Name: preparation of prokaryotic and eukaryotic organisms using chemical drying for morphological analysis in scanning electron microscopy (sem)
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM at JoVE.com

这项工作的资金来自中央密歇根大学研究和研究生办公室对 mls 的资助和对 mak 的暑期学者奖。蓝藻是由 zimba 和浮游生物实验室提供的, 该实验室是德克萨斯 a & m 大学科珀斯克里斯蒂海岸研究中心的一部分。优化子碱是由密歇根州立大学的 triemer 实验室提供的。

1. 准备和固定
<ol><li>准备蓝藻。<ol>
<li>在14:10 小时的光暗循环中,在24°c 的温度下在 f/2 介质中生长单藻培养物。将足够的培养物转移到 1.5 ml 的微离心管中, 使细菌颗粒在10分钟内沉淀后, 其大小约为0.05 毫升。取出培养基, 更换1.5 毫升的固剂 (1.25 戊二醛, 0.1 m 磷酸盐缓冲液 ph 7.0), 轻轻反转数次, 并在4°c 下孵育过夜。</li>
		<li>根据电池的大小, 用玻璃移?...

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在这里, 我们描述了一个协议使用 sem 来获取有关三种生物的外部形态特征的详细信息, 其他生物可以应用于检查许多类型的生物体或组织的特征。在协议的每个步骤中, 都可能出现潜在的误差点, 下文将对此进行详细讨论。
虽然这里给出的固定和洗涤量是具体的, 但一般来说, 固定和洗涤的体积应该是样品体积的5-10x。所有的固定、清洗和脱水时间都是基于我们的经验, 如果出?...

扫描电子显微镜 (sem) 使用高能电子的聚焦束从二次电子中生成显示样本1的形态和地形的图像。sem 可用于直接确定样品的物理尺寸、表面结构和三维形状, 并提供更大的分辨率和更大的景深相比, 光显微镜。另一种形式的电子显微镜 (em), 透射电子显微镜 (tem) 使用聚焦电子通过样品, 产生图像与内部结构的细节。虽然 tem 的分辨率高于光或 sem, 可用于解析单个原子这样小的结构, 但它有三个主要缺点: 广泛的样品制备、一个小的视野和一个浅景深 2,3。虽然其他可视化协议存在使用 sem 来检查特定的细胞, 细胞器,或组织4,5,6, 7, 8,9,10 、该协议的独特之处在于, 我们描述了可广泛应用于大量生物进行形态评估的方法。
sem 在研究无机材料方面有着广泛的应用, 包括纳米粒子11、12、聚合物13,以及在地质、工业和材料科学14 中的众多应用. 15,16。在生物学中, sem 长期以来一直被用作检测从单个蛋白质到整个生物体的生物样本的方法 17,18。扫描电镜具有特殊的价值, 因为形态表面细节可以用来为科学发现提供信息。sem 分析是一种快速、廉价、简单的方法, 可以获得关于各种生物样品的结构、大小和表面特性的详细信息。
由于 sem 通常在高真空 (10-6 torr 最小值) 下工作, 以支持高速电子的相干束, 因此样品室中不允许使用液体 (水、油、醇), 因为液体可防止真空形成。因此, 使用 sem 检测的所有样品都必须脱水, 通常使用分级乙醇系列, 然后采用干燥过程来去除乙醇。有几种方法来干燥生物组织用于扫描电镜, 包括空气干燥, 冻干, 使用临界点干燥 (cpd) 装置, 或使用 t-丁醇 (tba) 或六甲基二硅烷 (hmds)化学干燥19,20,21,22. 大多数情况下, 选择干燥方法是经验性的, 因为每个生物样本对每个干燥方法的反应可能不同。对于任何给定的示例, 所有这些方法都可能是适当的, 因此比较每种方法的优缺点对于选择适当的方法很有用。
虽然在室温或干燥炉 (60°c) 下空气干燥样品是最简单的方法, 但大多数生物样品显示干燥引起的损伤, 如枯萎和塌陷, 导致样品变形。冷冻干燥的过程也会去除样品中的水 (或冰), 但需要将样品进行闪存冷冻,并置于真空下, 通过升华过程去除冰, 从而有可能对样品造成损害。此外, 用户必须有权访问冻干机。sem 脱水样品最常用的方法是临界点干燥 (cpd)。在 cpd 中, 样品中的乙醇被液体二氧化碳 (co2) 所取代, 在被称为临界点的特定温度和压力条件下 (31.1°c 和 1, 073 psi), co2在不产生表面张力的情况下蒸发, 从而产生表面张力, 从而有效地保持样品的形态和结构特征。虽然 cpd 通常是标准方法, 但它有几个缺点。首先, 这一过程需要使用临界点干燥器, 这不仅昂贵, 而且还需要使用液体二氧化碳。其次, 可以干燥的样品的大小仅限于临界点干燥机的腔体大小。第三, 在 cpd 过程中液体的交换会导致湍流, 从而损坏样品。
与 cpd 相比, 化学干燥具有许多优势, 并可作为一种合适的替代品, 在 sem 样品制备中得到广泛应用。使用 tba 和 hmds 等化学脱水剂为其他方法提供了快速、廉价和简单的替代方法, 同时仍然保持样品的结构完整性。我们最近发现, 在成人嗜酸性视网膜组织23中使用 cpd 或 tba 作为干燥方法时, 组织的完整性或最终图像的质量没有差异.与 cpd 不同, tba 和 hmds 不需要干燥仪器或液体co2 , 也不限制要干燥的样品的大小。除了获得这些化学品外, 完成干燥过程只需标准的化学烟罩和适当的个人防护设备 (手套、实验室涂层和安全护目镜) 即可完成。虽然 tba 和 hmds 都是易燃的, 但 tba 的毒性和成本都比 hmds 低, 成本也更低 (约为 hmds 成本的 1.5倍)。
在这篇文章中, 我们描述了如何固定, 洗涤, 脱水, 干燥, 安装, 溅射外套, 和图像三种类型的生物体: 蓝藻(toxifilum mysicida, golenkina sp., 和一个未知的 sp), 两种黄酮类化合物属单诺莫诺莫莫莫莫尼卡(金银花和假花蝇) 和果蝇 (果蝇)。这些生物体的大小 (0.5μm 至4毫米) 和细胞多样性 (单细胞到多细胞) 范围很广, 但所有生物体都很容易适应 sem 分析, 只需进行样品制备所需的微小变化。该协议描述了使用化学脱水和扫描电镜分析来检查三种生物的形态细节的方法, 并将广泛适用于检查许多组织和组织类型。

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			<td height="40" style="height:40px;width:351px;">&#160;gold&#8211;palladium</td>
			<td style="width:373px;">Ted Pella, Inc.</td>
			<td style="width:395px;">91212</td>
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			<td height="40" style="height:40px;">Silver conductive adhesive 503</td>
			<td>Electron Microscopy Sciences</td>
			<td>12686-15</td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 8 &#956;m, polycarbonate</td>
			<td>Sigma</td>
			<td>WHA110614</td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.8 &#956;m, polycarbonate, black</td>
			<td>Sigma</td>
			<td>WHA110659</td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.2 &#956;m, polycarbonate</td>
			<td>Sigma</td>
			<td>WHA110606</td>
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			<td>Fisher Scientific</td>
			<td>08-732-100</td>
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			<td height="40" style="height:40px;">aluminum mounting stubs (12 mm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>75210</td>
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			<td>Electron Microscopy Sciences</td>
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			<td>77825</td>
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			<td style="width:373px;">Culture Collection of Algae at the University of Texas at Austin</td>
			<td style="width:395px;">https://utex.org/products/f_2-medium</td>
			<td>No Catalog number given - see link&#160;</td>
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			<td style="width:373px;">Bigelow - National Center for Marin Algae and Microbiota</td>
			<td style="width:395px;">https://ncma.bigelow.org/media/wysiwyg/Algal_recipes/NCMA_algal_medium_AF6_1.pdf</td>
			<td>No Catalog number given - see link&#160;</td>
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			<td height="40" style="height:40px;">Soil water medium</td>
			<td>Carolina Biological Supply Company</td>
			<td>153785</td>
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			<td height="40" style="height:40px;">Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td>
			<td>VWR</td>
			<td>97062-208</td>
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			<td height="40" style="height:40px;">Hummer 6.2 Sputter Coater</td>
			<td>Anatech USA</td>
			<td>http://www.anatechusa.com/hummer-sputter-systems/4</td>
			<td>No Catalog number given - see link&#160;</td>
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			<td height="40" style="height:40px;">Hitachi 3400N-II SEM&#160;</td>
			<td>Hitachi</td>
			<td style="width:395px;">https://www.hitachi-hightech.com/us/product_list/?ld=sms2&#38;md=sms2-1&#38;sd=sms2-1-2&#38;gclid=EAIaIQobChMIpq_jtJfj3AIVS7jACh2mdgkPEAAYASAAEgKAnfD_BwE</td>
			<td>The company doesn&#39;t appear to sell this model any longer&#160;</td>
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preparation of prokaryotic and eukaryotic organisms using chemical drying for morphological analysis in scanning electron microscopy (sem)

扫描电子显微镜 (sem) 是一种广泛使用的技术, 已被应用于研究生物标本, 从单个蛋白质到细胞, 组织, 细胞器, 甚至整个生物体。该协议侧重于两种化学干燥方法, 六甲基二硅烷 (hmds) 和 t-丁醇 (tba), 及其在原核生物和真核生物的成像使用 sem。在这篇文章中, 我们描述了如何固定, 洗涤, 脱水, 干燥, 安装, 溅射外套, 和图像三种类型的生物体: 蓝藻(toxifilum mysicida, golenkina sp., 和一个未知的 sp), 两种黄酮类化合物属单诺莫诺莫莫莫莫尼卡(金银花和假花蝇) 和果蝇 (果蝇)。本协议的目的是描述一种快速、廉价和简单的方法, 以获取有关样品的结构、大小和表面特性的详细信息, 这些信息可广泛应用于大量生物的形态评估。成功完成此协议将允许其他人使用 sem 通过将这些技术应用于他们的系统来可视化样本。

蓝藻是对全球碳、氧和氮循环28、29至关重要的原核生物群。在估计的6000种蓝藻中, 大多数有粘液鞘, 覆盖和连接细胞和其他结构31, 连同形状, 可以通过显微镜下解决32。细胞大小、形状和色素存在区分个别物种和水生栖息地, 可以想象为蓝藻 "开花"28。现代?...

Watch this Scientific Journal Video about Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM at JoVE.com

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

扫描电镜分析是帮助物种鉴定或表型鉴别的有效方法。该协议描述了检查三种具有代表性的生物体的具体形态细节的方法, 并将广泛适用于检查许多组织和组织类型的特征。

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

该协议侧重于两种化学干燥方法，六甲基二苯醚和T-丁基醇及其应用成像使用SEM的原核生物和真核生物。该技术的主要优点是，它不需要使用专门的干燥设备，如临界点干燥机来准备样品进行分析。该协议描述了使用化学脱水和SEM分析三种类型的生物体的方法，但将广泛适用于检查许多有机体和组织类型。演示这个程序将是麦迪逊孔，一个本科生从我的实验室。首先，通过隔夜固定来准备蓝藻。然后，将它们转移到含有聚碳酸酯 25 mL 过滤器的 10 mL 过滤装置中。用橡胶塞密封过滤装置的侧臂和漏斗，以遏制漏斗中的培养。拆下两个止动器后，通过轻轻吸尘过滤瓶来去除固定剂。要准备单细胞藻类，通过将3滴4%的三氧化二氮直接加入到之前被转移到10 mL过滤装置中的培养体中来修复它们。经过30分钟的孵育，通过轻轻吸尘过滤瓶，拆下两个塞子并取出固定剂。在室温下用 5 mL 0.1 M 磷酸盐缓冲液 pH 7.0 清洗固定 euglenoid 三次，每次洗涤 10 分钟，同时用塞子关闭烧瓶和漏斗以进行清洗。拆下两个塞子后，通过过滤瓶上的温和真空去除每个洗涤液。要使样品脱水，同时保持连续浸入状态，在漏斗中使用分级乙醇系列，在 5 mL 的体积内使用分级乙醇系列 10 分钟，同时保持烧瓶和漏斗与塞子关闭，以将乙醇控制在漏斗中。拆下两个塞子后，轻轻真空在过滤瓶上去除乙醇。将样品转移到一次性铝称重盘中，其中只含有足够 100%乙醇的样品。要使用 TBA 对黄酮进行化学干燥，首先用 1 比 1 的 TBA 溶液和 100% 乙醇替换铝盘中的 1 比 1 乙醇溶液 20 分钟。然后，将 1 比 1 解决方案替换为 100%TBA 20 分钟，然后重复此过程两次。取出 TBA，并更换足够的新鲜 100%TBA 来覆盖样品。将菜在四摄氏度下放置十分钟，以冷冻 TBA。将样品转移到真空干燥器中，并配有冷冻凝胶包，以保持 TBA 冷冻。使用旋转泵疏散和维护真空，使样品通过真空升华冷冻 TBA 干燥至少三个小时。要将蓝藻安装在 euglenoid 中，请标记 25 mm 铝制安装存根的底部，以指示顶部放置的白化病菌。然后将每个过滤器放在固定在存根顶部的碳粘合卡舌上。为了准备果蝇黑色素，将麻醉苍蝇浸入1 mL的固定剂中，在4摄氏度下停留两小时，确保它们完全被淹没。使用玻璃移液器拆下固定器。用1 mL 0.1 M磷酸盐缓冲液将固定的果蝇样品用1 mL 0.1 M磷酸盐缓冲液洗涤三次，在室温下将pH7.2洗涤10分钟。使用玻璃移液器去除每次洗涤，小心不要取出样品。然后，使用分级乙醇系列将样品在1 mL的微混管中脱水1分钟。使用玻璃移液器去除每次洗涤，小心不要取出样品。将样品放在 1.5 mL 微离心管中，只需足够的 100% 乙醇覆盖它。要使用 HMDS 对苍蝇进行化学干燥，首先用 1 到 2 的 HMDS 溶液和 100%乙醇溶液替换 100% 乙醇溶液 20 分钟。然后用 HMDS 的 2 比 1 溶液和 100%乙醇溶液更换溶液 20 分钟。最后，用 100%HMDS 替换此解决方案 20 分钟，然后重复此过程一次。将 1.5 mL 微型离心管和 HMDS 中的样品转移到一次性铝称重盘中。将 100%HMDS 更换为足够新鲜 100%HMDS 以覆盖样品。将样品直接放在铝制烟罩中，用松动的盖子（如盒子）干燥，以防止碎屑落在样品上，并允许其干燥 12 至 24 小时。要安装果蝇，请标记铝制安装存根的底部。使用精密钳子将干苍蝇放在解剖显微镜下固定在存根顶部的碳粘合卡上的所需位置。在存根的外边缘周围涂抹银导电胶。使用牙签将银漆连接到苍蝇，确保导电性，确保油漆不会触及所需的成像区域。将存根放在存根支架盒中，然后将打开的存根支架盒放在干燥器中，让银漆干燥至少三个小时。要准备溅射涂层装置，请执行 4 到 5 次气冲。根据要涂覆的样品，将计时器设置为手稿中描述的不同设置。溅射按照制造商的说明涂覆存根。最后，使用扫描电子显微镜进行图像采样，该显微镜包括一个二次电子探测器，其设置取决于所用样品。利用该协议成功制备了SEM的蓝藻，在图像中可以看到细胞的细节，包括表面的形状和纹理，以及细胞的粘液和投影。来自科罗拉多的丝丝淡水物种的SEM图像显示了可见的粘液护套。来自俄亥俄州的淡水细菌的SEM图像揭示了单个细胞，这些细胞有时形成由薄薄的粘液层聚集在一起的菌落。纤维样突起从单个细胞延伸。来自德克萨斯州沿海水域高盐度河口的未知丝质物种的图像显示了单个和分裂的细胞。SEM 用于可视化两种不同 Euglenoid 物种的皮条条。当比较单态性，和Aenigmatica与单态性伪热，可见的卵条的可见侧向排列，出现在后端形成尾巴，有助于在植物性终止。正常果蝇眼的SEM图像显示有秩序的刷毛和镜片阵列，但与阿尔茨海默病相关的蛋白质的表达创造了粗糙的眼睛表型。抑制或增强粗糙眼表型的基因可能在疾病进展中起生理作用。以下是要避免的潜在坑瀑布示例，包括由于干燥技术不当导致眼睛折叠结构的示例，以及图像采集过程中出现溅射编码不足导致电子充电的示例。这里描述的协议利用了非常受尊敬的共谋的生物体，但所有生物体都适合进行SEM分析，以收集有用的形态学信息，这些信息对于跨越生物学许多子学科的科学发现至关重要。不要忘记，使用 T-丁基醇、六甲基二甲苯和三氧化二氮可能很危险，应始终遵守适当的安全措施以保护用户，尤其是将处理这些化学品的学生。

Research

JoVE Journal

Biology

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

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

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

电子显微镜对胚胎的制备果蝇胚胎心管

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

科研

生物学

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

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

准备厚生物样品的观察扫描透射电子断层扫描

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

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

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

Miniaturized Sample Preparation for Transmission Electron Microscopy

透射电镜的小型化样品制备方法

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

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

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...