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

我々はSTORM顕微鏡へのアクセスのためにXufeng Wuに感謝します。この研究は、国立がん研究所（NCI）がん研究センターと国立心臓肺血液研究所（NHLBI）の教室内研究プログラムによって支援されました。

注意：使用前に関連するすべての物質安全性データシート（MSDS）を参照してください。このプロトコルで使用される化学物質のいくつかは有毒で発癌性があります。エンジニアリングコントロール（ヒュームフード、グローブボックス）および個人用保護具（安全眼鏡、手袋、ラボコート、全長ズボン、クローズトゥーシューズ）の使用を含む、プロトコールを実行する際は、適切なすべて...

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madSTORMのシーケンシャルマルチプレックス、ドリフト補正、アラインメントの手順により、細胞内の異種構造の正確かつ高度に多重化された可視化が可能になります。さらに、madSTORMは、非遠赤色色素9,12の色収差や準最適な光スイッチング/発光特性など、多色STORMの制限を回避します。溶出工程が逐次的に結合した抗体からの立体障害を有意に減少させるので、madSTORMを使用して、従来の技?...

光学顕微鏡の回折限界（約200nm）を克服するために、様々な超解像顕微鏡技術が開発されている。これらの中には、光活性化局在顕微鏡法（PALM）および確率的光学再構成顕微鏡法（STORM）を含む単一分子局在化顕微鏡（SMLM）と呼ばれる技術のカテゴリーがある。 SMLM技術は、オン（蛍光）状態とオフ（ダーク/光スイッチ）状態との間で切り替えることができるフルオロフォアの使用を共有し、単一分子1,2,3からの蛍光の連続的な局在化を可能にする。 市販の染料や顕微鏡との互換性のため、直接STORM（dSTORM）は広く採用されたSMLM技術となっています4 。 dSTORMは、回折線の中心を計算する際の不確かさとして定義される、約10nmの定位精度を日常的に達成することができるイオン制限点広がり関数（PSF）。しかしながら、局在化アルゴリズム5,6,7を用いて推定された高い精度にもかかわらず、単一分子の実際の位置の正確な決定は、多くの問題によって妨げられてきた。第1に、画像取得中の顕微鏡ステージの機械的移動は、位置特定精度に重大な不確実性を加える。 SMLM画像が何千ものタイムラプスフレームにわたって得られるので、顕微鏡ステージのナノスケールの動きは、最終的な超解像画像8の精度を著しく損なう可能性がある。画像取得中のステージの動きを補償するために、ステージドリフトは、画像自体からのビニングされたローカライゼーションの回帰ベースのフィッティング（相互相関）または基準マーカからの連続的なローカライゼーション（基準補正）1,9から一般的に推定される。ハウヴェこれらの方法は、各画像スタックに対して複数のパラメータの最適化を必要とし、機械的振動などの短い時間スケールでステージの動きを説明することができない。金ナノ粒子と多色蛍光ビーズは、SMLMの基準マーカーとして使用されてきましたが、光安定性ではなく、ドリフト補正後に使用された窒素空孔中心蛍光ナノダイヤモンド（FND） madSTORM 10で 。 回折限界に加えて、光学顕微鏡法はスペクトル限界によってさらに制限される。複数のターゲットを同時に視覚化するには、重複しないスペクトルプロファイルを有する蛍光プローブが必要であり、一般に、蛍光ベースの光学顕微鏡法を6色に、SMLMを2-3色にそれぞれ制限する（4,11,12）。さらに、非線形色収差は、多色画像の不整合を引き起こし、w多色の基準マーカー8,13を用いて広範囲のアライメント手順を必要とする。これらの限界を克服するために、以前の研究では、連続的に結合した蛍光体14,15,16,17,18,19の繰り返しの光退色または化学的消光を用いて複数の標的を画像化した。これらの方法は顕微鏡法のスペクトル限界を克服することができるが、蛍光漂白は有毒なプロセス20であることが知られており、漂白または消光の長期化は架橋の損失などの望ましくない副作用を引き起こす可能性がある。さらに、蛍光プローブの蓄積は、試料中の結合部位の立体的なブロッキングをもたらし、エピトープの大規模な多重化および頑強な標的化を妨げる可能性がある。このような立体的な干渉を避けるために、自由に拡散するタンパク質断片の確率的交換を用いて多重化を達成した21 。この方法は細胞構造の高密度標識化を可能にするが、ペプチド断片を単離するための広範な生化学的調製を必要とし、単一分子位置を特定することができず、市販のプローブを用いた大規模多重化を容易に促進しない。我々は、多重化、抗体サイズ限定dSTORM（madSTORM）イメージング、および正確なドリフト補正およびアライメントを達成するための蛍光ナノダイヤモンドの使用のための蛍光抗体の順次結合および溶出を記載する詳細なビデオプロトコルを提示する。

<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

単一分子局在化顕微鏡法を用いた画像化異種細胞構造は、不適切な局在精度および多重化能力によって妨げられている。蛍光ナノダイヤモンド基準マーカーを使用して、単一分子局在化顕微鏡法で高精度を得るために必要なドリフト補正およびアライメント手順を説明する。さらに、蛍光抗体の逐次結合および溶出を用いて複数の分子が同じ細胞内で標的化される新しい多重化戦略madSTORMが記載されている。 madSTORMは、T細胞受容体マイクロクラスターと呼ばれる膜結合多タンパク質構造内の異なる成分の位置を視覚化するために、活性化されたT細胞上で実証される。さらに、madSTORMのマルチタンパク質構造の可視化のための一般的なツールとしての応用について議論する。

連続的な溶出および染色法を使用して、活性化されたJurkat T細胞（ 図1 、検査図のアライメント）におけるマイクロクラスターおよび他の構造の多重化madSTORM画像を作製した。各疑似カラー画像は、ステップ1.1.1〜1.3.13で取得したmadSTORM画像の1ラウンドを表します。前に説明したように、多重標的間のクロストークを避けるために、視野は前の溶?...

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

我々は、蛍光標識された抗体の逐次結合および溶出を用いて、単一分子精度で異種ナノ構造内の複数の分子をイメージングする方法を実証する。

madSTORMを用いたT細胞の高度に多重化された超解像イメージング

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

マウスT細胞におけるT細胞受容体のレトロウイルス導入

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 ...

免疫・感染学

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

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

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

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

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

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

胚中心の高解像度生体内ストライプ照明顕微鏡

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

Jsubtomoを用いた電子Cryotomography再建からのウイルスのエンベロープ糖タンパク質スパイクの平均化

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

グラスサポート平面脂質二重層上のナチュラルキラー細胞免疫シナプスの超解像イメージング

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

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

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

構造化照明顕微鏡データの時空間画像相関分光法により定量化したT細胞における皮質アクチンの流れ

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