这项研究得到了生物和 #38 的支持; 国家研究基金会 (NRF) 和 #38 的医疗技术发展方案; 由韩国政府资助 (MSIP 和 #38; 保健) (No. 2016M3A9B6904244)。我们还感谢医学 Bioconvergence 研究中心的成员外切纯化。

1. 外切块的制备、切片、染色和成像
<ol>
	<li>将 HCT116 细胞培养上清液中的体, 在 10万 x g 为 1.5 h23进行离心。去除培养上清液, 在1° c 的 0.1 M 钠甲溶液 (pH 7.0) 中, 用1毫升2.5% 戊二醛仔细修复纯化的外切颗粒, 4 小时。对0.1 米甲钠, 溶解4.28 克胂酸在160毫升蒸馏水 (DW)。将 pH 值调整到7.4 与0.1 米 HCl, 然后用蒸馏水构成200毫升。</li>
	<li>在室温下用1毫升的0.1 米甲缓冲液去除固定剂并冲洗颗粒。重复三次, 每次变化持续10分钟。</li>
	<li>在4° c 的情况下, 用1毫升2% 锇氧化在1小时后修复样品。</li>
	<li>取下定影液, 冲洗三次, 每10分钟用0.1 米甲缓冲。</li>
	<li>用分级丙酮系列 (50%、60%、70%、80%、90%、95%、100%、分别) 在振动筛上孵育10分钟。</li>
	<li>除去丙酮, 孵育3:1 丙酮的溶液: 低粘度嵌入混合物30分钟。外切颗粒在试管里。</li>
	<li>去除培养基, 加入1:1 丙酮: 低粘度的嵌入混合物培养基, 然后孵育30分钟。</li>
	<li>去除培养基, 加入1:3 丙酮: 低粘度的嵌入混合物培养基, 然后孵育30分钟。</li>
	<li>去除培养基, 加入100% 低粘度的嵌入混合物, 在室温下过夜。</li>
	<li>将样品嵌入纯低粘度的嵌入混合物中, 并在65° c 下烘烤24小时。</li>
	<li>通过 ultra-microtome 准备 60 nm 厚度的剖面。</li>
	<li>双色染色, 2% 铀醋酸20分钟, 柠檬酸铅10分钟。 对于雷诺铅溶液, 添加1.33 克硝酸铅和1.76 克柠檬酸钠, 总共50毫升蒸馏水。</li>
	<li>在 80 kV 的透射电镜下观察网格。</li>
	<li>单击 "获取", 然后在 80 kV 的电子显微镜下, 在 CCD 摄像系统中单击 "文件" 和 "另存为"。按照曝光时间的自动设置进行。</li>
</ol>
2. 切片和成像的免疫染色 (图 1)
<ol>
	<li>使用 ultra-microtome 切割60纳米超薄切片, 并在镍网格上收集。</li>
	<li>孵育网格在50µL 滴0.02 米甘氨酸为10分钟到淬火游离醛组。</li>
	<li>在100μ l 的 DW 中冲洗三次, 每次10分钟, 在室温下孵育 1 h 在含有 1% BSA 的 PBS 中。</li>
	<li>孵育网格在 50-100 µL 滴抗 KRS 抗体24 (1:100 在 PBS 中含有 0.1% BSA) 为 1 h (如有必要, 此步骤应在夜间在4° c 进行。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。</li>
	<li>传输网格到下落 2nd抗体为 1 h (兔 IgG 共轭到10毫微米金微粒 (1:100) 在 PBS 包含 0.1% BSA)。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 每 10 min。</li>
	<li>双染色2% 铀醋酸20分钟在黑暗条件下, 并与雷诺的铅柠檬酸10分钟, 分别。</li>
	<li>单击 "获取", 然后在 80 kV 的 TEM 下, 在 CCD 摄像系统中点击 "文件" 和 "另存为"。曝光时间遵循自动设置。</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56482/56482fig1.jpg"> 
图 1: 样品制备和免疫的过程.(A)双固定外切的脱水和渗透低粘度嵌入混合树脂。(B)树脂嵌入。(C)超薄截面带金刚石刀。(D)设置免疫金标签。与超薄部分的网格放在液滴上的膜, 这是放置在湿纸巾。每节都用 50-100 µL 抗体的飞沫孵育, 然后用缓冲液冲洗。(E)双染色, 醋酸铀和柠檬酸铅。盖子上覆盖着铝箔, 用于重金属染色。(F)染色液由滤纸移除。<a href="//ecsource.jove.com/files/ftp_upload/56482/56482fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 阴性染色
<ol>
	<li>辉光放电的薄 formvar/碳膜覆盖200网状铜 EM 栅1分钟的辉光料器。</li>
	<li>1毫升2% 甲醛 (粉煤灰) 固定纯化体5分钟。 警告: 甲醛的烟雾是有毒的。所有工作应在通风油烟罩。</li>
	<li>在网格上加载 5-7 µL 外切悬浮液, 孵育1分钟。如果外切浓度过高, 稀释浓度为 1/2-1/5。</li>
	<li>立即染色与〜20滴过滤1% 铀醋酸盐 (UA) 解决方案在表面的 EM 网格的注射器。</li>
	<li>通过与过滤纸接触网格边缘, 删除网格上多余的 UA 解决方案。</li>
	<li>用一滴水迅速冲洗网格。此步骤将删除多余的染色液。</li>
	<li>用镊子把网格放在桌上, 用一个培养皿把网格部分盖住, 在室温下晾干10分钟。</li>
	<li>将网格存储在 EM 网格框中, 以便在80伏的 TEM 上进行观察。</li>
</ol>
4. 全山免疫
<ol>
	<li>辉光放电薄 formvar/碳膜涂层200网状铜 EM 网三十年代。</li>
	<li>1毫升2% 甲醛 (粉煤灰) 固定纯化体5分钟。 警告: 甲醛的烟雾是有毒的。所有工作应在通风油烟罩。</li>
	<li>在网格上加载 5-7 µL 固定外切溶液, 孵育5分钟, 100 µL PBS 三次, 每10分钟冲洗一次。</li>
	<li>治疗50µL 0.05 米甘氨酸10分钟, 以淬火游离醛组的网格。</li>
	<li>将网格转移到一滴阻塞缓冲器 (PBS 含有 1% BSA), 用于30分钟。</li>
	<li>孵育网格与 50-100 µL 抗 PD-L1 抗体 (1:100 在 PBS 包含 0.1% BSA) 为 1 h (如果需要, 这步应该执行在4° c 隔夜)。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。</li>
	<li>将网格传输到 1 h 的 2nd抗体的下落。</li></ol>抗鼠 IgG 共轭 9-11 nm 的黄金粒子稀释在1:100 在 PBS 含有 0.1% BSA。
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。用两个单独滴 (50 µL) 的 DW 洗涤网格。用2% 铀醋酸酯进行阴性染色, 描述从3.4 到3.8。</li>
	<li>将网格存储在 EM 网格框中, 以便在80伏的 TEM 上进行观察。</li>

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作者没有什么可透露的。

本文提出了一个协议, 以观察详细的外切的结构和标签的具体蛋白质。阴性染色被认为是外切成像17的最佳方法。这种传统的技术已经显示体的杯状结构。然而, 这个杯子形状是一种形式的人造制品, 可能发生由于干燥过程。低温透射电镜结果表明, 体在水溶液中具有完美的球形结构25,26。低温透射电镜技术是检测天然结构的一种非常有?...

胞外囊泡 (EVs) 是由细胞分泌的脂质双层囊泡, 其大小范围介于30和 300 nm 之间。EVs 是第一次报告在 1978年, 与证据从囊性霍奇金病患者的水泡1。据报道, 这些小泡的大小是40到120纳米。据报道, 在 1980年, EVs 参与了凝血系统和血栓形成, 在癌症患者的血管内产生了血块2。30年后, EVs 被报道是促进肿瘤侵袭、免疫逃逸和血管生成的重要因素3。此外, 在炎症、免疫紊乱、神经系统疾病和癌症等领域的细胞相互作用中, EVs 的功能已被研究为调节因子4。由于 EVs 包含特定的生物分子, 如蛋白质, mRNA, 和 rna5, 它们在诊断和治疗方面的潜在应用已被分析6,7。EVs 是一个由子群组成的类别, 包括体、prostasomes、oncosomes、dexosomes、微粒、promininosomes、argosomes 和外切样泡, 这取决于它们的细胞来源和生物学功能3。此外, 根据它们的生物, 这些 EVs 可分为微 (棚微、100-1000 nm) 和体 (30-300 nm)8,9。其中, 外切被报告为免疫应答的细胞通信器10、癌症11、12和传染性疾病13。
对体作为早期诊断的生物标记物的兴趣正在迅速增加, 体的纯化和鉴定必须伴有分子成像技术。不同的大小和形态的体, 根据它们的起源和功能14, 可以区分高分辨率的显微镜技术, 如电子显微镜。大多数体是由负染色透射电镜 (TEM)15,16,17, 这些结果是通过 immunolabeling 的一个特定的蛋白在整个芒泡的确认18. 有几个研究小组通过扫描电子显微镜、原子力显微镜、19、20、低温-TEM21、22来报告该结构。然而, 虽然这些技术是有用的研究外切结构, 他们是不足够的观察位置的特定蛋白质位于外切。因此, 我们介绍了一个协议的成像体与特定的蛋白质的标签。我们采用了块制备, 超薄切片和免疫, 以确定蛋白质的详细位置在外切。这是与阴性染色和整个芒免疫, 这是传统用于表征的外切。

<table border="0" cellpadding="0" cellspacing="0" width="583">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glutaraldehyde</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">16200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium cacodylate&#160;</td>
			<td>EMS</td>
			<td style="width:119px;">12300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Osmium tetroxide 1 g crystal</td>
			<td>EMS</td>
			<td style="width:119px;">19100</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">acetone</td>
			<td>JUNSEI</td>
			<td>1B2031</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Spurr medium</td>
			<td>TED PELLA</td>
			<td style="width:119px;">18108</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ultra-microtome</td>
			<td style="width:81px;">Leica</td>
			<td style="width:119px;">UCT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Uranyl acetate</td>
			<td>EMS</td>
			<td style="width:119px;">22400</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Lead citrate</td>
			<td>EMS</td>
			<td style="width:119px;">17900</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">Hitachi</td>
			<td style="width:119px;">H7600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">nickel grid</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">G200-Ni</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Copper grid</td>
			<td>EMS</td>
			<td style="width:119px;">G200-Cu</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">glycine</td>
			<td>SIGMA</td>
			<td>022K5404</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate buffer saline</td>
			<td style="width:81px;">SIGMA</td>
			<td style="width:119px;">P4417</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine serum albumin</td>
			<td style="width:81px;">Aurion</td>
			<td style="width:119px;">25557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">1st Antibody</td>
			<td style="width:81px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">purification in manually&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">329710</td>
			<td style="width:167px;">Whole mount Immumogold</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified Mouse IgG2b, &#954; Isotype Ctrl</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">401202</td>
			<td style="width:167px;">Whole mount Immumogold (Control)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2nd Anti-mouse igG conjugated 9-11 nm gold particle</td>
			<td style="width:81px;">sigma</td>
			<td style="width:119px;">G7652</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">silver paint</td>
			<td style="width:81px;">CANS</td>
			<td style="width:119px;">CTP100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">aluminum stub</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">75622</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FIB-SEM</td>
			<td style="width:81px;">Zeiss</td>
			<td style="width:119px;">AURIGA</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JEM2200FS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glow discharger</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JFC1100E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Carbon grid</td>
			<td>EMS</td>
			<td style="width:119px;">121119</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:119px;">19210</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated Copper Grid (200 meh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-CU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated&#160; Nickel Grid (200 mesh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-NI</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

sample preparation and imaging of exosomes by transmission electron microscopy

体是一种由体液分泌的纳米细胞胞外囊, 它能代表分泌它们的细胞的特征。分泌囊泡的含量和形态反映细胞的行为或生理状态, 例如细胞生长、迁移、分裂和死亡。体的角色可能高度依赖于大小, 体的大小从30到 300 nm 不等。最广泛使用的方法外切成像是阴性染色, 而其他结果是基于低温透射电镜, 扫描电镜, 和原子力显微镜。典型的外切的形态通过阴性染色评估是一个杯子形状, 但进一步细节还不清楚。通过结构研究的外切良好在医学和药学领域是必要的。因此, 功能相关的形态学应该通过电子显微镜技术来验证, 例如在外切的详细结构中标注特定的蛋白质。对体的结构、超薄切片图像和负染色图像进行了对比观察。在本协议中, 我们建议传输电子显微镜的成像体包括阴性染色, 全贴剂免疫染色, 块准备, 薄切片, 免疫金标签。

目前, 体被分为大小和形状类别的透射电子显微镜。图 2显示了负染色外切和免疫标签外切在整个安装状态。图 3显示了切片后的分段外切和免疫标记体。免疫金染色使用特异蛋白抗体, 用于积极识别外切和分类的蛋白质类型的外切。本协议采用全贴免疫和免疫塑料分段外切。
<img alt=...

Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

本协议描述了传输电子显微镜所需的各种技术, 包括阴性染色, 超薄切片的详细结构, 和免疫金标签, 以确定特定的蛋白质在体的位置。

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

sample-preparation-imaging-exosomes-transmission-electron

Research

JoVE Journal

Biology

38.6K 次观看.  Asan Medical Center. 本协议描述了传输电子显微镜所需的各种技术, 包括阴性染色, 超薄切片的详细结构, 和免疫金标签, 以确定特定的蛋白质在体的位置。

视频: Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

科研

生物学

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

该做什么和不该做什么低温电子显微镜的:入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.