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。

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

作者聚焦：使用石墨烯涂层网格提高 CryoEM 分辨率 

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。

用于冷冻电子显微镜的单层石墨烯涂层网格的制备

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

2.9K 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 

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

生物学

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

制备热塑性微流体通道

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

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, ...

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 ...