Name: correlative light- and electron microscopy using quantum dot nanoparticles
Uploaded: 2016-08-07T14:00:00
Description: Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

The authors wish to acknowledge the support of Xiao Juan Wu (Immunohistochemistry Laboratory) and the Department of Anatomical Pathology, Sydney South West Pathology Service (SSWPS), NSW Health Pathology, Liverpool, New South Wales, Australia.

1.組織の解剖と固定<ol><li> 組織切開 <ol><li>外科的に切除腫瘍標本または組織生検からの組織片を解剖。 注：この研究で使用した組織は、日常的にホルマリン中に固定したが、新鮮な組織にも適しています。私たちは確認エリアを選択し、日常的な組織学的染色および抗ソマトスタチン免疫染色（図示せず）の後ソマトスタチン産生腫瘍を含むように、解剖学的病理学者に?...

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この研究は、CLEM研究のためのユニバーサルプローブとしてQDの潜在的有用性を実証してきました。使用される585 nmのQDナノ粒子は、広視野光顕微鏡で見たときに明るく安定した蛍光を示し、容易にTEMにより観察しました。本著者の一つ前の研究では、超解像光学顕微鏡7に適するように量子ドットを示しています。その光安定性、拡張視聴期間と長いイメージング・エクスポージャー?...

相関軽および電子顕微鏡（CLEM）は、過渡ダイナミックイベント1、まれな事象2、3、複雑なシステム4の分析のための強力なアプローチです。一般的な要件は、単一の試料6の同じ構造が複数の顕微鏡モダリティにより撮像されることがあるが提起された課題に応じて、5利用可能な多くの異なる技術的な順列があります。 CLEMに対する当社の特定のアプローチは、アーカイブヒト病理組織の研究と十分に特徴付けられ、7以前に公開されている、ここで使用される場合のために開発されました。目的は、超微細構造レベルで見られる免疫細胞化学標識パターンの文脈を理解しやすくするために、蛍光光学顕微鏡を使用すること、第二に、単一の生検または外科的サンプルから分析データを最大化するために、まずました。 量子ドットナノ結晶（量子ドット）は、ユニバーサルマーカーシステムABLの可能性を提供しますeは両方によって観察される、蛍光光学顕微鏡および電子顕微鏡8、9、10。それらの結晶コア構造は、それらの発光スペクトル11から遠い波長の光によって励起されると、異なるサイズの量子ドットは、蛍光発光ピークの広い範囲を生成することができます。それらの原子量は、透過型電子顕微鏡、走査型透過電子顕微鏡（STEM）や電界放射型走査電子顕微鏡によって検出可能な電子密度をもたらすのに十分です。単一量子ドットは、標的分子12ごとにQDの最終的な感度を与える観察され得るように、それらは、特に免疫細胞化学研究に適しています。さらに、QDに応じて、それらがマッピングするのに適した個々の元素シグネチャーを有することができる使用しました。 ヒト病理サンプルは、翻訳生物医学研究のための重要な利点を提供します。外科的組織生検サンプルが日常的にバイオバンクのためにとappropに提出されますriate倫理クリアランスは、調査研究のためにアクセスすることができます。ヒト組織は、動物においてまたは疾患のインビトロモデルで発生する可能性があります関連性や解釈の問題を持っていません。しかし、病理試料の試料調製は、しばしば最適ではありません。不適切な固定液は、ホルマリンではなく、TEMや不適切なサンプリングのためのグルタルアルデヒドとして使用する固定液中に配置されている組織の遅れ、がある場合もあります。 CLEM法は、単一のヒトのサンプルから利用可能な診断および予後情報を最適化する可能性を秘めています。しかし、このようなミニ一重項酸素発生器（miniSOG）を用いたものなど、いくつかの新たに開発された相関アプローチが原因で、遺伝的に興味13の細胞内に符号化されるべきタグの必要性への病理学で使用することはできません。このような理由から、我々は相関免疫細胞化学的研究のための日常準備TEM組織のQD標識の有用性を検討しています。李からエッチングされたエポキシ樹脂またはアクリル樹脂のセクションに適用される量子ドットghtlyアルデヒド固定生検および組織サンプルは、単一のサンプルから相関蛍光光学顕微鏡とTEMデータを得る可能性を提供します。

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			<td height="21" style="height:21px;width:255px;">Sodium cacodylate</td>
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			<td height="21" style="height:21px;width:255px;">Osmium tetroxide</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C010</td>
			<td>Use only in fume hood</td>
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			<td height="21" style="height:21px;width:255px;">Uranyl acetate</td>
			<td style="width:117px;">Univar-Ajax</td>
			<td style="width:119px;">569</td>
			<td>Hazardous chemical</td>
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			<td height="21" style="height:21px;width:255px;">Ethanol 100%</td>
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			<td height="21" style="height:21px;width:255px;">Acetone 100%</td>
			<td style="width:117px;">Fronine</td>
			<td style="width:119px;">JJ006</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">ERL 4221</td>
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			<td style="width:119px;">C056</td>
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			<td height="21" style="height:21px;width:255px;">DER 732</td>
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			<td style="width:119px;">C047</td>
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			<td style="width:119px;">C050</td>
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			<td height="21" style="height:21px;width:255px;">Sodium metaperiodate</td>
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			<td style="width:119px;">10259</td>
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			<td height="21" style="height:21px;width:255px;">anti-somatostatin antibody</td>
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			<td style="width:119px;">A0566</td>
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			<td height="21" style="height:21px;width:255px;">Qdot 585 Streptavidin Conjugate&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">Q10113MP</td>
			<td></td>
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			<td height="42" style="height:42px;width:255px;">Biotinylated goat anti-rabbit IgG antibody</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">B7389-1ML</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Glutaraldehyde 50%</td>
			<td style="width:117px;">EMS</td>
			<td style="width:119px;">16320</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Normal goat serum</td>
			<td style="width:117px;">Invitrogen</td>
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			<td height="21" style="height:21px;width:255px;">PBS &#34;Dulbecco A&#34;</td>
			<td style="width:117px;">Oxoid&#160;</td>
			<td style="width:119px;">BR0014G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BSAc (10%)</td>
			<td style="width:117px;">Aurion</td>
			<td style="width:119px;">900.022</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:117px;">Pechiney PP</td>
			<td style="width:119px;">M</td>
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			<td height="21" style="height:21px;width:255px;">pH indicator strips (pH 2.0 - 9.0)</td>
			<td style="width:117px;">Merck</td>
			<td style="width:119px;">1.09584.0001</td>
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			<td style="width:117px;">Diatome</td>
			<td style="width:119px;">Ultra 45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Transmission electron microscope</td>
			<td style="width:117px;">FEI</td>
			<td style="width:119px;">Morgagni 268D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fluorescence light microscope</td>
			<td style="width:117px;">Carl Zeiss</td>
			<td style="width:119px;">Axioscope A1</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Grids 300 mesh nickel (thin bar)</td>
			<td style="width:117px;">Agar Scientific</td>
			<td style="width:119px;">G2740N</td>
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			<td height="21" style="height:21px;width:255px;">Ultramicrotome</td>
			<td style="width:117px;">RMC</td>
			<td style="width:119px;">Powertome</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">TEM camera control software</td>
			<td style="width:117px;">Soft Imaging System</td>
			<td style="width:119px;">AnalySIS</td>
			<td>Version 3.0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Image processing software</td>
			<td style="width:117px;">Adobe Systems Incorporated</td>
			<td style="width:119px;">Photoshop CS2</td>
			<td></td>
		</tr>
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</table>

correlative light- and electron microscopy using quantum dot nanoparticles

量子ドット（QD）ナノ粒子は、広視野蛍光光学顕微鏡および透過型電子顕微鏡（TEM）を用いて、ヒト病理組織の相関免疫細胞化学的研究のために使用することができる方法が記載されています。ストレプトアビジンコンジュゲートさ585 nmのカドミウムセレン（CdSeの）量子ドット（QD）とビオチン化二次抗体および可視化に続いて、我々はソマトスタチンに対する一次抗体を用いて、ヒトソマトスタチン腫瘍の超薄エポキシセクションを免疫標識されているプロトコルを、実証するために。切片を次に、広視野蛍光光学顕微鏡による観察のためにスライドガラス上に載置されたTEM試料グリッド上に搭載されています。光学顕微鏡は、腫瘍細胞の細胞質内の顆粒状のパターンを形成する明るいオレンジ色の蛍光として585 nmのQDラベルを明らかにする。ミッドレンジ拡大を低で光学顕微鏡により標識パターンを容易に認識することができ、非特異的またはバックグラウンドの標識のレベルを評価しました。これは非常に重要ですその後のTEMによる免疫標識パターンの解釈及び形態学的状況の評価のためのステップ。同じセクションは、その後、乾燥ブロットし、TEMにより観察されます。 QDプローブは、個々の分泌顆粒に含まれる非晶質材料に取り付けられるように見られています。画像は、相関分析のための光学顕微鏡で見て興味（ROI）の同じ領域から取得されます。各モダリティからの画像を対応する、次に対応する領域のTEM超微細構造上の蛍光データをオーバーレイするために配合することができます。

この研究のために使用されるソマトスタチノーマ腫瘍試料は、コラーゲンストーマ組織と混合管構造を形成する腫瘍細胞を含んでいました。蛍光光学顕微鏡によって、豊富な分泌顆粒に含まれる個々の腫瘍細胞は、ソマトスタチンホルモン陽性の標識化を示しました。核は、最小限の非特異的標識の検出可能（ 図1）のように暗い穴が登場しました。低倍...

Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

量子ドットナノ粒子を用いた相関光 - 及び電子顕微鏡

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

The overall goal of this method is to obtain correlative light and electron microscopy, or CLEM data, by combining fluorescence immunocytochemical information with ultra-structural morphology from transmission electron microscopy in a single image. This method can help answer key questions in the field of immunocytochemistry and pathology, such as what subcellular structure is a particular protein of interest associated with. The main advantage of this technique is that it uses the same quantum dot nanoparticle probe to obtain both the live microscopy signal from the target protein and the electron microscopy structural localization.After fixing, embedding and sectioning human somatostatinoma tumor tissue according to the text protocol, prepare a section adhesive solution by placing 20 centimeters of clear adhesive tape into a five milliliter bottle containing 2.0 milliliters of acetone and let it stand for 10 minutes. Remove the tape and dip a nickel grid into the solution, then remove the grid and allow it to air dry. Once dry, use the dull side of the grid to pick up an ultra thin section.It's critical to establish the correct etching time for the chosen resin formulation. 30 minutes works well for this formulation, but it may need to be recalculated for harder or softer formulations. Prepare one milliliter of fresh saturated sodium metaperiodate etching solution in distilled water and place droplets of the solution on a clean lab film surface.Place completely dry grids with sections onto the droplets and incubate at room temperature for 30 minutes. Then, transfer the grids to distilled water droplets for 60 seconds to wash them off. To carry out antibody and quantum dot, or QD labeling, on a clean lab film surface, pipette drops of 0.05 molar glycene and PBS.Place the grids on the droplets and incubate for 10 minutes to block residual aldehyde on the sections. To remove the aldehyde block, briefly blot the edge of the grid. Then place the grid on a droplet of one percent normal goat serum, or NGS, and one percent BSAC in PBS for 10 minutes.Prepare one milliliter of blocking solution by adding 10 microliters of NGS and 100 microliters of BSAC to 890 microliters of PBS. Then, condition the sections by placing them on a droplet of antibody diluent for 10 minutes. Next, use antibody diluent to dilute anti-somatostatin polyclonal antibody one to 10, and place the grids on droplets of the solution.Incubate in a moist chamber at room temperature for one hour. Following the incubation, remove the antibody reagent by blotting the edge of the grid. Then use antibody diluent to wash the sections two times for five minutes each.After diluting secondary antibody one to 10 and preparing drops, incubate the grids on the droplets at room temperature for one hour. Then, remove the antibody solution and wash the sections two times. Dilute streptavidin conjugated QDs one to 10 and incubate the samples on drops of the solution in the dark, at room temperature for one hour.Then use antibody diluent to wash the grids twice, each time for five minutes, and blot the edge of the grids. Next, wash the grids in distilled water for two minutes before blotting dry the edges. Place the immunostained grid section in a droplet of water on a glass slide and use a glass cover slip to cover it.To carry out fluorescence light microscopy, insert a filter cube with the following characteristics and use 365 nanometer LED illumination. Place the immunostained section on the immunofluorescence microscope stage. Use light microscopy to evaluate the labeling pattern, the level of any non-specific labeling and to establish that negative controls are clear.Next, identify ROIs in relation to grid bars. Then set a full colored digital camera to FL auto color and set white balance to 3, 200 K.Set the camera to automatic exposure mode with a gamma setting between 0.45 and 1.00 to optimize the appearance of the images, and analog gain set to one X.Click the camera icon in the ZEN Two Light software graphical interface to live. Then focus on the image and click Snap in the ZEN Two Light software graphical interface to capture the image to the frame store.Save the images as tf files to avoid compression and pixelation. Prepare a print showing the position of the ROI in relation to the grid bars or other significant tissue landmarks. Then remove the grid from the slide, wash in distilled water, and gently blot the edges dry.To carry out transmission electron microscopy, transfer the grid to the microscope for examination using an accelerating voltage of 100 kilovolts. Begin with low magnification of approximately 1, 400X to navigate around the grid and find the ROIs with those found with light microscopy. Observe individual QDs at 70, 000X magnification or above.They will appear as irregular crystalline structures with moderate electron density. Then, using a digital camera with a monochrome 1, 392 by 1, 040 pixel sensor in the imaging software's graphical interface, click the camera icon to on and acquire images. Move the camera icon to the off position to store the image in the frame store.To carry out CLEM imaging, use a print from the light microscopy imaging to locate corresponding ROIs by TEM. Tissue architectural features are useful for navigation around the section. Capture an image of the corresponding ROI by TEM.Next, to prepare a CLEM image, ensure that the TEM image is correctly oriented and enlarged to the same size and resolution as the light microscope image, in this case, 300 pixels per inch. Extend the size of the light microscopy image canvas to double its size. Then copy and paste the TEM image beside the fluorescence image.To create a fluorescence overlay in Photoshop CS2, click on the rectangular marquee tool, select the fluorescent image, and create a duplicate layer from it. Adjust the layer style blending options to 30 to 40%transparency. Then click on the move tool and drag the fluorescence overlay layer over the corresponding TEM image, and align the images.After aligning the images, flatten the image to produce the final overlay. Then use the rectangular marquee tool to select the new overlay image and copy it to a new canvas. Finally, save the image as a tf file.In this fluorescence microscopy image, individual somatostatinoma tumor cells contain abundant secretory granules and were positively labeled for the somatostatin hormone. The nuclei appeared as dark holes and at low magnification, variably intense granular orange QD fluorescence was seen in the cytoplasm. The ROI chosen by light microscopy was recognized and imaged using TEM by referring to a 20X light microscopy image as indicated here.At higher magnification, the cell demonstrates bright fluorescence and intense QD labeling in cytoplasmic granules. The overlaying of fluorescence data on TEM images, as in this example, suggests strong positive labeling corresponds to granules containing moderately electron dense material within the vesicle limiting membrane. Finally, as seen here at higher magnification, the moderately electron dense granules showed intense QD nanocrystal immunolabeling.Once mastered, this technique can be done in eight hours if performed properly. While attempting the procedure, it's important to remember to handle the grids carefully, by the edges only. Touching the section while picking up the grid may detach the section from the grid.Following this procedure, other methods like multiplex immunolabeling can be performed in order to answer additional questions, such as does your protein of interest co-localize with another protein. After its development, this technique paved the way for researchers in the field of pathology to explore protein localization in archival human tissue process for electron microscopy. After watching this video, you should have a good understanding of how to combine immunocytochemical information from fluorescence light microscopy with ultra structure from transmission electron microscopy.Don't forget that working with osmium tetroxide can be extremely hazardous, and precautions such as wearing protective equipment, working in a fume hood and proper waste disposal should be taken while performing this procedure.

Research

JoVE Journal

Bioengineering

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

相関ライト/電子顕微鏡のための低コストクライオ光学顕微鏡のステージの作製

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

生物工学

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

ダイナミックな相互作用の3次元構造解析のための相関顕微鏡

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

のためのツールと​​してのリポソームカプセル化された近赤外蛍光体の蛍光消光<em&gt;インビボ</em&gt;光学イメージング

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Production and Targeting of Monovalent Quantum Dots

生産と一価量子ドットの標的

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

強化された暗視野顕微鏡とハイパースペクトルマッピングにより組織学的試料中の金属酸化物ナノ粒子の同定

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学の活用方法を使用して E 型肝炎ウイルスのナノ粒子の表面化学

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

マウス心臓の研究のための光シート蛍光顕微鏡

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

相関光電子顕微鏡 (クレム) 追跡のウイルス蛋白質のイメージング関連するクライオ固定化細胞の構造

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

極薄ポリジメチルシロキサン マイクロ流体チップを用いた保水ライブ線虫個人の固定化

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...