我们感谢Xiao Fan博士在Scripps Research建立这些方法时进行的有益讨论。B.B.得到了休伊特医学研究基金会的博士后研究奖学金的支持。W.C.得到了美国国家科学基金会博士前奖学金的支持。D.E.P得到了美国国立卫生研究院（NIH）对G.C.L.NS095892的资助。该项目还得到了 NIH 对 GCL GM142196、GM143805 和S10OD032467的资助。

1. 制备石墨烯网格所需的材料和配件
注：石墨烯容易被污染，这会降低石墨烯涂层的效率和石墨烯网格的质量;因此，彻底清洁所有与石墨烯接触的材料非常重要。材料的准备和所有步骤都应在通风橱中进行。
<ol><li>收集将用于用石墨烯涂覆网格的必要材料（图2A）。</li>
	<li>用去离子 （DI） 水冲洗玻璃器皿数次，以去除任何灰尘、棉绒和油性残留物。</li>
	<li>使用一次性湿巾用 75% 乙醇清洁玻璃盖玻片，并使用空气除尘器去除任何污染物。 注意： 本协议中使用的夹紧 TEM 网格支架块最多可容纳 45 个网格。对于石墨烯网格的大批量生产，一次可以准备45个或更少的网格。但是，建议从小批量生产（四到六个网格）开始，直到在实验室中建立方法。</li>
</ol>2.0.2 M过硫酸铵（APS）水溶液的制备
注意：该 APS 溶液用作蚀刻剂，以在后续步骤中从石墨烯/铜片中去除铜 （Cu） 载体。始终准备新鲜的 APS 溶液。重复使用或旧的溶液将无法有效地蚀刻铜，并可能在后续步骤中在石墨烯上留下铜残留物。
<ol><li>用去离子水 （DI） 水冲洗 500 mL 烧瓶，然后加入 200 mL 去离子水，并使用最大设置微波加热约 1 分钟以使水脱气。</li>
	<li>将 9 g 过硫酸铵加入 200 mL 去离子水中，产生 0.2 M APS 的溶液。 注意： APS 有毒，建议在处理 APS 时穿戴个人防护设备 （PPE）。在经批准的废物处理厂处理 APS 废物中处理。</li>
	<li>用磁力搅拌器上的搅拌棒搅拌溶液，同时将烧瓶连接到通风橱下的真空源。 注意：APS 溶液脱气将有助于防止气泡的形成，气泡会降低步骤 6 中 Cu 蚀刻的效率。</li>
</ol>3. 用一张吸墨纸将石墨烯/铜转移到干净的盖玻片上
注意：我们在铜上使用石墨烯供应商的 15 x 15 厘米化学气相沉积 （CVD） 石墨烯薄膜。商业购买的单层石墨烯/铜片应在真空下储存。由于石墨烯是通过CVD方法在Cu的两面生长的，因此石墨烯供应商通常会进行质量检查并推荐更好的一面使用。在本协议中，我们将石墨烯的这一推荐侧称为“顶部”侧，而另一侧称为“背面”。
<ol><li>将一张吸墨纸切成大约 20 mm x 40 mm 的矩形。该吸墨纸用作石墨烯/铜片的填充物，将吸收用于涂覆石墨烯/铜片的过量甲基丙烯酸甲酯（MMA（8.5）MMA EL6）;因此，一定要将其切割成比要使用的石墨烯/铜片更大的尺寸。</li>
	<li>使用聚酰亚胺胶带将吸墨纸的四个角粘在干净的盖玻片顶部，该盖玻片将适合自制的旋涂机。 注意： 使用聚酰亚胺胶带是因为它很薄且易于取下，因此易于处理。</li>
	<li>从真空存储中取出一块石墨烯/铜片。</li>
	<li>使用干净无尘的剪刀小心地剪下一小块铜石墨烯片，足以完全覆盖要准备的网格数量。例如，对于以 5 x 5 阵列排列的 25 个网格，切割一块 18 mm x 18 mm 的网格。用干净、干燥的镊子将石墨烯/铜片放在吸墨纸上。确保石墨烯/铜片的背面朝下，注意不要意外翻转石墨烯/铜片，因为很难从背面辨别顶部。</li>
	<li>将石墨烯/铜片的四个角粘在吸墨纸/盖玻片上，最大限度地减少胶带与石墨烯/铜片之间的接触量，因为在下一步中，任何被胶带覆盖的区域都不会被MMA覆盖。 注意：静电放电会损坏石墨烯薄膜，因此，建议在处理石墨烯或石墨烯网格时保持电气接地并尽量减少静电荷的积累。这可以通过在处理石墨烯或石墨烯网格之前立即佩戴手腕接地带或触摸接地的金属物体来实现。</li>
</ol>4.在单层石墨烯/铜片上涂上一层薄薄的MMA（8.5）MMA EL6（MMA）
注意：在Cu被蚀刻掉后，这层MMA将支持石墨烯单层，以便在以后的步骤中处理石墨烯片。MMA涂层还可以使石墨烯薄膜可视化，因为单层石墨烯是透明的。
<ol><li>将带有胶带石墨烯/铜片的盖玻片放在通风橱内的自制旋涂机（可以使用计算机风扇组装）上（图2B），如Han等人25先前描述的那样。</li>
	<li>戴上护目镜，使用玻璃移液管在石墨烯片上加入两滴MMA，然后立即开始以最大速度旋转。</li>
	<li>旋转时，在中心再加两滴。旋转 1 分钟。 注意：确保已应用足够的MMA以完全涂覆石墨烯。如果不确定，请再加几滴。如果石墨烯没有完全涂覆，铜被蚀刻掉后，薄膜上会出现“孔洞”。</li>
	<li>在通风橱内风干 10 分钟。</li>
</ol>5.去除石墨烯/Cu片背面的石墨烯
注意：在进行后续步骤之前，必须去除在铜背面（未涂有MMA的一侧）生长的石墨烯，因为这种过量的石墨烯会降低Cu蚀刻的有效性。我们通过将石墨烯暴露在等离子体中来去除这种石墨烯，这可以使用任何通常用于制备用于生物样品制备的电磁网格的辉光放电装置来实现。
<ol><li>小心地取下胶带，然后用干净、干燥的镊子从盖玻片上取下涂有 MMA 的石墨烯/铜片。</li>
	<li>将MMA/石墨烯/铜片的一块，MMA面朝下，粘在干净无尘的玻璃盖玻片上。</li>
	<li>将装有MMA/石墨烯/铜片的盖玻片放入辉光放电装置中，并应用通常在制备用于阴性染色或冷冻电镜样品制备的网格时使用的设置。 注意：应避免长时间的辉光放电，以防止背面的铜氧化，这可能导致石墨烯薄膜上的氧化铜 （CuO）（纳米）颗粒受到污染。</li>
</ol>6. 在APS溶液中蚀刻掉MMA/石墨烯/铜片上的铜
<ol><li>在通风橱中，将 200 mL 新鲜制备的 APS 溶液倒入干净无尘的结晶盘 （150 x 75 mm） 中。</li>
	<li>从载玻片上取下MMA/石墨烯/铜片。跟踪哪一侧包含 MMA。</li>
	<li>要开始蚀刻，将等离子体处理的铜面朝下的 MMA/石墨烯/铜片放在 APS 溶液的表面上。用一块铝箔或盖子盖住宽玻璃烧杯，以防止灰尘进入。</li>
	<li>孵育3小时。如果大部分铜在1小时后没有蚀刻，请重复第2-6节中的步骤，然后继续下一步。 注意：如果大部分Cu在1小时后没有蚀刻，则薄膜的MMA /石墨烯侧很可能已放置在APS溶液的表面上，而不是Cu侧。Cu应在3小时后完全蚀刻，如果Cu完全蚀刻，MMA/石墨烯膜是无色的。石墨烯很容易被污染，所以一定要盖住任何容器，以避免表面上堆积棉绒或任何油腻的残留物，因为这会对石墨烯的质量产生负面影响。</li>
</ol>7.用去离子水冲洗MMA/石墨烯薄膜
<ol><li>在通风橱中，用去离子水填充干净无尘的结晶盘。</li>
	<li>用干净、无尘的玻璃盖玻片轻轻舀起石墨烯-MMA薄膜，该盖玻片相对于APS溶液表面呈~45°角。</li>
	<li>将载玻片与水面成近 90° 角放置，然后轻轻地将玻璃盖玻片放入水中，使 MMA/石墨烯慢慢从载玻片上滑落并漂浮在水面上。</li>
	<li>将MMA /石墨烯薄膜留在水面上1小时以洗掉任何APS。</li>
</ol>8.清洁要涂上单层石墨烯的网格
注意：石墨烯将转移到的网格必须尽可能干净，以最大限度地将石墨烯附着在网格箔表面上。商业购买的网格通常含有残留污染物，必须在石墨烯转移之前去除这些污染物。
<ol><li>清洁并干燥三个结晶盘，使其无灰尘。</li>
	<li>在通风橱中，将 200 mL 氯仿、丙酮和异丙醇 （IPA） 倒入三个结晶培养皿中的每一个。用铝箔覆盖结晶皿，以尽量减少有机溶剂的蒸发。 注意：氯仿和丙酮会引起皮肤刺激，吸入可能有毒。限制接触这些有机溶剂并穿戴个人防护装备。</li>
	<li>将夹紧TEM网格支架块的底部放在含有200mL氯仿的第一个结晶皿的底部。</li>
	<li>将每个网格从网格盒单独转移到网格支架块中的孔中，确保开窗膜面朝上。用铝箔盖住结晶皿，将其放在轨道振荡器上，轻轻摇动30分钟。</li>
	<li>将金属盖放在网格支架块上，这将在转移到下一个结晶盘的过程中固定网格。用弯曲的叉子和长镊子小心地将网格支架块从结晶盘中取出，并将其放置在含有 200 mL 丙酮的第二个结晶盘的底部。从网格支架块上取下盖子，在轨道振荡器上轻轻摇晃30分钟。</li>
	<li>将盖子放在网格支架块上，然后转移到含有 200 mL IPA 溶液的结晶盘中以清除丙酮残留物。从网格支架块上取下盖子，轻轻摇晃 20 分钟。</li>
	<li>单独将网格从网格支架块中取出，使开窗膜面朝上，转移到覆盖有吸墨纸的玻璃培养皿上。</li>
	<li>在通风橱下将网格干燥至少 30 分钟，确保网格被覆盖以防止灰尘落在上面。</li>
</ol>9. 将干净的网格转移到吸墨纸上，放在不锈钢丝网或穿孔托盘上的去离子水
注意：网格必须浸没在平坦表面上的去离子水下，以便石墨烯可以漂浮在水面上并降低到网格上。这可以使用商业网格涂层槽或培养皿和蠕动泵进行，如Palovcak等人所述，用于生成氧化石墨烯网格18。
<ol><li>将不锈钢网或托盘放入网格涂层槽中，并用去离子水冲洗。</li>
	<li>剪下比不锈钢网/托盘略小的吸墨纸，将其放在平台顶部，浸入去离子水中。 注意：吸墨纸比平台略小，以便移动和处理。</li>
	<li>轻轻地转移清洁的网格，使开窗膜面朝上，放在吸墨纸的顶部。网格可能是疏水的，因此请将网格垂直浸入水中，否则它们可能会因水张力而弯曲。将网格放置在方形阵列中，使它们尽可能彼此靠近，但不重叠（图2C）。</li>
	<li>网格现在已经准备好涂上石墨烯单层。用更多的去离子水填充网格涂层槽，使水面至少高于网格 5 毫米。</li>
</ol>10.将石墨烯转移到网格中
<ol><li>小心地用干净的盖玻片从结晶盘中舀出MMA /石墨烯薄膜，方法是将盖玻片以一定角度缓慢降低到槽中，距离石墨烯薄膜一定距离。将盖玻片放置在石墨烯-MMA片的下方，使片的边缘与盖玻片平行，然后将盖玻片垂直抬出水面，带上MMA/石墨烯薄膜。</li>
	<li>将盖玻片以~45°角下降到水中，将MMA/石墨烯膜转移到槽中，使MMA/石墨烯膜从盖玻片上分离并漂浮在水面上。</li>
	<li>在水位降低之前，将MMA/石墨烯薄膜直接放置在网格上方。使用吸头已熔化的玻璃巴斯德移液管密封开口，以小心地操纵MMA /石墨烯薄膜的位置。</li>
	<li>用注射器以大约 1.25 mL/min 的速度缓慢降低水位，使 MMA/石墨烯薄膜在落在滤纸上时完全覆盖网格表面 注意：当水位下降时，可能需要进一步操作石墨烯-MMA片，以保持其在网格上方的位置。</li>
	<li>使用一把干净、干燥的镊子将固定网格的吸墨纸提起到干净、干燥、无尘的培养皿中，或转移整个不锈钢平台。</li>
	<li>在通风橱下风干MMA /石墨烯网格至少30分钟。保持网格用铝箔或盖子覆盖，以防止灰尘颗粒污染。</li>
	<li>将网格转移到培养箱中，并在65°C培养箱中烘烤网格30分钟。</li>
	<li>从培养箱中取出网格，并在室温下盖住它们5分钟，以将网格冷却至室温。</li>
</ol>11. 移除 MMA 并清洁网格
注意：MMA必须在丙酮中彻底洗掉，以防止石墨烯涂层网格上残留任何MMA。
<ol><li>在通风橱中，在室温下准备两个含有 200 mL 丙酮的结晶培养皿和一个含有 200 mL 异丙醇 （IPA） 的结晶培养皿。用铝箔覆盖结晶皿，以尽量减少有机溶剂的蒸发。 注意：处理丙酮时请穿戴个人防护装备，因为它会引起皮肤刺激，如果吸入可能有害。</li>
	<li>将夹紧TEM网格支架块的底部放在含有200mL新鲜丙酮的第一个结晶皿的底部。</li>
	<li>将每个网格从印迹纸单独转移到网格支架块中的孔中，确保 MMA 侧朝上。用箔纸盖住结晶皿，将其放在轨道振荡器上，轻轻摇动30分钟。</li>
	<li>将金属盖放在网格支架块上，这将在转移到下一个结晶盘的过程中固定网格。用弯曲的叉子和长镊子小心地将网格支架块从结晶盘中取出，并将其放置在含有 200 mL 新鲜丙酮的第二个结晶盘的底部。从网格支架块上取下盖子，在轨道振荡器上轻轻摇晃30分钟。</li>
	<li>将盖子放在网格支架块上，然后转移到装有 200 mL IPA 溶液的结晶盘中，以清除丙酮残留物。从网格支架块上取下盖子，轻轻摇晃 20 分钟。</li>
	<li>将网格转移到覆盖有吸墨纸的小玻璃培养皿中，并将网格风干至少10分钟。用盖子盖住网格，以防止灰尘颗粒污染。</li>
	<li>网格已准备好使用或转移到真空干燥器内用铝箔包裹的网格盒中。 注意：将石墨烯网格存放在真空干燥器内可以防止环境条件下的疏水颗粒污染。这些网格在使用前可以存放长达几个月27.</li>
</ol>12.通过紫外线/臭氧处理使石墨烯网格具有亲水性
注：石墨烯具有极强的疏水性，这与冷冻电镜样品制备不兼容，因为印迹浸入方法需要亲水表面，一滴样品可以均匀地分布在上面。虽然传统的辉光放电器件可以配置为温和地脉冲等离子体，使石墨烯表面具有亲水性，但这些器件往往会破坏薄的石墨烯单层。先前表明，紫外线/臭氧清洁剂可用于对石墨烯25 的表面进行部分氧化，使其在冷冻电镜样品制备中具有亲水性，而不会损坏单层。
<ol><li>如果使用需要灌注的紫外线/臭氧系统，请打开系统并灌注灯 10 分钟（在此步骤中，确保没有网格暴露）。当紫外线/臭氧清洁剂启动时，从真空干燥器中取出网格并将它们转移到干净的盖玻片上。</li>
	<li>当UV / Ozone系统准备就绪时，将包含网格的盖玻片（石墨烯面朝上）放入UV /臭氧清洁剂中，并将网格暴露在臭氧气体中4分钟。</li>
	<li>暴露于臭氧后，立即使用网格进行冷冻电镜样品制备。 注意：如果使用需要灌注的紫外线/臭氧系统，则必须在灯灌注 10 分钟后立即将网格放入清洁器中，否则会太冷而无法产生足够的臭氧气体来氧化石墨烯用于样品制备。不要将网格暴露在臭氧气体中超过 6 分钟，因为它会破坏石墨烯层。</li>
</ol>

用石墨烯单层涂覆 TEM 网格

<ol>
	<li>Li, X., Zheng, S. Q., Egami, K., Agard, D. 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作者没有要披露的利益冲突。

将生物样品保存在薄薄的玻璃冰层中是高分辨率冷冻电镜结构测定的关键步骤。然而，研究人员经常会遇到与 AWI 相互作用引起的问题，AWI 引入了首选方向、复杂的拆卸、变性和聚集。此外，样品不能总是足够浓缩，以填充悬浮在开窗薄膜孔上的薄冰。一些研究小组已经开发出用单层石墨烯涂覆电磁网格的方法，以帮助克服其中一些限制24,25,26,27,28,29,30，并且石墨烯网格已经取得了巨大的成功。在这里，我们提供了在内部有效制备石墨烯网格批次并通过TEM检查石墨烯网格质量的分步说明。我们强调，在一些关键步骤中应特别小心，我们将在下面概述这些步骤。
石墨烯具有很强的吸引空气污染物的倾向。因此，在石墨烯网格制造过程中，重要的是要确保所有与石墨烯/铜片或网格接触的工具都是清洁无尘的。用于转移石墨烯的玻璃盖玻片可以通过用乙醇和去离子水冲洗或使用空气除尘器进行清洁。还建议在通风橱下工作，并始终用箔纸或干净的玻璃板覆盖石墨烯片和网格。网格上的灰尘或污染物可能会阻止石墨烯完全粘附在电磁网格上。在处理石墨烯或石墨烯涂层的网格时，重要的是电气接地，以防止静电放电损坏石墨烯薄膜。通过使用手腕接地带，每次处理石墨烯或石墨烯网格时触摸接地的金属物体和/或在握住镊子的手上不戴手套，可以避免静电放电24.
由于石墨烯的单层非常薄（碳原子的宽度），因此在石墨烯向网格的转移过程中，用有机层（如MMA或poly-MMA（PMMA））支撑石墨烯非常重要。PMMA是石墨烯转移使用最广泛的材料。然而，PMMA与石墨烯有很强的亲和力，往往会导致石墨烯薄膜上的聚合物污染。MMA用于该协议，因为它留下的残留污染物较少25。然而，PMMA和MMA的缺点是形成皱纹和裂缝，可以在石墨烯薄膜的某些区域观察到（图3B）。避免这些皱纹可能具有挑战性，因为它们通常发生在石墨烯生长过程中，通过CVD方法31。最近开发了一种生长无皱纹超平石墨烯的方法，其中铜箔被Cu（111）/蓝宝石晶片作为生长基板32取代。
根据我们的经验，最好购买石墨烯/铜片并在内部用MMA支撑石墨烯，而不是从制造商那里购买聚合物覆盖的铜石墨烯片，后者在铜蚀刻后会变脆，并且在后续步骤中难以处理。如前所述，我们用于 MMA 涂层的旋涂机可以使用当地计算机/五金店的零件廉价地制造25.
在MMA涂层的步骤中，重要的是用MMA覆盖Cu-石墨烯片上的整个石墨烯表面。铜被蚀刻掉后，MMA-石墨烯将变得半透明，缺乏MMA覆盖的区域将看起来像空洞。为了防止铜侧的MMA涂层，重要的是在涂层过程中在其下方放置一小块吸墨纸，以便吸收从CVD薄膜中旋转出来的任何多余的MMA。
蚀刻和冲洗后，MMA/石墨烯片可以使用带有注射器或蠕动泵的商用或自制槽系统来控制水位，从而准备好转移到电磁网格中。在转移步骤之前，重要的是在氯仿、丙酮和 IPA 的连续浴中彻底预冲洗网格。在65°C下烘烤石墨烯涂层的网格有助于保持石墨烯的完整性，并促进石墨烯对网格的吸附。最后，为了防止 MMA 污染网格，重要的是在丙酮浴中彻底去除 MMA 并在 IPA 中清洁网格。将在EM网格上观察到任何未洗涤的MMA残留物，并降低图像的信噪比（图3C）。可以重复丙酮-IPA洗涤过程，以进一步清洁石墨烯表面。
为了使石墨烯网格具有亲水性，我们将网格暴露在紫外线/臭氧下。不同型号的紫外线/臭氧清洗剂可能需要优化，以便在不损坏石墨烯的情况下为石墨烯层充分氧化，用于冷冻电镜样品制备。无论使用何种系统，在紫外线/臭氧处理后立即将这些网格用于冷冻电镜样品应用至关重要。使石墨烯网格亲水的替代方法在其他研究中进行了描述33,34。

单颗粒低温电子显微镜（cryoEM）是一种越来越适用的技术，用于研究生物大分子的三维结构。在过去十年中，电子显微镜光学、直接电子检测1 和计算机算法 2,3,4 的技术进步使冷冻电镜用户能够以近原子分辨率确定生化稳定的大分子复合物的结构 5,6,7,8 .尽管取得了这些进展，但保存冷冻电镜成像样本仍然存在明显的障碍，这阻碍了大多数生物样本被解析到如此高分辨率。
高分辨率冷冻电镜分析的样品制备涉及捕获大分子，这些大分子在玻璃化冰薄层中以各种方向均匀分布。冷冻的“印迹和浸入式”方法是用于在网格上生成生物样品薄膜的最广泛使用的方法，用于冷冻电镜研究9,10。这些方法包括将几微升样品溶液涂在含有亲水性开窗膜的电磁网格上，然后用滤纸吸干大部分样品，然后迅速将网格放入液态乙烷或乙烷-丙烷混合物的制冷剂中9。
虽然该方法已成功用于确定各种生物标本的结构，但所有常用的冷冻电镜标本制备方法都将标本暴露在疏水性空气-水界面 （AWI） 中，这通常会引入限制高分辨率结构测定的问题。已经确定，生物标本在暴露于AWI时具有很高的变性倾向，这可能导致复杂的聚集和拆卸11,12,13,14。此外，生物标本表面的疏水斑块导致颗粒在冰中采用首选方向12。在许多情况下，样品的单个疏水区域迫使所有颗粒在冰中采用单一方向，从而消除了产生可靠重建的能力。除了AWI的问题外，标本还可能表现出对开窗薄膜层表面的亲和力，从而限制了悬浮在孔内冰中的颗粒数量15。
已经开发了几种方法和技术解决方案，以减少与AWI或电影互动引起的这些问题16,17。一种流行的方法是在EM网格的开窗膜上涂上一层薄薄的（数十纳米）无定形碳。该涂层在颗粒可以吸附的孔中提供了一个连续的表面，其优点是可以部分屏蔽样品免受与AWI15,18,19,20的相互作用。然而，额外的碳层会增加成像区域中的背景信号量，引入噪声，从而影响可达到的分辨率，特别是对于小型（<150 kDa）样品。近年来，使用氧化石墨烯（GO）薄片在冷冻电镜网格上生产支撑膜已被证明比传统的无定形碳具有优势。GO薄片是通过石墨层的氧化产生的，产生单层石墨的假结晶片，由于其表面和边缘以羧基、羟基和环氧基团的形式含有大量的氧，因此具有亲水性。水悬浮液中的商业GO薄片价格低廉，并且有许多已发表的方法可用于EM网格18,21。然而，这些方法通常会导致网格仅部分被 GO 薄片覆盖，以及包含多层 GO 薄片的区域。此外，GO薄片为冷冻电镜图像提供了明显的背景信号，该信号接近于用薄的无定形碳22,23观察到的背景信号。
原始的单层石墨烯由单个碳原子的二维晶体阵列组成，与GO的不同之处在于它不会在电子显微镜下产生相差。因此，单层石墨烯可用于生成用于生物样品成像的不可见支撑层。单层石墨烯也比GO更强，可以作为单层应用于EM网格上，石墨烯涂层EM网格制造的最新进展使得在内部制备高覆盖率的单层石墨烯网格成为可能24,25,26,27,28,29,30.然而，尽管使用石墨烯涂层网格进行冷冻电镜结构测定有好处，但由于商业选择的高昂费用和内部生产的复杂性，它们并未被广泛使用。在这里，我们描述了一个分步指南，以有效生产覆盖有单层石墨烯的电磁网格，用于生物标本的冷冻电镜结构测定（图1）。通过遵循这一详细的方案，cryoEM研究人员可以在一天内可重复地制备数十个高质量的石墨烯支撑网格。使用配备有 LaB6 灯丝的低端透射电子显微镜 （TEM） 可以很容易地检查石墨烯涂层网格的质量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% EtOH</td>
			<td>Pharmco (190 pf EtOH)</td>
			<td>241000190CSGL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma Aldrich</td>
			<td>215589-500g</td>
			<td>Hazardous; use extreme caution</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C2432-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping TEM Grid Holder Block for 45 Grids</td>
			<td>PELCO</td>
			<td>16830-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer fan</td>
			<td>Amazon (Noctua)</td>
			<td>B07CG2PGY6</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>Bellco Glass</td>
			<td>1203J71</td>
			<td>Standard cover slips</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Pyrex</td>
			<td>3140-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronics duster</td>
			<td>Falcon Safety Products</td>
			<td>75-37-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Dust-off Air Duster</td>
			<td>Staples</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter papers</td>
			<td>Whatman</td>
			<td>1001-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tip tweezer</td>
			<td>Dumont</td>
			<td>0508-L4-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Pyrex</td>
			<td>4980-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fork</td>
			<td>Supermarket</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphene/Cu</td>
			<td>Graphenea</td>
			<td>N/A</td>
			<td>CVD monolayer graphene cu</td>
		</tr>
		<tr>
			<td>Grid Coating Trough</td>
			<td>Ladd Research Industries</td>
			<td>10840</td>
			<td>Fragile</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapton Tape</td>
			<td>Amazon (MYJOR)</td>
			<td>MY-RZY001</td>
			<td>Polyimide tape</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Long twzeer</td>
			<td>Cole Parmer Essentials</td>
			<td>UX-07387-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal grid holder</td>
			<td>Ted Pella</td>
			<td>16820-81</td>
			<td></td>
		</tr>
		<tr>
			<td>MMA(8.5)MMA EL 6</td>
			<td>KAYAKU Advanced Materials</td>
			<td>M31006 0500L 1GL</td>
			<td>Flammable</td>
		</tr>
		<tr>
			<td>Model 10 Lab Oven</td>
			<td>Quincy Lab, Inc.</td>
			<td>FO19013</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Pyrex</td>
			<td>3610-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner (Solarus 950)</td>
			<td>Gatan, Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fiskars</td>
			<td>194813-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Analog Orbital Shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>UltrAuFoil R1.2/1.3 - Au300</td>
			<td>Quantifoil</td>
			<td>N/A</td>
			<td>Holey gold grids</td>
		</tr>
		<tr>
			<td>Ultraviolet Ozone Cleaning Systems</td>
			<td>UVOCS</td>
			<td>model T10X10/OES</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of monolayer graphene-coated grids for cryoelectron microscopy

低温电子显微镜（cryoEM）已成为探测大分子复合物原子结构的有力技术。冷冻电镜的样品制备需要将标本保存在一层薄薄的玻璃冰中，玻璃冰通常悬浮在开窗支撑膜的孔内。然而，冷冻电镜研究的所有常用样品制备方法都将样品暴露在空气-水界面下，对样品产生强烈的疏水作用，通常会导致变性、聚集和复杂的解离。此外，样品区域和空气-水界面之间优选的疏水相互作用会影响大分子采用的取向，从而产生具有各向异性方向分辨率的 3D 重建。 
冷冻电镜标本吸附在单层石墨烯上已被证明有助于减轻与空气-水界面的相互作用，同时最大限度地减少背景噪声的引入。石墨烯载体还具有大幅降低冷冻电镜成像所需蛋白质浓度的优点。尽管这些支架具有优势，但由于商业选择的高昂费用以及与大规模内部生产相关的挑战，石墨烯涂层网格并未被冷冻电镜社区广泛使用。本文描述了一种制备几乎完全覆盖单层石墨烯的冷冻电镜网格的有效方法。

Single-particle cryogenic electron microscopy (cryoEM) is an increasingly applicable technology used to investigate the 3D structures of biomacromolecules. Technological advances in electron microscope optics, direct electron detection1, and computer algorithms2,3,4 over the past decade have enabled cryoEM users to determine the structures of biochemically stable macromolecular complexes to near-atomic resolution5,6,7,8. Despite these advances, there remain notable barriers to preserving samples for cryoEM imaging, which prevent the majority of biological specimens from being resolved to such high resolutions.
Sample preparation for high-resolution cryoEM analysis involves trapping macromolecules that are evenly distributed in a wide range of orientations within a thin layer of vitrified ice. The &#34;blot and plunge&#34; methods of freezing are the most widely used methods employed to generate thin films of biological samples on grids for cryoEM studies9,10. These methods involve applying a few microliters of sample solution to an EM grid containing a fenestrated film that has been made hydrophilic and subsequently blotting away the majority of the sample with filter paper before rapidly plunging the grid into a cryogen of liquid ethane or ethane-propane mixture9.
While this method has successfully been used to determine structures of a wide range of biological specimens, all commonly used cryoEM specimen preparation methods expose specimens to the hydrophobic air-water interface (AWI), which often introduces&#160;issues that limit high-resolution structure determination. It is established that biological specimens have a high propensity to denature when exposed to the AWI, which can lead to complex aggregation and disassembly11,12,13,14. Furthermore, hydrophobic patches on the surfaces of biological specimens cause particles to adopt preferred orientations in the ice12. In many scenarios, a single hydrophobic region of the sample forces all particles to adopt a singular orientation in the ice, thereby abolishing the ability to generate a reliable reconstruction. In addition to issues with the AWI, specimens may demonstrate an affinity for the surface of the fenestrated layer of film, limiting the number of particles suspended in the ice within the holes15.
Several methodological and technological solutions have been developed to diminish these issues arising from interactions with the AWI or the films16,17. One popular approach is to coat the fenestrated film of the EM grids with a thin (tens of nanometers) layer of amorphous carbon. This coating provides a continuous surface across the holes to which particles can adsorb, with the benefit of partially shielding the sample from interactions with the AWI15,18,19,20. However, the additional carbon layer elevates the amount of background signal in the imaged regions, introducing noise that can compromise attainable resolution, particularly for small (&#60;150 kDa) specimens. In recent years, the use of graphene oxide (GO) flakes to produce supporting films on cryoEM grids has been shown to have advantages over traditional amorphous carbon. GO flakes are produced through the oxidation of graphite layers, resulting in pseudo-crystalline sheets of monolayer graphite that are hydrophilic due to their substantial oxygen content in the form of carboxyl, hydroxyl, and epoxy groups on the surfaces and edges. Commercial GO flakes in aqueous suspensions are inexpensive, and there are numerous published methods for applying GO flakes to EM grids18,21. However, these methods often result in grids that are only partially covered with GO flakes, as well as regions that contain multiple layers of GO flakes. Further, GO flakes contribute a noticeable background signal to cryoEM images that is close to that observed with thin amorphous carbon22,23.
Pristine monolayer graphene, which consists of a single 2D crystalline array of carbon atoms, is distinct from GO in that it does not produce phase contrast in the electron microscope. Monolayer graphene thus can be used to generate an invisible support layer for imaging biological samples. Monolayer graphene is also stronger than GO and can be applied as a single monolayer on an EM grid, and recent advancements in the fabrication of graphene-coated EM grids have made it possible to prepare high-coverage monolayer graphene grids in-house24,25,26,27,28,29,30. However, despite the benefits of using graphene-coated grids for cryoEM structure determination, they are not widely used due to the prohibitive expense of commercial options and the complexity of in-house production. Here, we describe a step-by-step guide to effectively producing EM grids covered with a monolayer of graphene for cryoEM structure determination of biological specimens (Figure 1). By following this detailed protocol, cryoEM researchers can reproducibly prepare dozens of high-quality graphene support grids in a single day. The quality of the graphene-coated grids can be readily examined using a low-end transmission electron microscope (TEM) equipped with a LaB6 filament.

作者聚焦：使用石墨烯涂层网格提高 CryoEM 分辨率 

用于冷冻电子显微镜的单层石墨烯涂层网格的制备

Currently optimally preserving biological samples is a major challenge in obtaining high resolution CryoEM structures. During the sample preparation process, specimens are exposed to hydrophobic air-water interface, which has denaturing effects on the samples and can cause stable samples to adopt a preferential orientation in the ice, which prevents high resolution structure determination. Coating CryoEM grids with a thin layer of carbon of like soft graphene oxide, has been commonly used to address issues related to interactions with air-water interface.However, this layers often increase background noise in our images, which compromise the resolutions of our structure. Coating the grid with a monolayer of graphene accomplishes similar protection without introducing significant background noise. Using graphene-coated CryoEM grids can help overcome issues related to interactions with air-water interface, however, commercial graphene grids are expensive and many labs consider graphene grids to be too complicated to prepare in-house.Researchers in the CryoEM field can now use this step-by-step protocol to reproduce fully produced dozens of high quality graphenes in one day.

这里描述的石墨烯网格制造协议的成功执行将导致完全涂有单层石墨烯的电磁网格。可以使用任何TEM检查网格的石墨烯覆盖率。由于单层干净的石墨烯在透射电镜中几乎不可见，因此必须使用显微镜的衍射模式对其进行检查，并观察与构成石墨烯的碳原子的六边形组织相对应的布拉格斑点（图3A）。偶尔观察到单层石墨烯的一些皱纹是正常的，这些皱纹是在MMA涂层过程中引入的（图3B）。人们还可以通过在石墨烯覆盖的孔的中心获取高倍率图像来检查石墨烯上存在的污染程度（图3C）。如果使用高分辨率检测器采集，则该图像的傅里叶变换应包含对应于2.14 Å处碳-碳间距的布拉格斑点（图4C）。单层碳原子不能产生足够的电子散射来产生相差，因此干净的石墨烯图像不会在图像的傅里叶变换中呈现与对比度传递函数相关的Thon环。然而，石墨烯网格在生产后很难防止污染，并且EM网格的清洗不足或石墨烯涂层后MMA的去除将导致网格上出现明显的污染物，这些污染物在真实空间图像中可见（图3C）。如图4所示，石墨烯网格对样品具有集中效应，当将0.5 mg / mL的脱铁蛋白应用于有（图4A）和没有石墨烯载体（图4B）的孔金网格时观察到。先前已经描述了类似的石墨烯制造方案，以高分辨率25,27 解析度求解蛋白质（如脱铁蛋白）的冷冻电镜结构。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65702/65702fig01.jpg"> 图 1：制备石墨烯涂层冷冻电镜网格的示意图。 图示了石墨烯网格制造过程中的关键步骤。缩写：cryoEM=低温电子显微镜;MMA = 甲基丙烯酸甲酯;APS = 过硫酸铵。 <a href="https://www.jove.com/files/ftp_upload/65702/65702fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65702/65702fig02.jpg"> 图2：制作石墨烯网格所需的材料 。 （A） 对冷冻电镜网格进行涂覆的必要材料进行了相应的标记。（B） 将石墨烯/铜片贴在载玻片上的吸墨纸上的涂布机的特写视图。旋涂机可以通过从当地计算机/五金店购买零件来组装。（C） 连接到可用于控制水位的注射器的网格涂层槽的特写视图。网格放置在不锈钢网的吸墨纸上。吸墨纸有助于操纵网格的位置，以便石墨烯片可以与之匹配。 <a href="https://www.jove.com/files/ftp_upload/65702/65702fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65702/65702fig03.jpg"> 图 3：石墨烯网格的代表性衍射图图像和明场图像，显示皱纹或 MMA 污染。 （A）当在透射电镜中以衍射模式成像时，覆盖有单层石墨烯的电磁栅格将显示对应于石墨烯六边形晶格的布拉格峰。对应于 2.14 Å 碳-碳间距的布拉格峰用圆圈圈表示。（B）单层石墨烯网格的明场图像，在石墨烯单层中有一些皱纹（用箭头表示）。（C）MMA污染的单层石墨烯的明场图像（用箭头表示）。比例尺 = 100 nm （B，C）。缩写：EM=电子显微镜;MMA = 甲基丙烯酸甲酯。<a href="https://www.jove.com/files/ftp_upload/65702/65702fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65702/65702fig04.jpg"> 图 4：石墨烯覆盖的金网格上的脱铁蛋白：（A） 石墨烯覆盖的金网格上 0.5 mg/mL 脱铁蛋白的冷冻电镜显微照片。（B）当使用不含石墨烯的多孔金网格制备时，在相同浓度下成像的脱铁蛋白在低得多的浓度下可见。（C） 0.5 mg/mL 脱铁蛋白在石墨烯覆盖的金网格上的冷冻电镜显微照片的 FFT，布拉格峰对应于六方石墨烯晶格。比例尺 = 100 nm （A，B）。缩写：cryoEM=低温电子显微镜;FFT = 快速傅里叶变换。<a href="https://www.jove.com/files/ftp_upload/65702/65702fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Enhancing CryoEM Resolution Using Graphene-Coated Grids at JoVE.com

在这里，我们描述了一种将单层石墨烯应用于电子显微镜网格的方案，以及如何准备它们以用于冷冻电镜结构测定。

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample ...

目前，以最佳方式保存生物样品是获得高分辨率 CryoEM 结构的主要挑战。在样品制备过程中，样品暴露在疏水性空气-水界面中，这对样品有变性作用，并可能导致稳定的样品在冰中采用优先取向，从而妨碍高分辨率结构测定。在冷冻电镜网格上涂上一层薄薄的碳，如软氧化石墨烯，通常用于解决与空气-水界面相互作用相关的问题。然而，这些层通常会增加图像中的背景噪声，从而影响我们结构的分辨率。用单层石墨烯涂覆网格可以实现类似的保护，而不会引入明显的背景噪声。使用石墨烯涂层的冷冻电镜网格可以帮助克服与空气-水界面相互作用相关的问题，然而，商业石墨烯网格价格昂贵，许多实验室认为石墨烯网格过于复杂，无法在内部制备。CryoEM领域的研究人员现在可以使用这种分步协议在一天内复制完全生产的数十种高质量石墨烯。

fabrication-monolayer-graphene-coated-grids-for-cryoelectron

Research

JoVE Journal

Biology

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

1.9K 次观看.  Scripps Research. 目前，以最佳方式保存生物样品是获得高分辨率 CryoEM 结构的主要挑战。在样品制备过程中，样品暴露在疏水性空气-水界面中，这对样品有变性作用，并可能导致稳定的样品在冰中采用优先取向，从而妨碍高分辨率结构测定。在冷冻电镜网格上涂上一层薄薄的碳，如软氧化石墨烯，通常用于解决与空气-水界面相互作用相关的问题。然而，这些层通常会增加图像中的背景...

视频: 作者聚焦：使用石墨烯涂层网格提高 CryoEM 分辨率 

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.

科研

生物学

Coating Single-Layer Graphene-Copper Sheet with MMA and Cleaning the Grids

To begin, inside a fume hood, place a cover slip with the taped graphene sheet on a homemade spin coater. Wearing safety goggles, use a glass pipette to add two drops of methyl methacrylate, or MMA, onto the graphene sheet, and immediately start spinning at maximum speed. While spinning, add two more drops in the center and spin for one minute.After cleaning and drying three crystallizing dishes, pour 200 milliliters of chloroform, acetone, and isopropyl alcohol into each dish. Place the base of the clamping TEM grid holder block at the bottom of the first dish containing 200 milliliters of chloroform. Ensuring the fenestrated film side is facing up, transfer each grid individually from the grid box to a well in the grid holder block.Cover the dish with aluminum foil and place it on an orbital shaker to gently shake for 30 minutes. Next, place the metal lid on the grid holder block to secure the grids for transfer to the next crystallizing dish. Using a bent fork and a long tweezer, carefully lift the grid holder block out of the dish and place it at the bottom of the second dish containing 200 milliliters of acetone.Remove the lid from the grid holder block and gently shake it on an orbital shaker. After 30 minutes, place the lid on the grid holder block and transfer it to a dish containing 200 milliliters of isopropyl alcohol to remove acetone residue. Remove the lid from the grid holder block and gently shake it for 20 minutes.Individually transfer the grids with the fenestrated film side up from the grid holder block to a glass Petri dish covered with blotting paper.

用MMA涂覆单层石墨烯铜片并清洁网格

Transferring Grids to a Perforated Tray Under DI Water, Transferring Graphene to the Grids, Removing MMA, and Cleaning the Grids

Transfer the cleaned grids with the fenestrated film side facing up onto the wet blotting paper. Submerge the hydrophobic grids vertically into the water to prevent bending due to water tension. Arrange the submerged grids in a square array, keeping them close but not overlapping.Fill the grid coating trough with more deionized water, ensuring the water surface is at least five millimeters above the grids. To scoop out the MMA graphene film from the dish, slowly lower the cover slip into the dish at an angle, some distance away from the graphene film. Position the cover slip underneath the graphene MMA sheet and align its edges parallelly with the sheet.Then, lift the cover slip vertically from the water, bringing the MMA graphene film with it. Transfer the MMA graphene film to the trough by lowering the cover slip into the water at a 45-degree angle so that the film detaches from the cover slip and floats on the water surface. Position the MMA graphene film above the grids before the water level decreases.Using a Pasteur pipette whose tip has been melted closed, carefully manipulate the MMA graphene film's position. Using a syringe, slowly lower the water level such that the MMA graphene film perfectly lands on the grids and completely covers their surfaces. Using a pair of clean, dry tweezers, lift the blotting paper holding the grids to a clean, dust-free Petri dish.Place the base of the clamping TEM grid holder block at the bottom of the first crystallizing dish containing 200 milliliters of fresh acetone. Individually, transfer each grid with the MMA side facing up from the blotting paper to a well in the grid holder block. Cover the dish with foil, and gently shake on an orbital shaker for 30 minutes.Secure the grids by placing the metal lid on the grid holder block. Then, using a bent fork or long tweezers, carefully lift the block out of the dish and place it at the bottom of the second dish containing 200 milliliters of fresh acetone. After removing the lid from the block, gently shake it on an orbital shaker for 30 minutes.Place the lid back on the grid holder block and transfer it to the dish containing 200 milliliters of isopropyl alcohol. Remove the lid before gently shaking for 20 minutes. Transfer the grids to a small glass Petri dish covered with blotting paper and air dry the grids for 10 minutes.In TEM diffraction modes, EM grids covered with a monolayer of graphene show Bragg peaks corresponding to the hexagonal lattice of the graphene. Graphene grids have a concentrating effect on a sample as observed when comparing apoferritin applied to holy gold grids with and without the graphene support. A fast Fourier transform of the cryoEM micrograph of apoferritin on graphene-covered gold grids also showed Bragg peaks corresponding to the hexagonal graphene lattice.