación precisa de la 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

La creación de imágenes de estructuras celulares heterogéneas mediante la microscopía de localización de moléculas individuales se ha visto dificultada por la precisión de localización y la capacidad de multiplexación inadecuadas. Utilizando marcadores fiduciales fluorescentes de nano-diamante, describimos los procedimientos de corrección y alineación de deriva requeridos para obtener alta precisión en microscopía de localización de molécula única. Además, se describe una nueva estrategia de multiplexación, madSTORM, en la que se dirigen múltiples moléculas en la misma célula usando unión secuencial y elución de anticuerpos fluorescentes. MadSTORM se demuestra en una célula T activada para visualizar las ubicaciones de los diferentes componentes dentro de una membrana-bound, multi-proteína estructura llamada el microcluster de células T receptor. Además, se discute la aplicación de madSTORM como una herramienta general para la visualización de estructuras multi-proteína.

Se utilizó el método secuencial de elución y tinción para producir la imagen madSTORM multiplexada de microclusters y otras estructuras en una célula T de Jurkat activada ( Figura 1 , alineaciones de figuras de comprobación). Cada imagen pseudo-coloreada representa una ronda de formación de imágenes madSTORM adquirida en los pasos 1.1.1 a 1.3.13. Como se ha descrito anteriormente 10 , el campo de visión debe ser monitorizado ...

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

Demostramos un método para la imagen de múltiples moléculas dentro de nano-estructuras heterogéneas con una sola molécula exactitud utilizando secuencial vinculante y elución de fluorescently marcó anticuerpos.

Multiplexado de alta resolución de imágenes de células T utilizando 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

Transducción retroviral de receptores de células T en ratones las 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 ...

Investigación

Inmunología e Infección

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 imágenes de células T en los ganglios linfáticos murino Rebanadas con campo amplio y microscopios 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

Expansión de la sangre periférica humana de células T γδ con 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

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

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

Muy resuelto Microscopía intravital rayas-iluminación de centros germinales

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

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando 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-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

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

Análisis de<em&gt; Yersinia enterocolitica</em&gt; Efector translocación en las células huésped Usando beta-lactamasa Efector fusiones

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 de actina de flujo en células T cuantificados por espacio-temporal de correlación Imagen Espectroscopia de Estructurado Iluminación Microscopía de datos

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

Diferenciación in vitro de las células CD4 + humano FOXP3 + células T reguladoras (Induced iTregs) de T naive CD4 + células utilizando un protocolo de TGF-β que contienen