Name: mitochondria and endoplasmic reticulum imaging by correlative light and volume electron microscopy
Uploaded: 2019-07-20T12:30:00
Description: Watch this Scientific Journal Video about Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy at JoVE.com

这项研究得到了KBRI基础研究计划的支持,该计划由韩国脑研究所资助,由科学和ICT部(19-BR-01-08)和韩国国家研究基金会(NRF)资助,由韩国政府(MSIT)资助(NO.2019R1A2C1010634. SCO1-APEX2和HRP-KDEL质粒由玄武瑞(首尔国立大学)提供。TEM数据是在KBRI的大脑研究核心设施获得的。

1. 具有SCO1-APEX2和HRP-KDEL质粒载体的带图案网格培养皿和细胞转染的细胞培养
<ol><li>种子 1 x 105 HEK293T 细胞,将它们放入 35 毫米玻璃网格底培养皿中,在 Dulbecco 的改性 Eagle 介质 (DMEM) 中,在 37°C 下含有 5% CO2的加湿气氛中,辅以 10% 胎儿牛血清,100 U/mL青霉素和100 U/mL链霉素。</li>
	<li>在细胞播种后的第二天,当细胞生长到50%-60%的汇合性时,根据制造商的说明(SCO1-APEX2 cDNA 0.5 μg | </...

<ol>
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使用EM以纳米分辨率确定特定蛋白质的细胞定位对于了解蛋白质的细胞功能至关重要。通常,有两种技术可以研究通过EM定位靶蛋白。一种是免疫黄金技术,自1960年以来一直用于EM,另一种是使用最近开发的遗传编码标签16的技术。传统的免疫金技术使用抗体结合的金颗粒或量子点来显示标记蛋白的位置。然而,由于对高质量抗体的要求和受树脂和固定性抗体影响的抗体的渗透效率,该?...

线粒体和内质视网膜(ER)是膜结合的细胞器。它们之间的联系对于它们的功能是必要的,并且与其网络相关的蛋白质已经描述了1。线粒体和ER之间的距离已报告约100纳米使用光显微镜2;然而,最近的超分辨率显微镜3和电子显微镜(EM)4的研究表明,它相当小,在大约10-25纳米。超分辨率显微镜的分辨率低于EM,需要进行特定的贴标。EM是一种合适的技术,用于线粒体和ER之间的连接结构研究,实现足够高分辨率的对比度。然而,缺点是有限的z轴信息,因为薄部分必须大约60纳米或更薄的传统传输电子显微镜(TEM)。对于足够的EM z轴成像,可以使用三维电子显微镜(3DEM)5。然而,这涉及到整个细胞的数百个薄序列部分的制备,这是非常棘手的工作,只有少数熟练的技术人员已经掌握。这些薄部分被收集在易碎的圆锥膜涂层单孔TEM网格上。如果薄膜在一个梁上断裂,则无法进行序列成像和体积重建。串行块面扫描电子显微镜 (SBEM) 是 3DEM 的一种常用技术,它使用扫描电子显微镜 (SEM) 真空室内的破坏性组合切片,使用金刚石刀 (Dik-SBEM) 或聚焦电束 (FIB-SEM)6.然而,由于这些技术并非在所有设施中都可用,我们建议阵列断层扫描7使用串行切片和 SEM。在阵列断层扫描中,使用超微缩的序列截取部分被转移到玻璃盖玻片上,而不是TEM网格,并通过光显微镜和SEM8进行可视化。为了增强背散射电子 (BSE) 成像的信号,我们使用了一个组合 EM 染色协议,该协议使用四氧化二氮(OsO 4) 固定细胞,其基质硫二氢化物 (TCH)9,使我们能够在不嵌入后双重染色。
此外,线粒体标记SCO1(细胞色素c氧化酶组装蛋白1)*抗坏药过氧化物酶2(APEX2)10分子标记用于在EM水平上可视化线粒体。APEX2约为28 kDa,来自大豆抗坏血球过氧化物酶11。它被开发为显示特定蛋白质在EM水平的详细位置,就像绿色荧光蛋白标记蛋白在光显微镜中使用一样。APEX2在存在过氧化氢(H2O2)的情况下,将3,3' -二氨基苯甲酸(DAB)转换成标签部位的不溶性亲血性聚合物。APEX2 可用作 EM 中传统抗体标记的替代品,在整个细胞的深度进行蛋白质定位。换句话说,APEX2标记的蛋白质可以通过特定的渗透11可视化,无需免疫金标记和超低温切片后的渗透。马萝卜过氧化物酶(HRP)也是一个敏感标签,催化H2O2依赖性聚化DAB成局部沉淀,提供EM对比度后处理与OsO4。ER靶肽序列HRP-KDEL(裂化-白脂-葡-绿露)12应用于整个细胞内的ER可视化。为了评价我们使用遗传标记和组染色的协议,同时使用渗透效应,在rOTO群染色中比较了膜对比度,并且不使用每个遗传标记。 虽然具有阵列断层扫描和 DAB 染色的 3DEM 分别用于其他目的,但我们的协议是独一无二的,因为我们将 3DEM 阵列断层扫描和 DAB 染色相结合,用于线粒体和 ER 标记。具体来说,我们在同一部分展示了五个带有APEX标记基因和无APEX标记基因的细胞,这有助于研究基因改造对细胞的影响。

<table border="0" cellpadding="0" cellspacing="0" style="width:869px;" width="868">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glutaraldehyde</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">16200</td>
			<td style="width:349px;">Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Paraformaldehyde</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">19210</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Sodium cacodylate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">12300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Osmium tetroxide 4 % aqueous solution</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">16320</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Epon 812</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">14120</td>
			<td>EMbed 812- 20 ml/ DDSA- 16 ml/ NMA- 8 ml/ DMP-30 - 0.8 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Ultra-microtome Leica ARTOS 3D</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">ARTOS 3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Uranyl acetate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">22400</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lead citrate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">17900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">35mm Gridded coverslip dish</td>
			<td style="width:123px;">Mattek</td>
			<td style="width:123px;">P35G-1.5-14-CGRD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glow discharger</td>
			<td style="width:123px;">Pelco</td>
			<td style="width:123px;">easiGlow</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Formvar carbon coated Copper Grid</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:123px;">01805-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hydrochloric acid</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">258148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fugene HD</td>
			<td style="width:123px;">Promega</td>
			<td style="width:123px;">E2311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glycine</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">G8898</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:275px;">3,3&#8242; -diaminobenzamidine (DAB)</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">D8001</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen peroxide solution</td>
			<td style="width:123px;">Merck</td>
			<td style="width:123px;">107210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate(II) trihydrate</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">P3289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">0.22 um syringe filter</td>
			<td style="width:123px;">Sartorius</td>
			<td style="width:123px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiocarbonyldihydrazide</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">223220</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Potassium hydroxide</td>
			<td style="width:123px;">Fluka</td>
			<td style="width:123px;">10193426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">L-aspartic acid</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">A9256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Ethanol</td>
			<td style="width:123px;">Merck</td>
			<td style="width:123px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Transmission electron microscopy</td>
			<td style="width:123px;">FEI</td>
			<td style="width:123px;">Tecnai G2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Indium tin oxide (ITO) coated glass coverslips</td>
			<td style="width:123px;">SPI</td>
			<td style="width:123px;">06489-AB</td>
			<td>fragil glass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isopropanol</td>
			<td style="width:123px;">Fisher Bioreagents</td>
			<td style="width:123px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Diamond knife</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">AT-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Inveted light microscopy</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:123px;">ECLipse TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Scanning electron microscopy</td>
			<td style="width:123px;">Zeiss</td>
			<td style="width:123px;">Auriga</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondria and endoplasmic reticulum imaging by correlative light and volume electron microscopy

细胞细胞器,如线粒体和内质视网膜(ER),创建一个网络来执行各种功能。这些高度弯曲的结构被折叠成各种形状,形成一个动态网络,根据细胞条件。利用超高分辨率荧光成像和光显微镜,尝试对线粒体和ER之间的网络进行可视化;然而,有限的分辨率不足以详细观察线粒体和ER之间的膜。透射电子显微镜为细胞细胞器的观察提供了良好的膜对比度和纳米级分辨率;然而,在评估高度弯曲的细胞器的三维(3D)结构时,非常耗时。因此,我们利用增强的抗甲酰氨基甲酸酯过氧化物酶2和马萝卜过氧化物酶染色技术,通过相关的光电子显微镜(CLEM)和体积电子显微镜技术观察线粒体和ER的形态。应用了一种组染色方法、超薄序列切片(阵列断层扫描)和体积电子显微镜来观察三维结构。在此协议中,我们建议将CLEM和3D电子显微镜相结合,对线粒体和ER进行详细的结构研究。

图 1描述了此协议的计划和工作流。协议需要7天;但是,根据在 SEM 成像上花费的时间,这可能会增加。对于细胞转染,应控制细胞的汇合,以免覆盖整个网格板的底部(图1A)。高细胞密度可以阻止在光学显微镜和EM观察过程中识别感兴趣的细胞。我们使用表示APEX2和HRP的基因标记质粒在培养皿中的众多培养细胞中选择有效的转染细胞。我们培养了HEK293T细胞,并确认了SCO1链接APEX2(线...

Watch this Scientific Journal Video about Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy at JoVE.com

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

我们提出了一个方案,研究线粒体和内质神经在全细胞的分布后,使用相关的光和体积电子显微镜,包括抗坏血蜡过氧化物酶2和马萝卜过氧化物酶染色后,同一部分中具有或没有靶基因的细胞的序列切片,并通过电子显微镜进行序列成像。

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 ...

这种体积电子显微镜技术用于进行生物研究，需要观察三D结构。在重拍图像之前提供杆、外野手视图和样本存储。Fx2 Asan 设计了催化脱功能的路径阶段，以增加目标结构的电子测试，并可用于定位特定目标。这些粒子结合了相关光和三D电子显微镜技术，研究测量神经细胞器和宿主细胞之间的相互作用。转染后16至24小时，用250微升30至37摄氏度固定溶液轻轻清洗培养。快速更换洗涤液 1.5 毫升新鲜固定溶液，并将培养剂放在冰上 30 分钟。在孵育结束时，每次洗涤用一毫升冰冷0.1摩尔钠化乳缓冲液冲洗细胞，洗涤1毫升。上次洗涤后，在盘中加入一毫升冰冷 0 至 4 摄氏度 20 毫升甘油溶液，在冰上孵育 10 分钟，然后每次洗涤用 1 毫升新鲜、冷的 0.1 摩尔钠二甲酸酯缓冲液进行三次五分钟洗涤。接下来，用500微升新准备的DAB溶液给电池贴上标签。在冰上孵化细胞约5至45分钟。当在倒置的显微镜下看到浅棕色污渍时，用一毫升新鲜、冷的 0.1 摩尔钠二甲酸酯缓冲液冲洗细胞三次，每次洗涤10分钟。然后，使用相差倒置显微镜以 100 倍或更高的放大率可视化 DAB 染色，并标记感兴趣区域的玻璃底部。对于电子显微镜块样品制备，首先在4摄氏度下用1毫升2%的微量三氧化氮固定一小时。在固定结束时，用三个五分钟的洗涤和一毫升的新鲜蒸馏水每次洗涤冲洗细胞。在用一毫升过滤的TCH溶液治疗细胞之前，使用20分钟。在孵育结束时，在蒸馏水中清洗细胞三次，正如刚刚演示的，用30分钟的孵育在2%的三氧化二氮中修复细胞。在第二次固定结束时，用蒸馏水清洗细胞三次，并在4摄氏度的高温下过夜，用1毫升水化1%醋酸酯覆盖培养物。第二天早上，用蒸馏水洗三次细胞，然后用一毫升60摄氏度的染色，在60摄氏度的烤箱中加热沃尔顿的铅阿斯巴酸溶液30分钟。在孵育结束时，用蒸馏水清洗细胞三次。并在20分钟2毫升乙醇等分的分级系列中孵育该培养。最后100%乙醇暴露后，在三到一乙醇的一毫升中孵育细胞30分钟，在室温下将混合物嵌入低粘度。在孵育结束时，将一毫升一对一乙醇的介质更换为低粘度嵌入混合物介质，在室温下再孵育30分钟。接下来，用一千分之一至三乙醇将介质更换为低粘度嵌入混合介质，在室温下再加热30分钟。然后，在一夜之间用100%低粘度嵌入介质的一毫升代替该介质，然后在100%低粘度嵌入混合物中嵌入样品，在60摄氏度下24小时。对于透射电子显微镜分析，第二天，使用超微原子获得90纳米厚的部分，并在200千伏的透射电子显微镜下观察网格。在开始样品制备之前，用异丙醇轻轻搅拌的氧化钛涂层玻璃盖玻片清洁 30 至 60 秒。搅拌结束时，排出多余的酒精，将盖玻片放在无尘环境中。当盖玻片干燥时，使用等离子涂布器发光放电盖玻片一分钟。然后将盖玻片插入基板支架，并将基板支架放入刀套中。要获取用于扫描电子显微镜的样品，请将嵌入的样品块插入超微子的样品架中。将支架设置为修剪块。使用剃须刀刀片修剪目标位置周围的多余的树脂，使块面获得梯形或矩形形状。前导和后缘应严格平行。获得大量串行部分并不容易。但是，您必须前缘和后缘完全平行。接下来，将样品架插入超微体臂中。将钻石刀放在刀架中，用过滤蒸馏水填充刀船。然后，小心地按下支架的手柄以调整托架位置，使盖玻片的边缘靠近刀。获得样品后，停止截面过程，慢慢打开管子的夹紧螺钉并排空水船。丝带收集过程完成后，用夹紧装置的手柄拆下基板并干燥带状物。使用基因标记质粒表达感兴趣的蛋白质，允许在标记蛋白染色后识别转染靶细胞。然后，可以使用相关光电子显微镜进一步可视化标记的细胞培养。例如，SCO1-APEX2 产生的暗渍仅在线粒体膜间空间中观察到，而不是在线粒体的矩阵空间中观察到。使用SCO1-APEX2和HRP-KDEL质粒的代码转染细胞在线粒体膜间空间和内质神经中表达高度密集的电子信号。然而，在仅与SCO1-APEX2转运的细胞中，没有观察到内质性神经染色。对于通过扫描电子显微镜进行串行成像，使用大面积的背散射电子探测器创建整个阵列图像的概览。接下来，感兴趣的区域将放在第一节中，并传播到所有其他部分。最后，包含五个目标单元的感兴趣区域用五纳米成像像素进行成像。放大显示详细的亚细胞结构，如高尔吉仪器，线粒体，和内皮神经。串行成像清楚地显示，内质性沉默线粒体接触发生在不同的z平面上。您应该对如何使用相关光和电子显微镜定位特定目标结构以调查特定身份修饰的影响有一个很好的理解。这些染色技术与体积电子显微镜相结合，可用于更大规模的EM研究细胞相互作用，特别是在欧元病毒中。请记住，谷胱甘肽，也四氧化二氮和TCH是非常有毒的，所以一定要穿防护服，并在使用这些材料时在烟罩工作。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

SCIENTISTS IN ACTION

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

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 ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

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 ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

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 ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

快速网格准备时间解决低温电子显微镜

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

单颗粒冷冻电子显微镜：从样品到结构

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...