Name: imaging of extracellular vesicles by atomic force microscopy
Uploaded: 2019-09-11T14:00:00
Description: Watch this Scientific Journal Video about Imaging of Extracellular Vesicles by Atomic Force Microscopy at JoVE.com

作者感谢国家科学基金会（奖励号IGERT-0903715）、犹他大学（化学工程系种子赠款和研究生研究奖学金）和斯科尔科沃科学研究所的财政支持。与技术（科技奖学金）。

1. 将EV与生物流体分离
<ol><li>通过一种既定方法隔离电动汽车，如差分超离心8、降水或尺寸排除色谱学9。</li>
	<li>确认是否存在预期的表面和发光生物标志物，以及没有表明制剂交叉污染的生物标志物。通过电子显微镜确认分离粒子的脂质双层形态。 注：在隔离外生体时，由纳米粒子跟踪分析（NTA）或动态光散射测量的水动力大小分布应处于?...

<ol>
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M., Hanssen, E., Lawson, V. A., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-infected+cells+regulate+the+release+of+exosomes+with+distinct+ultrastructural+features.">Prion-infected cells regulate the release of exosomes with distinct ultrastructural features.</a> FASEB Journal. 26, 4160-4173 (2012).</li><li>Briegel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+architecture+of+bacterial+chemoreceptor+arrays.">Universal architecture of bacterial chemoreceptor arrays.</a> Proceedings of the National Academy of Sciences. 106, 17181-17186 (2009).</li><li>Akagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Chip+Immunoelectrophoresis+of+Extracellular+Vesicles+Released+from+Human+Breast+Cancer+Cells.">On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells.</a> PLOS ONE. 10, e0123603 (2015).</li><li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural-mechanical+characterization+of+nanoparticle+exosomes+in+human+saliva,+using+correlative+AFM,+FESEM,+and+force+spectroscopy.">Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy.</a> ACS Nano. 4, 1921-1926 (2010).</li><li>Radeghieri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+human+amniocytes+express+hTERT,+which+is+distributed+between+nucleus+and+cytoplasm+and+is+secreted+in+extracellular+vesicles.">Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles.</a> Biochemical and Biophysical Research Communications. 483, 706-711 (2017).</li><li>Woo, J., Sharma, S., Gimzewski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Isolation+Methods+on+a+Nanoscale+Surface+Structure+and+Its+Effect+on+the+Size+of+Exosomes.">The Role of Isolation Methods on a Nanoscale Surface Structure and Its Effect on the Size of Exosomes.</a> Journal of Circulating Biomarkers. 5, 11 (2016).</li><li>Deegan, R. 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从生物流体、表面扫描和图像分析中固定 EV 是开发中 EV 在液体中的 AFM 表征协议的基本步骤。与图像表面积和固定在基板上的囊泡的表面浓度一起，可适应AFM成像的囊泡数量。鉴于EV和外兆体18的负zeta电位，我们提倡将EV从液体样品静态固定到AFM基板。当表面充满正电荷时，固定是有效的。在EV固定之前，可能需要向基材提供正表面电荷，如云母-一般公式KAl 2（AlSi3O10...

细胞外囊泡 （EV） 存在于所有体液中，包括血液、尿液、唾液、牛奶和羊水。外体体形成一个区域类别的EV与其他EV不同，通过内生，内生子途径的标记，和所有EV中最小的尺寸。外体体的大小经常被报告，在研究之间有很大的变异性。大小调整结果与方法相关，反映了不同分析技术用于估计EV尺寸1，2的物理原理的差异。例如，纳米粒子跟踪分析 （NTA） 是使用最广泛的尺寸表征技术，它估计了 EV 的大小作为流体动力学直径，该直径描述了溶液中 EV 对布朗流动性的阻力。囊泡的流体动力学直径越大，意味着它在液体中的流动性较低。囊泡周围的冠状层，由表面蛋白质和其他固定在膜表面或吸附的分子组成，严重阻碍流动性，增加EV的流体动力学尺寸。相对而言，这种增加对于外体体3来说特别大，如图1所示。
低温透射电子显微镜（低温-TEM）成像是描述囊泡大小和形态在其水合状态的明确技术。然而，仪器的高成本和使用它所需的专业知识正确地激发了对可成像水合电动汽车的替代技术的探索。在采集的低温-TEM 图像中观察到或特征的 EV 数量相对较少，是该技术的另一个显著缺点。
原子力显微镜（AFM）通过扫描基板上的探针来栅格显示表面粒子的图像，从而可视化水合或干燥EV4、5、6的三维地形。本研究概述了协议对AFM的EV特征的基本步骤。在液体中成像囊泡之前，必须将其固定在基板上，通过系绳到功能化表面、困在过滤器中或静电吸引力7。在带正电基板上的静电固定是一种特别方便的选择，用于对已知具有负Zeta电位的外体体进行固定。然而，固定表面细胞外囊泡的同样的静电力也会扭曲其形状，这使得成像后数据分析变得至关重要。我们通过描述基于固定在表面上的外体体变形形状的AFM数据估计溶液中球状囊泡大小的算法来阐述这一点。
在已开发的协议中，介绍了囊泡强稳定处理程序，并随后介绍了在水合或干燥状态下执行原子力成像所需的步骤。确定了影响固定囊泡表面浓度的因素。指导如何对溶液中不同浓度的EV样品进行静电固定。讨论了基于足够数量的固定囊泡估计不同生物物理特性的经验概率分布的实验条件的选择。给出了AFM数据成像后分析的例子。具体来说，描述了一种算法，用于根据固定性EV的AFM特征来确定溶液中囊泡的大小。具有代表性的结果表明AFM的囊泡大小与低温-TEM成像结果的一致性。

<table border="0" cellpadding="0" cellspacing="0" style="width:581px;" width="581">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">AFM/STM Controller&#160;</td>
			<td style="width:92px;">Bruker</td>
			<td style="width:93px;">Multimode Nanoscope V</td>
			<td style="width:173px;">This AFM controller supports imaging of biological samples in liquid and air.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">AFM/STM metal specimen discs (10 mm)</td>
			<td>TedPella</td>
			<td align="right">16207</td>
			<td>Metal specimen disc on which a mica disk is attached by a double-sided tape or other means.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">AFM/STM Mica discs (10 mm)</td>
			<td>TedPella</td>
			<td align="right">50</td>
			<td>Highest quality grade V1 mica, 0.21mm (0.0085&#8221;) thick. Interleaved, in packages of 10. Can be mounted on AFM/STM discs. Available in four diameters</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">AFM probe for imaging in the air</td>
			<td>Bruker</td>
			<td>TESP-V2</td>
			<td>High quality etched silicon probes for tapping mode and non-contact mode for scanning in the air.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">AFM probe for soft sample imaging in liquid</td>
			<td>Bruker</td>
			<td>MLCT</td>
			<td>Soft silicon nitride cantilevers with silicon nitride tips, which are well-suited for liquid operation.&#160; The range in force constants enables users to image extremely soft samples in contact mode as well as high load vs distance spectroscopy.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Double sided tape</td>
			<td>Spectrum</td>
			<td>360-77705</td>
			<td>Used to fix the mica disk on the metal specimen disc.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ExoQuick-TC</td>
			<td>System Biosciences</td>
			<td>EXOTC50A-1</td>
			<td>ExoQuick-TC is a proprietary polymer-based kit designed for exosome isolation from tissue culture media.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glass probe AFM holder for imaging in liquid</td>
			<td>Bruker</td>
			<td>&#160;MTFML-V2</td>
			<td>This glass probe holder is designed for scanning in fluid with the MultiMode AFM.&#160; The holder can be used in peak force tapping mode, contact mode, tapping mode, and force modulation.&#160; The probe is acoustically driven by a separate piezo oscillator for larger amplitude modulation.&#160; The holder is supplied with two ports, required fittings, and accessories kit for adding and removing fluids.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gwyddion</td>
			<td>Czech Metrology Institute.</td>
			<td>Version 2.52</td>
			<td>Open Source software for visualization and analysis of data fields obtained by scanning probe microscopy techniques.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Lint-free blotting paper</td>
			<td>GE Healthcare Whatman&#160;</td>
			<td>Grade GB003 Blotting Paper</td>
			<td>Use this blotting paper to remove NiCl2 after the modification of the mica&#39;s substrate.&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Lint-free cleanroom wipes</td>
			<td>Texwipe</td>
			<td>AlphaWipe TX1004</td>
			<td>Use these polyester wipes for surface cleaning.&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nickel(II) chloride (NiCl2)</td>
			<td>Sigma-Aldrich</td>
			<td align="right">339350</td>
			<td>Powder used to make 10 mM NiCl2 in DI water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phosphate Buffered Saline (1x)</td>
			<td>Gibco</td>
			<td align="right">10010023</td>
			<td>PBS, pH 7.4</td>
		</tr>
	</tbody>
</table>

imaging of extracellular vesicles by atomic force microscopy

外体和其他细胞外囊泡 （EV） 是分子复合物，由脂质膜囊泡、其表面装饰（由膜蛋白和其他分子）和从母细胞（包括RNA）继承的多种发光含量组成，蛋白质和DNA。EV的流体动力学尺寸的表征，取决于由表面装饰形成的囊泡和其冠状层的大小，已成为常规。对于最小EV的外生体，水动力和囊泡大小之间的相对差异显著。低温透射电子显微镜（低温-TEM）成像（低温-TEM）成像对囊泡尺寸的表征仍然是一项挑战，因为仪器的成本、执行样品制备、成像和样品制备所需的专业知识数据分析，以及图像中经常观察到的少量粒子。原子力显微镜（AFM）是一个广泛提供且易于获取的替代方法，它可以生成关于细胞外囊泡的三维几何形状、尺寸和其他生物物理特性的通用数据。开发的协议指导用户使用此分析工具，并概述了 AFM 分析 EV 的工作流程，其中包括水合或干燥形式的成像 EV 的样品制备、静电固定基板上的囊泡、数据采集、分析和解释。具有代表性的结果表明，EV在修改后的云母表面的固定是可预测的、可定制的，并允许用户获得大量囊泡的尺寸调整结果。根据AFM数据进行囊泡尺寸的尺寸与低温-TEM成像一致。

电动汽车的表面固定是成像序列中的关键步骤。外体体静电表面固定（已知具有负齐塔电位）将在云母基板被修改为具有正表面电荷后发生。如果不使用NiCl2进行表面正变化的处理，则发现基板上的EV固定作用无效。图10A中的高度图像，在含有2.59 x10 10囊泡的MCF-7体外体样品后在空气中获得，每mL的PBS囊泡在未修改的云母表面孵育了12小时，显示在表面后...

Watch this Scientific Journal Video about Imaging of Extracellular Vesicles by Atomic Force Microscopy at JoVE.com

Imaging of Extracellular Vesicles by Atomic Force Microscopy

描述了一个分步程序，用于从液体样品中分离体和细胞外囊泡的无标签固定，并通过原子力显微镜（AFM）进行成像。AFM 图像用于估计溶液中囊泡的大小，并描述其他生物物理特性。

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

该协议概述了以水合和干燥形式对细胞外囊泡进行 AFM 成像的简单准备、静电固定、表面扫描、体形识别以及数据分析和解释。该技术的主要优点是方便的静电固定囊泡在皮肤的表面和后成像分析，以考虑到形状变形造成的固定。获得的囊泡尺寸结果与黄金标准 CryoTEM 成像一致，该成像技术仍然成本高昂且具有挑战性。如果您是 AFM 的新用户，请先从干样品的表征开始，然后再进行水合样品。因为有很多其他因素会影响水合样品的采集。首先，将细胞外囊泡与生物流体分离，如随附的文本协议所述。接下来，将云母盘牢固地连接到磁性不锈钢试样盘上。使用锋利的剃须刀切割云母盘，露出新的材料层。在室温下，用100微升的十毫摩尔镍II氯化物溶液处理云母的顶面十秒钟。这会将表面电荷从负向正。用无绒擦拭或印迹纸将镍二氯化物溶液打上。然后，用去维化的水洗三次云母表面，然后用干氮流干燥。将 AFM 试样盘与附加表面修饰的云母放入培养皿中。接下来，用PBS稀释外体，获得每毫升溶液40亿到40亿颗粒的浓度。使用纳米颗粒跟踪分析验证稀释颗粒浓度。通过从移液器中排空稀释的外体溶液的一百微升，在云母表面形成一个酶滴。然后将盖子盖在培养皿上，用副芬膜密封，以减少样品蒸发。在冰箱里孵育十二小时。孵育后，小心吸吸80%至90%的样品，而不干扰表面。此时，外显体将在云母基材上静电固定。成像水合样品时，用 PBS 冲洗表面三次。注意在整个冲洗过程中保持样品水分。用 PBS 清洗云母表面后，取出 80% 至 90% 的液体和移液器 40 微升新鲜 PBS 以覆盖样品。水合样品已准备好进行成像。成像干燥 EV 时，用去电化水冲洗基材三次，将盐从表面去除。尽可能多地吸气液体后，在不接触表面的情况下，用干氮流干燥其余液体。要对干燥细胞外囊泡进行成像，请选择一个悬臂，用于在攻丝和非接触式成像模式下在空中扫描，然后安装到探头支架上。将样品放在 AFM 阶段。磁性不锈钢试样盘将固定样品在舞台上。将探针支架放入 AFM 中。留出时间进行准备和阶段以热平衡。使用攻丝模式扫描 5x5 微米以 1 赫兹的扫描速率在 512 行中拉的面积。获取高度和相位图像，因为它们提供有关样品的地形和表面属性的免费信息。扫描时间将随着图像区域和选择形成图像的行数而增加，但扫描速率也会减少。由于快速扫描速率可能会影响图像质量，因此栅格速度应该是采集时间和图像质量之间的平衡。要成像水合囊泡，请选择适合扫描软水合样品的悬臂，然后将悬臂安装到专为液体扫描而设计的探针支架上。用 PBS 将悬臂的尖端弄湿，以减少扫描过程中向液体引入气泡的可能性。然后，将样品固定到 AFM 阶段。样品热平衡后，在攻丝模式下对水合云母表面进行成像。获取高度和相位图像。要分析拍摄的图像，请先转到"数据处理"选择 SPM 模式，然后选择"模型提示"，然后选择用于扫描样本的图尖的几何形状和尺寸，然后单击"确定"，通过执行曲面重建来校正尖端侵蚀伪影。打开图像。从菜单中，选择"数据过程"，然后选择"SPM模式"，然后选择"提示"，然后选择"确定""下一步"，选择"数据处理"，然后选择"平面"，然后选择"平面"来对齐成像平面，通过从扫描数据中删除基板中的倾斜度来匹配实验室 XY 平面。通过选择"数据过程"，然后选择"对齐行"来对齐图像中的行，然后选择"对齐行""几个对齐选项可用，包括中位数，这是一种算法，用于查找每条扫描线的平均高度并从数据中减去它。接下来，转到"数据处理"，然后选择"删除疤痕"，这将删除常见的扫描错误，称为疤痕'以零高度对齐云母表面，转到数据处理'菜单，并在"级别"下拉菜单中选择"平展基础"。通过进入颗粒菜单并使用"阈值标记"来识别扫描表面上的额外细胞囊泡。该算法将表面固定外体识别为从零表面基板突出的颗粒，其高度高于用户所选阈值。选择一到三纳米范围内的阈值。这将消除大部分后场干扰。最后，使用从谷物菜单访问的可用分布算法对已识别的囊泡进行几何和尺寸特征化。导出 Gwydion 的 AFM 数据，由其他计算工具和自定义计算机程序进行专业分析。氯化镍表面的改性导致额外的细胞囊泡的固定化，这需要时间。固定囊泡的表面浓度在孵育24小时后过于密集，而12小时的孵育导致的外体更少，扫描数据更容易准确分析。此 AFM 图像显示了水合的 MCF7 外体静电固定在经过修改的云母表面。相应的 AFM 相位图像确认高度图像中的颗粒是软纳米颗粒，应视为膜囊泡的软纳米颗粒。此处显示了同一行中三个囊泡的高度数据。这些轮廓说明了外显子对改性云母的正电荷表面的静电吸引力造成的扁平形状。形状失真在固定囊泡及其横截面的放大视图中是明显的。要估计溶液中外显体球状大小，上可与表面固定膜和球形膜包络所包围的体积相匹配。溶液中球状囊泡的大小分布由 561 个固定囊泡的 AFM 数据确定。CryoTEM 图像中的囊泡大小与 AFM 结果一致。在成像经过赞美的外体之前，重要的是要记住用PBS彻底冲洗表面。这将删除入站外体，并防止它们附着在 AFM 提示中。成像干燥外显体时，请务必使用 DI 水来上升基材。当基板干燥时，DI 洗涤将防止表面形成盐晶体。如果存在，盐晶体将使图像处理成为一项艰巨的任务。

Research

JoVE Journal

Biochemistry

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

科研

生物化学

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Isolation of Tissue Extracellular Vesicles from the Liver

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...