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.组织剥离和固定<ol><li> 组织剥离 <ol><li>从手术切除肿瘤样品或组织活检解剖组织片。 注意：在此研究中使用的组织进行常规福尔马林固定，但新鲜组织也是合适的。我们选择了确认，并报告通过解剖病理学家以包含常规的组织学染色和抗促生长素抑制素的免疫染色（未示出）之后生长抑素瘤的区域。 </li><li>使用组织片不高于约1.0 立方毫米大。 </li></ol></li><li> ?...

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这项研究表明量子点的潜在效用为CLEM研究通用探头。由宽视场光学显微镜观察和透射电镜容易观察时使用的585纳米的纳米QD显示明亮，稳定的荧光。先前的研究由本作者之一显示量子点还适合于超分辨率光镜7。他们的耐光性是延长时间观看和长成像曝光特别有用。量子点也可以被用于以相同的激发波长下不同尺寸的探头的同时发射不同颜色的光谱多路复用免疫组织化学。 <p class="jove_co...

相关轻型和电子显微镜（CLEM）是一种瞬态动态事件1，罕见的事件2,3和复杂系统4的分析功能强大的方法。有可用5取决于问题的许多不同技术的排列被要求然而一个共同要求是一个单一的样品6中相同的结构由多个显微镜模式成像。我们对特殊CLEM方法被用于存档人体病理学组织的研究，用在这里得到了很好的表征，此前公布的7的情况下开发的。其目的是首先从单个活检或手术样品其次最大化的分析数据，以使用荧光光学显微镜帮助阐明在超微结构水平看到的免疫细胞化学标记图案的情况下。 量子点纳米晶体（量子点）提供了一个通用的标记系统ABL的潜力由两个被看作即，荧光光镜和电镜8，9，10，它们的结晶核结构允许不同尺寸的量子点，当通过光波长远离其发射光谱11激发而产生的广泛的荧光发射峰。其原子量是足以产生电子密度是通过透射电子显微镜，扫描透射电子显微镜（STEM）或场发射扫描电子显微镜检测。它们特别适合于免疫细胞化学研究，作为甚至单个量子点可观察到给予每靶分子12 1 QD的最终的灵敏度。此外，根据不同的QD使用它们可具有适于映射的个体元素的签名。 人体病理学样品提供翻译生物医学研究显著的好处。外科组织活检标本例行提交的生物样本库和appropriate道德间隙可以为研究报告进行访问。人体组织不具有可发生在动物或在疾病的体外模型相关性或解释的问题。然而，病理学样品的样品制备通常不是最佳的。可存在于组织中的延迟被放置在固定剂，不适当的固定剂使用诸如福尔马林而非戊二醛用于TEM和不适当的采样。 CLEM方法必须优化可用从单个人类样本的诊断和预后信息的可能性。然而，一些新开发的相关的方法，例如那些采用微型单线态氧发生器（miniSOG）不适用于在病理学使用由于需要对标签被遗传编码到感兴趣13的细胞。为此，我们探索了常规准备TEM组织进行相关的免疫细胞化学研究的QD标签的效用。量子点应用于蚀刻环氧树脂或从李丙烯酸树脂切片ghtly醛固定活检组织样品提供从单个样品获得相关荧光光学显微镜和TEM数据的可能性。

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			<td height="21" style="height:21px;width:255px;">Sodium cacodylate</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C0205</td>
			<td style="width:167px;">Harmful chemical</td>
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		<tr height="21">
			<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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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol 100%</td>
			<td style="width:117px;">Fronine</td>
			<td style="width:119px;">JJ008</td>
			<td></td>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ERL 4221</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C056</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DER 732</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C047</td>
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			<td height="21" style="height:21px;width:255px;">NSA</td>
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			<td style="width:119px;">C059</td>
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			<td height="21" style="height:21px;width:255px;">DMAE</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C050</td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Sodium metaperiodate</td>
			<td style="width:117px;">Analar BDH</td>
			<td style="width:119px;">10259</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-somatostatin antibody</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">A0566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody diluent</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">S3022</td>
			<td></td>
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		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Normal goat serum</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">PCN5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:117px;">Pechiney PP</td>
			<td style="width:119px;">M</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Micromoulds</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">RL063</td>
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			<td height="21" style="height:21px;width:255px;">Diamond knife</td>
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ultramicrotome</td>
			<td style="width:117px;">RMC</td>
			<td style="width:119px;">Powertome</td>
			<td></td>
		</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>
	</tbody>
</table>

correlative light- and electron microscopy using quantum dot nanoparticles

描述了一种方法，即量子点（QD）纳米颗粒可用于使用宽视场荧光光镜和透射电子显微镜（TEM）人体病理学组织相关的免疫细胞化学研究。为了证明我们已经用初级抗体对促生长素抑制素免疫标记人生长抑素瘤的超薄环氧部分的协议，其次是生物素化的二级抗体和可视化用链霉缀585nm的镉 - 硒（硒化镉）量子点（QDs）。的部分被安装在TEM样品网格，然后通过宽视场荧光光镜放置在载玻片观察。光镜显示585纳米量子点标记为明亮的橙色荧光形成肿瘤细胞的细胞质中的颗粒图案。在通过光学显微镜低中档放大率标签图案可以容易地识别和非特异性或背景标记的水平进行评估。这是一个关键步骤，通过TEM免疫标记图案的后续解释和评价形态上下文。然后相同部分被吸干并用TEM观察。 QD探针被视为要附加到包含在个别分泌颗粒无定形物质。图像是从减光镜进行相关性分析可见感兴趣区域（ROI）的同一区域收购。从每个模态对应的图像随后可以被混合以覆盖上相应的区域的TEM超微结构荧光数据。

用于该研究的生长抑素瘤样品包括形成具有胶原造口组织混合导管结构的肿瘤细胞。通过荧光光镜，含有丰富的分泌颗粒个体肿瘤细胞表现为生长抑素激素阳性标记。细胞核出现暗孔以最小非特异性标记检测的（ 图1）。在低倍率，可变地激烈粒状橙色荧光被认为在这些充分表征的肿瘤细胞的细胞质中。荧光的橙色由QD使用的大小决定。在这项研究中，我们使?...

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.

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

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

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

科研

生物工程

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

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

生产和目标单价量子点

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

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

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

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

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

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

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

光片荧光显微术在小鼠心脏研究中的应用

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

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

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

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

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

相关轻型和电子显微镜使用量子点纳米粒子