此处描述的协议源自 Jiang 等人 （2020） 发表的文章。这项工作得到了科技部的资助（973项目编号2011CB812502和2014CB849902）和北京市政府的资助。

本实验中使用的所有耗材都列在 材料表中。当前协议的分步工作流程如图 1 所示。
1. 蓝宝石盘上的细胞培养
<ol><li>将 3 mm x 0.16 mm 蓝宝石盘转移到含有 1 mL 乙醇的 2 mL 离心管中，并在超声波清洗器中超声处理 10 分钟。</li>
	<li>在酒精灯火焰中燃烧每个蓝宝石圆盘，直到表面上没有可见的沉积物。 注意：通过上述方法，蓝宝石盘可以多次回收。</li>
	<li>使用耐溶剂笔在每个蓝宝石圆盘的一侧标记一个数字（图 1A）。 注意：使用的记号笔需要事先进行测试，以确定它是否耐丙酮和甲醇溶剂，以防止标记丢失。</li>
	<li>将标记的蓝宝石圆盘放在 35 mm 培养皿的底部，标记的一面朝上。</li>
	<li>在紫外线下用蓝宝石盘对细胞培养皿进行灭菌30分钟。</li>
	<li>用镊子翻转蓝宝石光盘（标记的一面朝下），并在紫外线下再消毒 30 分钟。现在，含有蓝宝石盘的培养皿已准备好进行细胞培养。 注意：蓝宝石盘也可以通过高压灭菌进行灭菌。</li>
	<li>将细胞接种在上面制备的蓝宝石盘上，并在37°C，95%湿度和5%CO2 的细胞培养箱中生长至80%-90%汇合度（图1B）。 注意：表达MTn标签的稳定HeLa细胞系如Jiang等人26中所述产生。</li>
</ol>2. 高压冷冻
注意：本实验使用液氮。使用液氮时，请使用适当的安全程序和个人防护设备，以防止冻伤和窒息。
<ol><li>根据制造商的说明在实验前至少2小时准备高压冷冻机。</li>
	<li>将 A 型 HPF 铝载体（凹槽：0.025/0.275 mm）转移到含有 1 mL 乙醇的 2 mL 离心管中，并在超声波清洗器中超声处理 10 分钟。</li>
	<li>在覆盖有定性滤纸（中速）的培养皿中干燥载体。</li>
	<li>将预清洁的载体浸泡在1-十六碳烯中。 注意：在当前的实验中，不建议将BSA用作冷冻保护剂，因为它会干扰随后的金纳米颗粒合成。</li>
	<li>使用细镊子从培养皿中取出带有细胞的蓝宝石盘;用定性滤纸（中速）触摸标记的表面以去除多余的介质。 注意：水的导热系数很低，残留水过多会影响冷冻效果。保留最少的水以防止细胞变干。</li>
	<li>将蓝宝石圆盘安装到 HPF 试样支架中，并用含有 1-十六烯的 0.025 毫米深铝载体快速盖住圆盘（图 1C）。</li>
	<li>用定性滤纸（中速）吸出过量溶液。</li>
	<li>加载试样支架进行高压冷冻（图1D）。 注意：试样加载过程需要尽可能快（1 分钟内），以尽量减少由于温度、湿度、pH 值和氧含量变化而导致的生理变化。</li>
	<li>在泡沫冷冻箱中的液氮下卸载蓝宝石盘载体组件，并在使用前将组件存放在液氮中的冷冻管中。 注意：协议可能在此处暂停。冷冻管中的标本可以在液氮杜瓦瓶中储存多年。</li>
</ol>3. 冷冻替代固定（FSF）和补液（图1E）
<ol><li>用液氮填充自动冷冻替代机（AFS），并将腔室冷却至-90°C。 注意：本实验使用液氮。使用液氮时，请使用适当的安全程序和个人防护设备，以防止冻伤和窒息。</li>
	<li>在通风橱中的20mm圆形聚丙烯容器（图1E）中制备2mL的0.01%单宁酸（w / v）丙酮（FSF溶液1），并将其冷冻在液氮中。</li>
	<li>使用一对预冷的镊子将标本（蓝宝石圆盘载体组件）装入装有LN2下的冷冻FSF溶液1的容器中。</li>
	<li>将容器转移到预冷的AFS室中进行FSF处理，温度为-90°C。</li>
	<li>将样品在-90°C下保持1小时;然后，用预冷镊子将蓝宝石圆盘与载体分开，并确保蓝宝石圆盘的标记侧面朝下。 注意：镊子必须充分预冷以防止温度变化。蓝宝石圆盘应易于分离。</li>
	<li>在-90°C下再保持8-10小时;然后，在3小时内加热至-60°C。</li>
	<li>用预冷的丙酮替换容器中的FSF溶液1，孵育1小时。重复这种丙酮洗涤2次。</li>
	<li>在3小时内加热至-30°C。在此期间，在2 mL离心管中制备并预冷2mL 0.01%乙酸铀酰丙酮溶液（FSF溶液2，由10%乙酸铀酰在甲醇中稀释）。 注意：当前实验中使用的乙酸铀酰具有放射性和剧毒;必须按照适当的安全程序进行处理，并作为危险化学废物处理。</li>
	<li>用FSF溶液2替换丙酮，并在-30°C下孵育3小时。</li>
	<li>用预冷的丙酮替换容器中的FSF溶液2，并在-30°C下孵育30分钟。 重复这种丙酮洗涤2次。</li>
	<li>在2小时内从-30°C加热至4°C。</li>
	<li>用 pH 5.5 的 2 mL 含有 1 mM CaCl 2 和 1 mM MgCl 2 的0.2 M HEPES 缓冲液替换丙酮。</li>
	<li>更换缓冲液一次，并在室温下孵育1小时或在4°C孵育过夜。 注意：方案可以在这里暂停一晚或持续至少6小时，直到在步骤5中再次冷冻标本。</li>
</ol>4. 哺乳动物细胞基于ANSM的AuNP合成（图1F）
<ol><li>用PBS-A缓冲液洗涤3次，每次5分钟。</li>
	<li>在室温下将蓝宝石盘转移到含有 1 mL PBS-A 缓冲液的 35 mm 培养皿中。 注意：始终保持蓝宝石圆盘的标记面朝下，并避免蓝宝石圆盘重叠并擦掉细胞。</li>
	<li>通过将 4.28 μL 2-巯基乙醇加入含有 1 mL PBS-A 缓冲液的 2 mL 离心管中并在通风橱中充分混合来制备还原溶液。 注意：2-巯基乙醇有毒，有刺激性气味;必须按照适当的安全程序进行处理。</li>
	<li>用1mL还原溶液替换培养皿中的PBS-A缓冲液，并在室温下孵育1小时。</li>
	<li>使用前准备黄金前体。<ol><li>将 80 μL 10 mM HAuCl4 中的 ddH 2 O 加入含有 1 mL 还原溶液的2mL 离心管中，并立即涡旋。 注意：解决方案可能会变得浑浊。</li>
		<li>将 80 μL 500 mM D-青霉胺的 ddH 2 O 加入上述溶液中，并立即涡旋。 注意：解决方案可能会再次变得清晰。</li>
	</ol></li>
	<li>用步骤4.5中制备的1mL金前体溶液替换培养皿中的溶液，并在4°C下孵育2小时。 注意：一毫升金前体足以与大约1×106 个细胞（在35毫米培养皿上汇合）反应。当AuNPs明显变小时，需要更大体积的前体。</li>
	<li>称取 0.0038 g NaBH 4 到 1.5 mL 离心管中，加入 1 mL 冰冷的 ddH2O，并在使用前涡旋制成新鲜的 100 mM NaBH4。</li>
	<li>将 20-100 μL 的 100 mM NaBH 4 加入步骤4.6 的溶液中，立即摇匀混合，并在室温下孵育 5 分钟。 注意：新鲜准备 100 mM NaBH4 以立即使用。加入NaBH4后，溶液的颜色会逐渐变红，然后加深。如果没有观察到颜色变化，则在很大程度上表明实验失败。</li>
	<li>用 2 mL PBS-A 缓冲液替换溶液以停止反应。 注意：在30分钟内停止反应至关重要，因为长时间的反应会逐渐分解AuNP。</li>
</ol>5.高压冷冻和冷冻替代固定（图1C-F）
注意：标本再次是HPF和FSF，程序与前面在第2节和第3节中描述的程序几乎相同，但进行了一些修改。
<ol><li>在丙酮中制备1%四氧化锇和0.1%乙酸铀酰（FSF溶液3，由4%四氧化锇丙酮溶液和10%乙酸铀酰甲醇稀释）。 注意：四氧化锇和乙酸铀酰是剧毒化学品;它们必须按照适当的安全程序进行处理，并作为危险化学废物处理。</li>
	<li>使用一对预冷镊子将标本（蓝宝石圆盘载体组件）装入装有LN2下的冷冻FSF溶液3的容器中。</li>
	<li>将容器转移到预冷的AFS室中进行-90°C的FSF处理，并按如下方式运行FSF程序：在-90°C下保持8-10小时;然后，在3小时内加热至-60°C;在-60°C下保持3小时;然后，在3小时内加热至-30°C;在-30°C下保持3小时;然后，在2小时内加热至4°C。</li>
	<li>当温度达到4°C时，将试样转移到通风橱中，并在室温下保持15分钟。</li>
	<li>用丙酮洗涤3次，每次15分钟。</li>
	<li>在室温下在丙酮中保持至少2小时。</li>
</ol>6. 树脂浸润、包埋和聚合
<ol><li>使用前，请根据制造商的说明准备环氧树脂混合物。 注意：当前实验中使用的环氧树脂在聚合之前是有毒的;必须按照适当的安全程序进行处理。未聚合的树脂在处置前应作为危险化学废物处理或聚合。</li>
	<li>将蓝宝石盘转移到平底包埋胶囊中，并在室温下用树脂浸润至少 30 分钟。 注意：保持蓝宝石圆盘的标记侧面朝下。</li>
	<li>将样品在60°C的烘箱中聚合至少18小时。 注意：聚合后，协议可以在此处暂停。聚合的试样块可以在干燥的容器中储存多年。</li>
</ol>7. 超薄切片
<ol><li>使用剃须刀小心修剪聚合的试样块，露出蓝宝石盘。</li>
	<li>将带有蓝宝石圆盘的块尖端浸入液氮中几秒钟，然后在室温下解冻。重复冻融动作几次，将光盘从块上拆下。 注意：避免长时间冷冻，因为这可能会使试样块破裂。用刀片轻轻地拆下蓝宝石圆盘，避免过度用力，以免损坏样品。</li>
	<li>将块面修剪为梯形形状以进行超薄切片（图1G）。</li>
	<li>用超薄切片机获得70-100nm超薄切片（图1H）。</li>
	<li>在 200 目六角形铜网格上选取截面。 注意：分段后，协议可以在此处暂停。腰带上的部分可以在干燥的容器中存放多年。如果需要，网格上的切片可以用2%铀酰丙酮染色，以获得更好的膜对比度。应避免铅染色，因为它会掩盖纳米金颗粒信号。</li>
</ol>8. 透射电镜成像（图1I）
<ol><li>使用透射电子显微镜检查铜栅上的超薄切片。通常，11 kX 至 30 kX 的放大倍率范围适用于可视化细胞中的 AuNP，并且需要适当的散焦值以确保 2-3 nm AuNP 可见。</li>
</ol>

直接合成金纳米颗粒用于蛋白质定位分析

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K., 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=Metallothionein+as+a+clonable+tag+for+protein+localization+by+electron+microscopy+of+cells.">Metallothionein as a clonable tag for protein localization by electron microscopy of cells.</a> Journal of Microscopy. 260 (1), 20-29 (2015).</li><li>Nishino, Y., Yasunaga, T., Miyazawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+metallothionein+tag+enabling+efficient+protein+detection+by+electron+microscopy.">A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy.</a> Journal of Electron Microscopy. 56 (3), 93-101 (2007).</li><li>Bouchet-Marquis, C., Pagratis, M., Kirmse, R., Hoenger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallothionein+as+a+clonable+high-density+marker+for+cryo-electron+microscopy.">Metallothionein as a clonable high-density marker for cryo-electron microscopy.</a> Journal of Structural Biology. 177 (1), 119-127 (2012).</li><li>Fukunaga, Y., 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=Electron+microscopic+analysis+of+a+fusion+protein+of+postsynaptic+density-95+and+metallothionein+in+cultured+hippocampal+neurons.">Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons.</a> Journal of Electron Microscopy. 56 (4), 119-129 (2007).</li><li>Diestra, E., Fontana, J., Guichard, P., Marco, S., Risco, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+proteins+in+intact+cells+with+a+clonable+tag+for+electron+microscopy.">Visualization of proteins in intact cells with a clonable tag for electron microscopy.</a> Journal of Structural Biology. 165 (3), 157-168 (2009).</li><li>Risco, C., 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=sensitive,+high-resolution+detection+of+protein+molecules+in+eukaryotic+cells+using+metal-tagging+transmission+electron+microscopy.">sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy.</a> Structure. 20 (5), 759-766 (2012).</li><li>Ding, S. -. W., 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=Noncanonical+role+for+the+host+Vps4+AAA++ATPase+ESCRT+protein+in+the+formation+of+tomato+bushy+stunt+virus+replicase.">Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of tomato bushy stunt virus replicase.</a> PLoS Pathogens. 10 (4), e1004087 (2014).</li><li>Jiang, Z., 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=Direct+synthesis+of+gold+nanoparticles+on+cysteine-rich+tags+in+yeast+cells.">Direct synthesis of gold nanoparticles on cysteine-rich tags in yeast cells.</a> Protocol Exchange. , (2020).</li><li>Jiang, Z., 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=Direct+synthesis+of+gold+nanoparticles+on+cysteine-rich+tags+in+mammalian+cells.">Direct synthesis of gold nanoparticles on cysteine-rich tags in mammalian cells.</a> Protocol Exchange. , (2020).</li><li>Jiang, Z., 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=Genetically+encoded+tags+for+direct+synthesis+of+EM-visible+gold+nanoparticles+in+cells.">Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.</a> Nature Methods. 17 (9), 937-946 (2020).</li><li>Brust, M., Walker, M., Bethell, D., Schiffrin, D. J., Whyman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+thiol-derivatised+gold+nanoparticles+in+a+two-phase+liquid-liquid+system.">Synthesis of thiol-derivatised gold nanoparticles in a two-phase liquid-liquid system.</a> Journal of the Chemical Society, Chemical Communications. 7, 801-802 (1994).</li></ol>

作者声明不存在利益冲突。

该研究在这里展示了一种强大的可克隆EM标记技术，用于以超微结构分辨率在细胞环境中对蛋白质分子进行单分子可视化。直接在富含半胱氨酸的基因编码标签上合成的AuNPs提供了目标蛋白的明确和精确的定位。高压冷冻和冷冻替代技术很好地保留了生物样品的超微结构。综上所述，这里介绍的可克隆电子显微镜标记技术为在细胞超微结构环境中使用EM原 位 定位和识别单个分子提供了强大?...

在后基因组时代，绿色荧光蛋白（GFP）作为光学显微镜单分子报告基因的发展彻底改变了现代生命科学研究领域1，2。几十年来，电子显微镜（EM）一直是直观观察细胞超微结构的强大工具，分辨率为纳米级分辨率3;然而，蛋白质分子的精确鉴定和定位仍然具有挑战性。
最常用的EM标记技术是基于抗原 - 抗体反应的免疫电子显微镜（IEM）标记技术。然而，尽管在IEM标记领域已经开发了许多技术，包括预包埋IEM和后嵌入IEM（在树脂切片或水合冷冻切片上），但它仍然存在标记效率低（<10%）4，5，这与样品制备和抗体质量有关。为了克服这些限制，开发基因编码标签具有巨大的应用潜力。
近年来，EM标签的两种主要类型已被彻底探索。一种类型是DAB染色法，该方法利用APEX2等标签将3，3'-二氨基联苯胺（DAB）氧化为亲渗透聚合物，用于EM可视化6，7，8，9，10，11，12。它可以标记亚细胞区域中的高丰度蛋白质，但不适用于单分子计数。另一种类型利用金属结合蛋白，如铁蛋白13和金属硫蛋白（MT）14，15，16，17，18，19，20，21，22，23，24，25，26，在用于电磁可视化的原位。只有后者具有单分子可视化和计数的真正潜力。铁蛋白的分子尺寸太大（~450 kD），无法用作有前途的标签，而MT的小尺寸（~5 kD）及其通过其20个半胱氨酸结合各种离子的能力引起了极大的关注。一些实验室试图通过直接与Au+孵育来标记纯化的MT融合蛋白或表达MT的细胞。这些尝试初步证明了MT标签可以结合金离子形成高对比度信号，但没有一个真正实现细胞中单个蛋白质的有效鉴定，并且它们并不广泛适用14，15，16，17，18，19，20，21，22，23。
基于ANSM的AuNP合成技术涉及直接在富含半胱氨酸的标签（例如MT，MT变体MTn和MTα，AFP）上合成2-6 nm大小的AuNP，作为EM可视化的电子致密标记，是细胞中蛋白质标记和单分子检测的第一个可靠且适用的方法24，25，26.它允许在分离的标签融合蛋白上特异性合成AuNPs，并在非固定或化学固定的原核（大肠杆菌）和真核（S. pombe）细胞中实现了前所未有的标记效率。然而，在哺乳动物细胞甚至组织等更先进的系统中实施相同的协议涉及额外的挑战，例如更复杂的细胞内氧化还原稳态和更脆弱的细胞结构。
本研究提出了一种可克隆的EM标记技术，该技术将基于ANSM的新型AuNP合成技术与HPF / FSF-再水化-HPF / FSF样品制备方法相结合，能够明确单分子鉴定HeLa细胞中ER膜，ER腔和线粒体基质中的标记蛋白。目前的方法综合了标记效率高、信噪比高、单分子标记、通用性强等特点，在生命科学研究中具有广阔的应用前景。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025 mm/0.275 mm Aluminum carrier</td>
			<td>Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 M HEPES buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve HEPES (0.2 M) in 980 mL of ddH2O, then add 10 mL of 100 mM MgCl2 and 10 mL of 100 mM CaCl2 (final concentration 1 mM), respectively, adjust pH to 5.5</td>
		</tr>
		<tr>
			<td>1.5 mL MaxyClear snaplock microtube</td>
			<td>Axygen Scientific</td>
			<td>MCT150C</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL polypropylene screw cap microtubes</td>
			<td>Biologix</td>
			<td>81-0204</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mesh hexagonal copper grid</td>
			<td>Tedpella inc</td>
			<td>G200HEX</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Amresco</td>
			<td>0482-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Corning</td>
			<td>430928</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Beiijng Tong Guang Fine Chemicals Company</td>
			<td>31025</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated freeze substitution machine</td>
			<td>Leica</td>
			<td>AFS2</td>
			<td></td>
		</tr>
		<tr>
			<td>Customized 3.05 mm x 0.66 mm specimen holders for HPF</td>
			<td>Beijing Wulundes Biotech Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-penicillamine</td>
			<td>TCI</td>
			<td>P0147</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium</td>
			<td>GBICO</td>
			<td>C11965500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>GBICO</td>
			<td>10099-141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom embedding capsule</td>
			<td>Tedpella inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foam cryobox</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar 15/95 resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>15800</td>
			<td></td>
		</tr>
		<tr>
			<td>HAuCl4</td>
			<td>Sigma</td>
			<td>4022-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>sigma&#160;</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>HPF machine</td>
			<td>Wohlwend&#160;</td>
			<td>HPF compact01</td>
			<td></td>
		</tr>
		<tr>
			<td>Methonal&#160;</td>
			<td>Beiijng Tong Guang Fine Chemicals Company</td>
			<td>12397</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma</td>
			<td>480886-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>Electron Microscopy Sciences</td>
			<td>19110</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-A buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve NaH2PO4 (1.125 mM), Na2HPO4 (3.867 mM), NaCl (100 mM) in 1 L of ddH2O, adjust to pH 7.4</td>
		</tr>
		<tr>
			<td>Qualitative filter paper (medium speed)</td>
			<td>Beyotime Biotechnology</td>
			<td>FFT08</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire discs</td>
			<td>Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solvent resistant pen</td>
			<td>Electron Microscopy Sciences</td>
			<td>62053-B</td>
			<td></td>
		</tr>
		<tr>
			<td>SPI-Pon 812 resin</td>
			<td>SPI Inc</td>
			<td>02659-AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>FEI</td>
			<td>Tecnai G2 spirit</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>GBICO</td>
			<td>C25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramictotome</td>
			<td>Leica</td>
			<td>FC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct synthesis of em-visible gold nanoparticles in cells for protein localization analysis with well-preserved ultrastructure

以超微结构分辨率分析细胞中蛋白质分子的精确定位，对于研究所有生物体的各种生理或病理过程具有重要意义。因此，开发可用作电子显微镜探针的可克隆标签具有重要价值，就像荧光蛋白在光学成像领域发挥了至关重要的作用一样。最近发现了自成核抑制机制（ANSM），它允许在富含半胱氨酸的标签上特异性合成金纳米颗粒（AuNPs），例如金属硫蛋白（MT）和防冻蛋白（AFP）。
基于ANSM，开发了一种电子显微镜标记技术，该技术能够以前所未有的标记效率对原核和真核细胞中的标记蛋白进行特异性检测。这项研究说明了在具有良好保存的超微结构的哺乳动物细胞中检测MTn（一种缺乏醛反应性残基的工程MT变体）融合蛋白的方案。在该协议中，使用非醛固定剂（如单宁酸，乙酸铀酰）进行高压冷冻和冷冻取代固定，以保持近乎天然的超微结构并避免醛交联对标签活性的损害。
在基于ANSM的AuNP合成之前，使用简单的一步复液。结果表明，标记蛋白靶向内质网（ER）膜和管腔等多种细胞器，线粒体基质高效、特异性高。这项研究为生物学家提供了一个强大的协议，以解决细胞超微结构背景下单分子水平上的大量生物学问题。

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

Protein detection and localization are essential in biological research. Our protocol describes a novel EM-labeling technology using genetically-encoded tags which allows single-molecule visualization in situ with well-preserved ultrastructure. It is crucial to avoid over-fixation.The labeling method is based on the activity of cysteines in the tags, which are highly sensitive to aldehyde cross-linking. Our protocol avoids use of aldehyde fixatives and preserves the tag activity and ultrastructure. at the same time.EM labeling has always been challenging, suffering from poor morphology or inefficient labeling. Our protocols synthesizes in situ nanoparticles directly on individual proteins to provide clear and precise localization of the target proteins, with a high signal-to-noise ratio, high efficiency, and specificity. We have so far achieved excellent labeling in isolated proteins, E.coli cells, yeast cells, and HeLa cells.We will explore and optimize the method for application to tissue samples. Combining the prevailing cryo-FIB and cryo-EM technologies will allow us to address important biological questions.

基于ANSM的AuNP合成技术是用TEM26标记和检测MT标记蛋白的非常有用的工具。为了验证其在哺乳动物细胞中的稳健性，在Hela细胞中产生了三种表达EGFP-MTn-KDEL，Ost4-EGFP-MTn或Mito-acGFP-MTn的稳定细胞系。KDEL是一个规范的C端内质网（ER）保留/检索序列，其将融合蛋白EGFP-MTn-KDEL维持在ER腔内或核包膜（NE）的核周空间内。Ost4是寡糖转移酶复合物的一个亚基，寡糖转移酶复合物是一种位于ER和N...

Watch this Scientific Journal Video about Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure at JoVE.com

本协议描述了一种可克隆的电子显微镜标记技术，用于使用基于新型自成核抑制机制的金纳米颗粒合成技术检测细胞中的金属硫蛋白标记蛋白。

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various ...

蛋白质检测和定位在生物学研究中至关重要。我们的协议描述了一种使用基因编码标签的新型EM标记技术，该技术允许具有保存完好的超微结构的单分子原位可视化。避免过度固定至关重要。标记方法基于标签中半胱氨酸的活性，半胱氨酸对醛交联高度敏感。我们的方案避免使用醛固定剂，并保留标签活性和超微结构。与此同时。EM标记一直具有挑战性，存在形态不良或标记效率低下的问题。我们的方案直接在单个蛋白质上原位合成纳米颗粒，以提供目标蛋白质的清晰和精确定位，具有高信噪比、高效率和特异性。到目前为止，我们已经在分离的蛋白质、大肠杆菌细胞、酵母细胞和HeLa细胞中实现了出色的标记。我们将探索和优化应用于组织样品的方法。结合流行的冷冻FIB和冷冻电镜技术将使我们能够解决重要的生物学问题。

direct-synthesis-em-visible-gold-nanoparticles-cells-for-protein

Research

JoVE Journal

Biology

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

817 次观看.  National Institute of Biological Sciences, Beijing. 蛋白质检测和定位在生物学研究中至关重要。我们的协议描述了一种使用基因编码标签的新型EM标记技术，该技术允许具有保存完好的超微结构的单分子原位可视化。避免过度固定至关重要。标记方法基于标签中半胱氨酸的活性，半胱氨酸对醛交联高度敏感。我们的方案避免使用醛固定剂，并保留标签活性和超微结构。与此同时。EM标记一直具有挑战性，存在形态?...

视频: 作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

科研

生物学

﻿Preparing Sapphire Discs for Cell Culture

Preparing Sapphire Discs for Cell Culture  

Transfer three millimeters by 0.16 millimeters sapphire discs to a two-milliliter centrifuge tube containing one milliliter ethanol and sonicate it in an ultrasonic cleaner for 10 minutes. Burn each sapphire disc in an alcohol lamp flame until there are no visible deposits on the surface. Label one side of each sapphire disc with a number using a solvent-resistant pen.Place the labeled sapphire discs on the bottom of a 35-millimeter culture dish with the labeled side facing up. Sterilize the cell culture dish with sapphire discs under ultraviolet light for 30 minutes. Flip the sapphire discs with tweezers, ensuring that the labeled side is facing down, and sterilize under ultraviolet light for an additional 30 minutes.Seed the stable HeLa cell lines expressing MTn tags on the prepared sapphire discs and grow to 80 to 90%confluency in a cell incubator.

制备用于细胞培养的蓝宝石盘

High-Pressure Freezing (HPF), Freeze-Substitution Fixation (FSF) and Rehydration of Sapphire Disc-Carrier Assembly

Prepare a high pressure freezing or HPF machine, at least two hours before starting the experiment according to the manufacturer's instructions. Transfer Type A HPF aluminum carriers to a two-millimeter centrifuge tube containing one milliliter ethanol and sonicate them in an ultrasonic cleaner for 10 minutes. Dry the carriers in a Petri dish covered with qualitative filter paper.Soak the pre-cleaned carriers in 1-hexadecene. Use fine tweezers to pick up a pre-prepared sapphire disk from the HeLa cell culture dish. Touch the labeled surface with qualitative filter paper to remove the excess medium.Mount the sapphire disk into the HPF specimen holder and quickly cap the disk with a 0.025-millimeters-deep aluminum carrier containing 1-hexadecene. Aspirate excess solution with qualitative filter paper. Load the specimen holder for high-pressure freezing.Unload the sapphire disk carrier assembly under liquid nitrogen in a foam cryo box. Prepare two milliliters of the freeze-substitution fixation or FSF solution one in a 20-millimeter round polypropylene container in a fume hood and freeze it using liquid nitrogen. Use a pair of pre-cooled tweezers to load the sapphire disks and carrier assemblies into the container with frozen FSF solution one under liquid nitrogen.Transfer this container to a pre-cooled automated freeze substitution machine chamber for FSF processing at minus 90 degrees Celsius. Keep the specimens at minus 90 degrees Celsius for one hour, then separate the sapphire disks from the carriers with pre-cooled tweezers, making sure that the marked sides of the sapphire disks are facing down. Keep the specimens at minus 90 degrees Celsius for an additional 8 to 10 hours before warming up to minus 60 degrees Celsius within three hours.Replace the FSF solution one in the container with pre-cooled acetone and incubate at minus 60 degrees Celsius for one hour. Repeat this acetone wash twice. Warm up to minus 30 degrees Celsius within three hours.Meanwhile, prepare and pre-cool two milliliters of FSF solution two in a two-milliliter centrifuge tube at minus 30 degrees Celsius. Replace the acetone with FSF solution two and incubate at minus 30 degrees Celsius for three hours. Then replace FSF solution two in the container with pre-cooled acetone and incubate for 30 minutes at minus 30 degrees Celsius.Repeat this acetone wash twice. Warm for minus 30 to 4 degrees Celsius within two hours. Replace the acetone with two milliliters of 0.2 molar HEPES buffer.Change the buffer once and incubate at room temperature or four degrees Celsius overnight.

蓝宝石盘载体组件的高压冷冻 （HPF）、冷冻替代固定 （FSF） 和再水化

Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs

Take a pre-prepared HPF and FSF treated sapphire disc containing stable HeLa cells, wash it with PBS-A buffer for five minutes, and repeat this process three times. Transfer the sapphire discs to a 35-millimeter culture dish containing one milliliter of PBS-A buffer at room temperature. Replace the PBS-A buffer in the culture dish with one milliliter of reducing solution and incubate for one hour at room temperature.Prepare the gold precursor in one milliliter of reducing solution and immediately mix by vortexing. Add 80 microliters of 500-millimolar D-Penicillamine in double distilled water to the gold precursor solution. And immediately vortex.Replace the solution in the cultured dish with one milliliter of gold precursor solution and incubate for two hours at four degrees Celsius. Add 20 to 100 microliters of the freshly prepared sodium borohydride solution to the gold precursor solution, immediately shake to mix well, and incubate for five minutes at room temperature. Replace the solution with two milliliters of PBS-A buffer to stop the reaction.Then prepare the epoxy resin mixture according to the manufacturer's instructions, transfer the sapphire discs to flat bottom embedding capsules, and infiltrate with resin for at least 30 minutes at room temperature. Polymerize the specimen at 60 degrees Celsius in an oven for at least 18 hours. Prepare ultra-thin sections of the polymerized specimen by trimming the specimen blocks carefully using a razor to expose the sapphire discs.Dip the block tips with sapphire discs into liquid nitrogen for several seconds and thaw room temperature. Repeat the freeze-thaw action several times to detach the discs from the blocks. The EGFP-MTn-KDEL protein appeared as two to five-nanometer sized gold nanoparticles exclusively distributed in the peripheral ER lumen and in the perinuclear space of the nuclear envelope.The well-preserved ultra structure enabled the single molecule identification of tagged proteins and facilitated the analysis of organelle interactions, such as ER mitochondria interactions. Nanoparticles of the Ost4-EGFP-MTn protein delineated the ER membrane in the outer membrane of the nuclear envelope. Likewise, the Mito-acGFP-MTn expressing cells exhibited specific labeling in the mitochondrial matrix.