Name: sample preparation for in situ cryotomography of mammalian cells
Uploaded: 2023-12-15T13:30:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Optimizing Grid Preparation for Enhanced Cryoelectron Tomography at JoVE.com

我们要感谢 Mansky 实验室提供极速冻设备。这项工作的一部分是在明尼苏达大学的表征设施中进行的，该设施通过材料研究科学与工程中心（MRSEC;奖项编号 DMR-2011401） 和国家神经科学课程倡议 （NNCI;奖项编号 ECCS-2025124） 计划。我们要感谢病毒环境中的HIV行为中心（B-HIVE;1U54AI170855-01）和杜克大学HIV结构生物学中心（DCHSB;U54AI170752） 中心。

1. 电网准备
注：在实验设计中，计划每次浸入冷冻期间最多 8-12 个网格，每孔最多 4-5 个网格。超过此值将导致非常长的冷冻过程，这可能会导致细胞压力增加、冰污染和用户错误。
<ol><li>辉光放电<ol><li>在辉光放电装置中，用穿孔碳支撑膜加载适当数量的网格，以 15 mA 的电流在 0.39 mBar 气氛下以 0.39 毫秒的电流进行辉光放电。</li>
		<li>确保电网的碳面朝上。完成后，将网格转移到衬有滤纸的干净容器中（图1A1）。 注意：只要材料对电池无毒（不包括任何铜网格），就可以使用其他网格。Finder 网格对于关联研究非常有用。也可以使用其他材料制成的网格。许多实验室在 SiO2 支持网格 8,9 上生长细胞取得了良好的结果。</li>
	</ol></li>
	<li>依从性治疗<ol><li>将网格、纤连蛋白、PBS、板和镊子转移到生物安全柜中。通过在无菌表面（例如 6 孔板的盖子）上微量移液两个大大小的贴壁溶液点，用 20 μg/mL 牛纤连蛋白处理网格的两侧。确保点足够大以包裹整个网格（建议约 100 μL）。 注：在用选定的贴壁溶液处理之前，可以选择在此处对网格进行光微图案化，以优化网格正方形中心细胞的附着。降低单元格附着到条形图的能力将增加可成像单元格的数量。这是涉及冷冻FIB研磨的实验的理想选择。</li>
		<li>使用 5/15 镊子操作网格。确保在溶液中处理网格的两侧。 注意：可以使用其他粘合剂溶液（纤维蛋白原、胶原蛋白、其他细胞外基质成分等）。这里使用纤连蛋白，因为它在多种细胞系中都能获得良好的结果。（U2OS、HeLa、Vero、Calu-3、Tzm-bl）。5/15 镊子非常有用，因为它的几何形状可以提高 6 孔板、35 mm 培养皿和微型载玻片周围的可操作性。这样可以减少网格上的机械应力，并形成更完整的碳膜。确保在开始之前将所有必需的材料都放在生物安全柜中，因为这样可以减少网格污染的可能性。</li>
	</ol></li>
	<li>附加网格<ol><li>一旦用牛纤连蛋白处理，网格就会变得粘稠。使用 5/15 镊子，将网格的非碳表面轻轻接触空盘/盘子的底部，然后打开镊子。网格应易于附着在新表面上（图1A2）。 注意： 尽量避免将网格放在盘子/盘子的直接中心或边缘。添加细胞时，细胞密度有在板中心积聚的趋势。这可能导致网格具有过高的细胞密度。或者，这里可以使用3D打印的网格支架，而不是将细胞直接连接到培养皿/板12的表面。</li>
	</ol></li>
	<li>孵化<ol><li>将网格在纤连蛋白贴壁溶液（20μg/ mL）中孵育30分钟。为确保网格在此过程中不会变干，每15分钟将1-2滴更粘附的溶液直接滴在网格顶部（图1A3）。 注意：孵育期将根据所使用的贴壁溶液而有所不同。对于牛纤连蛋白，推荐时间为 30 分钟。</li>
	</ol></li>
	<li>洗涤<ol><li>孵育期过后，通过微量移液去除多余的粘附溶液。通过将PBS滴微量移液液直接在网格顶部来清洗网格。重复此操作 2-3 次。（图 1A4）。</li>
	</ol></li>
	<li>灭菌<ol><li>使用紫外线对生物安全柜中的网格进行消毒1小时。将网格放置在尽可能靠近紫外线源的位置，以最大限度地提高灭菌效果（图1A5）。为确保网格在此过程中不会变干，每 10 分钟将 1-2 滴 PBS 直接滴在网格顶部。 注意：步骤 1.4-1.6 可以同时执行。</li>
	</ol></li>
</ol>2. 播种网格
<ol><li>细胞计数：<ol><li>使用机械或酶法分离和计数细胞。在计数之前确定用于接种的最佳细胞数。对于接种在 6 孔板中的 U2OS，每孔使用 6 x 104 至 1.6 x 105 个细胞之间的任意位置（图 1B1）。 注：接种的细胞数量将根据细胞类型、孔的表面积、使用的培养皿或微载玻片、接种和浸入冷冻之间的时间以及实验设计而变化。测试一系列细胞数量，以确定在骤降冻结时将导致每个网格方块产生 0.25-1 个细胞的条件。需要注意的是，高细胞密度会降低传热速度，并可能影响玻璃化过程。为了解决这个问题，一些实验室已经发现使用细胞过滤器取得了成功，这种过滤器的作用是防止细胞团块形成13。最后，该协议导致细胞随机附着在碳层上。如果需要有针对性的附件，可以使用光微图案化（参见步骤 1.3）14。</li>
	</ol></li>
	<li>添加细胞和生长培养基：通过微量移液管将细胞添加到孔中，避免气泡（图1B2）。在 6 孔板中，建议总孔体积为 1.5-2.0 mL。 注意： 建议在添加整个体积之前小心地弄湿网格周围。这将有助于防止网格在溶液中分离和浮动。轻轻地左右移动板，以促进孔上的细胞均匀分布。不要以圆周运动旋转板，因为这会导致孔中心分配过多的细胞密度。</li>
	<li>孵育：根据实验设计，将载玻片在37°C下孵育适当的时间，这将取决于下游应用。对于本例（HIV分子克隆的转染），孵育16-24小时（图1B3）。 注意：较长的孵育时间将导致更多的细胞分裂周期。因此，请相应地调整接种的细胞起始数量。</li>
</ol>3. 转染
<ol><li>制备转染混合物：<ol><li>制备适当的阳离子脂质体试剂，用于将质粒转染到所选细胞系中。</li>
		<li>在该转染示例中，每孔（在 6 孔板中）使用 1 μg 总质粒 DNA，以 1：3 的比例转染 HIViΔEnv 质粒与 psPAX2 质粒。对于每个孔，将 1 μg DNA 稀释到 50 μL 无血清 DMEM 中。通过反复微量移液或轻轻旋转板来匀浆混合物。</li>
		<li>对于每个孔，在无血清DMEM中稀释3μL转染试剂，然后通过微量移液或轻轻旋转再次匀浆混合物。将整个稀释的转染试剂混合物加入稀释的DNA混合物中（步骤3.1.2），并在室温（RT）下孵育15分钟（图1C1）。 注意：当该混合物孵育时，用 1.5 mL 新鲜 DMEM 替换孔中的培养基。小心不要将格栅从井底移开。</li>
	</ol></li>
	<li>为了应用转染混合物，将步骤3.1.3中的100μL混合物逐滴移液到每个孔中，将液滴集中在网格上以确保接触（图1C2）。转染后16-24小时更换生长培养基（图1C3）。</li>
</ol>4.骤降冻结
注意：将细胞保持在37°C，直到需要印迹。此外，在整个过程中，请务必记下电网的碳侧。
<ol><li>细胞密度<ol><li>在倒置光学显微镜中检查网格，并注意每个网格上的细胞密度。 注意：每个网格方块少于 0.25 个单元格（或每 4 个网格方块一个单元格）的网格被视为“空”。每个网格方格有 0.25-1 个像元的网格被认为是“正常”的。每个格子方块具有 1 个以上像元的格网被视为“已满”。</li>
		<li>记下每个网格上的细胞密度，以便在速冻期间稍后调整印迹时间。将2-3mL PBS加入空板/培养皿中，并加热至37°C。</li>
	</ol></li>
	<li>准备基准点<ol><li>为了制备用于下游冷冻断层扫描应用的金基准点，将 50 μL 10 nm 胶体金珠溶液移液到干净的 1.5 mL 试管中。向试管中加入 2 μL 1 mg/mL BSA，并通过反复微量移液匀浆。</li>
		<li>将试管以15,000-20,000× g 离心15分钟。微量移液器在不干扰沉淀的情况下尽可能多地去除上清液。 注意：颗粒会非常松散。</li>
		<li>向沉淀中加入 50 μL PBS 并重悬。再次以15,000-20,000× g 离心管15分钟。</li>
		<li>使用 10 μL 吸头，直接吸出 4 μL 沉淀，并将其转移到干净的 1.5 mL 管中（图 1D1）。每个网格使用 1 μL 洗涤和浓缩的金。 注意：使用的基准点类型取决于下游应用。对于 TEM 断层扫描，请使用 10 nm 金珠。对于软 X 射线断层扫描，请使用 200 nm 金珠。对于相关显微镜检查，请使用荧光乳胶珠。对于涉及冷冻FIB研磨的实验，基准点通常不是必需的，因为研磨过程会破坏它们。</li>
	</ol></li>
	<li>杂交<ol><li>将环境室设置为95%的湿度和30°C（图1D1）。使用 5/15 镊子选择网格。</li>
		<li>用PBS清洗网格并将它们转移到插入镊子上（图1D2）。固定后，取出 5/15 镊子并滑动夹子amp 在插入镊子上。</li>
		<li>将网格插入切入式冷冻机，保持夹子牢固（图 1D3）。向网格的背面加入1μL金基准点（图1D4）。将 2-3 μL PBS 添加到网格的碳侧。使用自动印迹法印迹网格的背面，并将冷冻液浸入液态乙烷中（图1D5）。 注：建议每个网格的体积在 3-4 μL 之间，以获得最佳印迹效果。洗涤后，网格可能会保留不同量的 PBS。调整增加的 PBS 体积，以考虑网格上预先存在的液体潴留。建议将黄金基准点添加到网格的背面。在前面加法会导致在插入点附近产生金基准点池，而从后面加法会导致更均匀的解决方案。印迹持续时间取决于网格的细胞牙科。空格格分别为 2 秒、正常格栅为 3 秒、全格格为 4 秒。可用的速冻仪类型可能决定了印迹的方法：徕卡GP2速冻仪是该协议中使用的速冻机，默认情况下能够进行自动单面印迹。Thermo Fisher Vitrobot 可进行自动双面印迹，但这通常会损坏附着在碳侧的细胞。也可以在 Vitrobot 中进行手动印迹，也可以使用手动重力柱塞。</li>
	</ol></li>
</ol>

用于原位细胞冷冻断层扫描的电子显微镜网格的制备

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作者声明没有利益冲突。

在这里，我们提供了一种可访问、灵活且可重复的方案，用于在原 位 冷冻电子断层扫描应用中的电子显微镜网格上接种细胞。该方法可以很容易地适应下游应用和/或实验要求的需求。除了极大的灵活性外，我们还描述了一种工作流程，该工作流程可以优化和减少网格接种中的常见缺陷，特别是对碳层的广泛破坏、低细胞密度和薄细胞突起的结构完整性差。
尽管此处描述的协议确实提供了几种替代方案，但应遵循一些关键步骤来优化一般结果。网格细胞接种的最大问题之一是网格从孔或微载玻片上分离和漂浮。因此，重要的是在两侧用粘附溶液完全润湿网格，并防止其在孵育期间干燥。如果使用 3D 打印的网格支架，请注意，对这些支架进行多次介质更换可能会产生浮动网格，因为被困在网格下方的空气会迫使其离开支架。 
我们选择的镊子还提高了网格质量，因为它提供了一种几何上有利的方法来操纵网格，而不会造成会损坏碳层的大量网格弯曲。在坠入之前，将细胞保持在37°C下尽可能长的时间可以减少细胞的痛苦，并改善网格上薄的可成像细胞的数量。最后，从金侧进行印迹将保护细胞免受可能导致脆弱细胞结构受损的苛刻机械力的影响。
虽然不包括在该协议中，但网格光微图案化已被证明通过优化可成像细胞与网格方块14中心的附着来增加可成像细胞的数量。最后，3D打印的网格支架最近被用于通过限制直接的网格操作来减少网格损坏12。
需要注意的是，该协议针对细胞的薄边缘和突起的成像进行了优化，用于冷冻断层扫描的应用。我们建议根据协议中的建议对各种情况进行故障排除，以便为所选的下游应用找到最佳结果。总体而言，该协议提供了一种可靠而通用的方法，可以将细胞接种到网格上，可以根据特定需求进行调整。

原位细胞冷冻断层扫描是一种强大的技术，无需化学固定即可研究细胞中的生物学相关结构。通过将细胞连接到EM网格并在制冷剂中对网格进行浸入冷冻，感兴趣的物体在其自然细胞环境中被冷冻，而不会从细胞内水中形成结晶冰1,2。化学固定和结晶冰的形成都会破坏相关分子的结构，例如蛋白质和脂质，从而降低使用这些技术获得的图像的生物学准确性3,4。在断层扫描中，使用电子显微镜以增量角度对网格进行成像，然后使用这些图像来构建成像的目标区域的三维表示5。原位冷冻断层扫描可与其他显微镜技术一起用于综合和相关成像，例如冷冻荧光成像、软 X 射线断层扫描和 cryoFIB/SEM（低温聚焦离子束/扫描电子显微镜）6,7,8,9,10,11 .与任何单一显微镜技术相比，集成多种技术可以获得更多关于结构或过程的信息。
尽管原 位 细胞冷冻断层扫描具有所有优点，但由于各种原因，样品制备可能具有挑战性。由于它们的脆弱性，强行操纵电子显微镜网格会导致损坏，特别是薄碳层很脆弱，容易撕裂，从而减小网格的可成像面积。由于尺寸小，电子显微镜网格也难以操作，并且容易与用于生长细胞的孔或微载玻片表面分离。由于井或微载玻片的几何形状，操作这些网格可能很困难。网格准备不当（例如，允许它们漂浮）会导致细胞密度低并减少潜在成像区域的数量，特别是当细胞不容易附着在网格本身时。对于直接细胞冷冻断层扫描，细胞必须扩散得非常薄，这可能会因多种原因而中断，包括温度不当或网格处理粗糙。
通过各种优化，本文介绍的技术旨在处理在制备冷冻断层扫描电子显微镜网格时出现的这些最常见的陷阱。使用 5/15 角镊子可以操纵孔板或微载玻片内的网格。在电镀前将纤连蛋白溶液涂在网格的两侧，可降低浮动网格的可能性，这有利于确保网格具有足够的细胞密度，并且网格不太可能因操作而损坏。通过将网格保持在 37°C 下孵育直到冷冻前，我们还确保细胞保持在舒适的环境中，以防止细胞缩回其薄边缘。从背面吸干网格还可以防止机械力对细胞的损坏。总之，这些措施提高了原 位 细胞冷冻断层扫描研究样品制备的成功率，增加了这种成像方法的可及性。

<table><tbody><tr><td>10 nm colloidal gold bead solution</td><td>Sigma-Aldrich</td><td>741957</td><td></td></tr><tr><td>6 well multidish, 100/CS</td><td>Fisher Scientific</td><td>FB012927</td><td></td></tr><tr><td>Allegra V-15R Benchtop Centrifuge, IVD 120 V 60 Hz</td><td>Beckman-Coulter</td><td>C63125</td><td></td></tr><tr><td>Au G300F1 with R2/2 Quantifoil carbon</td><td>Quantifoil</td><td>TEM-G300F1-AU</td><td></td></tr><tr><td>Bovine serum albumin</td><td>MilliporeSigma</td><td>A9647</td><td></td></tr><tr><td>BRAND&amp;nbsp;counting chamber BLAUBRAND&amp;nbsp;Neubauer improved</td><td>Sigma-Aldrich</td><td>BR717805-1EA</td><td></td></tr><tr><td>DMi1 Inverted Microscope</td><td>Leica</td><td>22A00G119</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium - high glucose, no glutamine</td><td>Gibco</td><td>11-960-044</td><td></td></tr><tr><td>Dumont 5/15 tweezer</td><td>Electron Microscopy Sciences</td><td>0103-5/15-PO</td><td></td></tr><tr><td>EM GP2</td><td>Leica</td><td>587085</td><td>Automated plunge freezer</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A5209</td><td></td></tr><tr><td>Fibronectin from bovine plasma, cell culture grade</td><td>MilliporeSigma</td><td>F1141</td><td></td></tr><tr><td>GenJet version II in vitro DNA transfection reagent</td><td>SignaGen Laboratories</td><td>SL100489</td><td></td></tr><tr><td>GlutaMAX I 100x</td><td>Fisher Scientific</td><td>35050061</td><td>Media supplement</td></tr><tr><td>Neslab EX-211 Heating Circulator</td><td>Neslab</td><td>Out of production</td><td>Water bath for media warming</td></tr><tr><td>Original Portable Pipet-Aid Pipette Controller</td><td>Drummond Scientific</td><td>4-000-100</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Pelco easyGlow device</td><td>Pelco</td><td>91000S</td><td>Glow discharge device</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td>Media supplement</td></tr><tr><td>Pipetman P1000, 100&amp;ndash;1000 &amp;micro;L, Metal Ejector</td><td>Gilson</td><td>F144059M</td><td></td></tr><tr><td>Pipetman P2, 0.2&amp;ndash;2 &amp;micro;L, Metal Ejector</td><td>Gilson</td><td>F144054M</td><td></td></tr><tr><td>Pipetman P20, 2&amp;ndash;20 &amp;micro;L, Metal Ejector</td><td>Gilson</td><td>F144056M</td><td></td></tr><tr><td>Whatman number 2 filter paper, 55 mm</td><td>Whatman</td><td>28455-041</td><td>Blotting paper</td></tr></tbody></table>

sample preparation for in situ cryotomography of mammalian cells

原位 细胞冷冻断层扫描是一种强大的技术，用于在天然冷冻水合细胞环境中研究复杂物体，使其与细胞生物学和病毒学高度相关。将冷冻断层扫描与其他显微镜模式相结合的潜力使其成为综合和相关成像的完美技术。然而，原 位 细胞断层扫描的样品制备并不简单，因为细胞不容易附着在电子显微镜网格上并拉伸。此外，网格本身很脆弱，如果处理得太用力，可能会破裂，从而导致可成像区域的丢失。当用镊子操纵网格时，组织培养皿的几何形状也会带来挑战。在这里，我们描述了克服这些（和其他）挑战的技巧和窍门，并为贴壁哺乳动物细胞的 原位 细胞冷冻断层扫描和相关成像制备高质量的样品。随着冷冻显微镜技术的不断进步，这项技术在促进我们对复杂生物系统的理解方面具有巨大的前景。

在HIViGFPΔEnv和psPAX2共转染后，所有网格在碳层中的撕裂最小。在与转染试剂孵育 24 小时后，使用相光显微镜和荧光显微镜对网格进行成像（图 2）。模拟网格和共转染网格上的细胞在多个网格方块中都包含活细胞。
psPAX2 编码 HIV-1 的所有结构蛋白和酶蛋白，无需任何荧光标记。HIViGFPΔEnv 与 psPAX2 相似，但具有 GFP 标记的 HIV Gag 蛋白的代码。两种质粒都是ΔEnvelope。共转染导致荧光 HIV-1 颗粒的天然样组装和出芽，使其成为在生物安全 1 级条件下进行 HIV CLEM 研究的绝佳系统。共转染网格显示一部分细胞表现出绿色荧光，表明共转染成功。模拟网格上没有细胞表现出荧光，进一步验证了使用 HIViGFPΔEnv 和 psPAX2 的共转染。使用基于光学的显微镜观察网格后，将网格冷冻并移至液氮中长期储存。
图 3 描述了使用相同实验方法但使用略有不同的质粒构建体产生的网格的结果。使用不同的HIV克隆（HIVmCherryΔEnv和NL4-3ΔEnvGFP）以1：6的比例共转染含U2OS的网格。由于使用了更高质量量的荧光标记质粒，这些网格能够观察到更多的转染细胞，在使用 cryoCLEM 和 cryoET 捕获图像时具有优势。使用 cryoCLEM，使用冷冻荧光显微镜为每个网格生成完整的网格图谱，以记录所有共转染细胞的位置。在已知细胞位置的情况下，进行冷冻ET。收集完整的低倍率网格图谱，并与冷冻荧光时收集的荧光图谱叠加（图3A）。在细胞位点收集冷冻造影，捕捉病毒生命周期的复杂细节，包括HIV从细胞中组装和出芽（图3B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65697/65697fig01.jpg"> 图 1：网格上的单元格播种工作流程。 在冷冻电镜网格上接种细胞的整个过程的示意图。该过程分为四个主要步骤，包括 （A） 在孔中制备用于接种的网格，（B） 向每个孔中添加适量的细胞，（C） 可选的细胞转染用于荧光成像，以及 （D） 网格的浸入冷冻以允许样品的玻璃化。 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig01large.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/65697/65697fig02.jpg"> 图 2：使用 HIViGFPΔEnv 和 psPAX2 共转染 U2OS。 U2OS 细胞与含 GFP 的 HIViGFPΔEnv 和 psPAX2 以 1：3 的比例共转染。通过相差和荧光显微镜对网格进行成像。显示具有GFP表达的细胞表明共转染成功。比例尺：500μm。比例尺（插图）：100 μm。 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig02large.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/65697/65697fig03.jpg"> 图 3：潜在的下游基于冷冻的方法 。 （A） 共转染 U2OS 细胞的 cryoCLEM 图像。绿色细胞代表产生HIV的细胞，用于测量共转染成功。红色点状物代表 mCherry 标记的 HIV-1 Gag。比例尺：250μm。比例尺（插图）：25 μm （B） 从 U2OS 细胞质膜萌芽的多个 HIV 颗粒的冷冻 ET 图像。比例尺：50纳米 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Optimizing Grid Preparation for Enhanced Cryoelectron Tomography at JoVE.com

作者聚焦：优化增强型冷冻电子断层扫描的网格制备 

该方法为原 位 细胞冷冻断层扫描和相关光电子显微镜 （CLEM） 的电子显微镜 （EM） 网格的制备提供了一种可访问且灵活的协议。

哺乳动物细胞 原位 冷冻断层扫描的样品制备

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

本研究的范围是开发一种既可访问又可靠可重复的样品制备方法。通过实现这一点，直接细胞冷冻电子断层扫描的力量可用于回答各种生物学问题。电网准备的挑战是广泛的。细胞不容易附着在网格上，操作会导致碳层损坏，从而导致可成像网格区域的丢失。此外，在不注意适当的样品制备条件的情况下，可以减少薄细胞突起的发生率。目前的协议通常需要专门的设备来解决接种的主要瓶颈，例如分别用于细胞附着和操作的微图案化和 3D 打印支架。该协议为解决方案提供了更大的可访问性，同时仍然保持了这些专用工具用于下游应用的灵活性。

sample-preparation-for-in-situ-cryotomography-of-mammalian-cells

Research

JoVE Journal

Biochemistry

Sample Preparation for In Situ Cryotomography of Mammalian Cells

2.9K 次观看.  University of Minnesota. 本研究的范围是开发一种既可访问又可靠可重复的样品制备方法。通过实现这一点，直接细胞冷冻电子断层扫描的力量可用于回答各种生物学问题。电网准备的挑战是广泛的。细胞不容易附着在网格上，操作会导致碳层损坏，从而导致可成像网格区域的丢失。此外，在不注意适当的样品制备条件的情况下，可以减少薄细胞突起的发生率。目前的协议通常需要专门的设备?...

视频: 作者聚焦：优化增强型冷冻电子断层扫描的网格制备 

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

科研

生物化学

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

细菌细胞的电子Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

生物学

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

低温结构照明微观数据收集来自低温保存细胞

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

哺乳动物细胞原位冷冻断层扫描的样品制备

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硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工

细菌细胞的电子Cryotomography

Cryosectioning方法小鼠结肠黏膜显微切割

对小鼠乳腺冰冻切片间接免疫荧光

高通量，矿化组织的多图像Cryohistology

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

通过冷冻软X射线断层扫描对细胞进行3D制图描述

低温结构照明微观数据收集来自低温保存细胞

哺乳动物细胞 原位 冷冻断层扫描的样品制备

副本

摘要

探索更多视频

此视频中的章节

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

细菌细胞的电子Cryotomography

Cryosectioning方法小鼠结肠黏膜显微切割

对小鼠乳腺冰冻切片间接免疫荧光

高通量，矿化组织的多图像Cryohistology

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

通过冷冻软X射线断层扫描对细胞进行3D制图描述

低温结构照明微观数据收集来自低温保存细胞

哺乳动物细胞原位冷冻断层扫描的样品制备

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工