Name: sample preparation method of scanning and transmission electron microscope for the appendages of woodboring beetle
Uploaded: 2020-02-03T12:00:00
Description: Watch this Scientific Journal Video about Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle at JoVE.com

我们感谢北京农业职业学院、中国农科院原子能应用研究所、北京林业大学生物研究中心和山根教授的慷慨援助。中国科学院动物学研究所张。该研究得到了国家重点研发项目（2017YFD0600103）、国家自然科学基金（授权号31570643、81774015）、中国公益林科学研究（201504304）、内蒙古农业大学高层次人才研究启动计划（203206038）和内蒙古自治区高等教育研究项目（NJZZ18047）、内蒙古自治区林雪"双一"建设工程（170001）。

注意：在使用前，请查阅试剂的材料安全数据表。样品制备过程中使用的几种化学品具有毒性、诱变性、致癌性和/或再毒性。使用个人防护设备（手套、实验室外套、全长裤子和闭趾鞋），在处理样品时在烟罩下工作。
1. SEM 样品制备和成像
<ol><li>样品固定和清洁<ol>
<li>在发生C.卡拉干纳的区域工作，吸引成年人进入田间陷阱，诱饵植物吸引剂，如异磷16...

<ol>
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本文介绍了一种木钻甲虫扫描和透射电子显微镜的样品制备方案。以昆虫附属项为代表研究课题，对传统的样品制备方法进行了一些改进。
从固体表面分离的液体油被乳化成小液滴，小液滴可以很好地分散并悬浮在洗涤介质中，以减少在物体表面的再沉积。表面活性剂的洗涤性能包括所有基本特性，如润湿性、渗透性、乳化特性、分散性、溶解性11、12、13?...

扫描电子显微镜是许多形态学研究中的重要工具，SEM显示表面结构1，2。透射电子显微镜的吸引力是，它可用于研究纳米尺度上的多种生物结构，从细胞结构和细胞器的超微结构，到大分子复合物和蛋白质的结构。TEM显示内部结构3，4，5。
科洛普特拉是最大的昆虫群，包括大约182个家族和35万种昆虫。大多数的科林托普兰昆虫，特别是木质甲虫，以植物为食，其中许多是森林和果树的重要害虫，对树木造成毁灭性的破坏。目前，基于化学生态学理论的病虫害防治工作已日益受到重视。高效、低毒、无污染信息素控制方法已成为一种有效的方法。昆虫的感性形态学和超微结构研究是昆虫化学生态学研究的重要组成部分。扫描和透射电子显微镜（SEM和TEM分别）对它们的形态学和内部解剖学的研究有很大的效果。然而，在电子显微镜（EM）昆虫样品的制备过程中，观测点的客观性和真实性可能会受到影响。一般来说，昆虫的SEM样品制备需要清洗、组织固定、脱水、转移、干燥和溅射涂层10。由于林甲虫生活的环境复杂，人体表面往往有各种各样的污染物，其附属物往往有许多细长的鼻孔或毛刺。特别是，一些木钻孔机不能从实验室饲养，直接收集在现场，然后放入固定流体，以确保新鲜度，随后在实验室洗涤。如果样品先固定然后清洗，显然要清除碎屑要困难得多，因为谷醛会强烈地将其固定在样品上。Tween 20 是表面活性剂 11、12、13、14，在洗涤过程中起着重要作用，包括降低水的表面张力和提高洗衣表面水的润湿性。在这项研究中，将Tween 20添加到固定溶液和PBS清洁溶液中，以降低液体的表面张力，防止污垢沉积在木质甲虫的体表面，使SEM的车身表面更清洁。
使用TEM，可以对昆虫的不同器官进行切片，以揭示昆虫体内的清晰结构，从而为分析昆虫功能提供依据。当昆虫，如木钻甲虫，是大，其身体壁有相当程度的硬化，所以固定可能无法完全饱和器官组织内的昆虫体内。补间20可以增强污垢的分散和悬浮能力。在这项研究中，Tween 20被添加到固定剂中，以增强固定流体渗透到虫体壁的木刺甲虫，避免表皮11、12、13的变形和坍塌。此外，使用一般的切片技术，很难准确定位不同类型的感菜，特别是对于一些小的森西拉15。本研究在传统TEM样品制备的基础上，结合荧光显微镜和SEM，确定昆虫感菌在嵌入块中的位置，从而提高切片精度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anatomical lens</td>
			<td>Chongqing Auto Optical 
			limited liability company</td>
			<td>1425277</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon adhesive tape</td>
			<td>SPI Supplies, Division of Structure Probes, Inc.</td>
			<td>7311</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon tetrachloride</td>
			<td>Sigma</td>
			<td>56-23-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>GilderGrids</td>
			<td>G300</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate</td>
			<td>Sinopharm group chemical reagent co., LTD</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T. Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat embedding molds</td>
			<td>Hyde Venture (Beijing) Biotechnology Co., Ltd.</td>
			<td>70900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>LEICA</td>
			<td>DM2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>111-30-8</td>
			<td>Anhydrous EM Grade</td>
		</tr>
		<tr>
			<td>Isophorone</td>
			<td>Sigma</td>
			<td>78-59-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Sigma</td>
			<td>512-26-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate</td>
			<td>Its group chemical reagent co., LTD</td>
			<td>7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective micrometer</td>
			<td>Olympus</td>
			<td>0-001-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma</td>
			<td>541-09-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Aldrich</td>
			<td>1998</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Gillette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resin</td>
			<td>Spurr</td>
			<td>ERL4221</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lianhui</td>
			<td>GB/T19001-2008</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>Hitachi</td>
			<td>S-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel desiccant</td>
			<td>Suzhou Longhui Desiccant Co., Ltd.</td>
			<td>112926-00-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Small brush</td>
			<td>Martol</td>
			<td>G1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter ion instrument</td>
			<td>Hitachi Koki Co. Ltd., Tokyo, Japan</td>
			<td>E-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>EZ4 HD</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>Hitachi</td>
			<td>H-7500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Tianjin Damao Chemical Reagent</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>GT Sonic</td>
			<td>GT-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Sigma</td>
			<td>6159-44-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation method of scanning and transmission electron microscope for the appendages of woodboring beetle

本报告描述了扫描和传输电子显微镜观察的样品制备方法，通过制备木钻甲虫、氯磷酸二甲AA和Wang（2012年）的附属物来证明这两种电子显微镜。扫描电子显微镜（SEM）样品制备方案基于样品化学固定、一系列乙醇浴中的脱水、干燥和溅射涂层。通过在固定液和洗涤溶液中加入Tween 20（聚氧乙烯山梨酸盐），在SEM中更干净地清洗了木钻甲虫的昆虫体表面。本研究的透射电子显微镜（TEM）样品制备涉及一系列步骤，包括固定、乙醇脱水、嵌入树脂、使用荧光显微镜定位、切片和染色。修复与Tween 20能够穿透昆虫体壁的木钻甲虫比没有Tween 20更容易，随后更好的固定组织和器官在体内，从而产生清晰的传输电子显微镜观察昆虫感性超结构。此制备的下一步是使用荧光显微镜确定嵌入树脂块中的样品中的昆虫敏菌的位置，以提高目标感镜定位的精度。这提高了切片精度。

使用使用 Tween 20 的清洁和固定溶液，比没有补间 20 的更清洁 SEM 图像（图 3）。补间20固定溶液将谷醛固定溶液渗透到组织中。微管结构清晰可见。没有Tween20（图4），样品内部结构的TEM图像被模糊了。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59251/59251fig3a.jpg"> 图3：SEM下...

Watch this Scientific Journal Video about Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle at JoVE.com

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

为了观察昆虫的超结构，在研究中提出了扫描和透射电子显微镜（SEM和TEM，分别为SEM和TEM）的样品制备方案。在SEM中加入Tween 20，以避免样品变形。 荧光显微镜有助于提高TEM的切片精度。

木钻甲虫附着物扫描和透射电子显微镜的样品制备方法

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

木鸟甲虫他们的身体表面总是很难在SEM。Tween-20 被添加到固定和洗涤溶液中。在TEM中，Tween-20的木孔甲虫的身体表面可以增强固定性渗透到身体壁中，更好地固定体内的组织和器官。荧光显微镜有助于提高切片精度。该技术有助于洗涤身体表面和渗透到木孔甲虫的身体壁。因此，它将清除SEM和TEM的愿景。荧光显微镜有助于提高TEM的切片精度。演示这个程序的将是来自我们实验室的研究生张燕茹。在氯丙酮卡拉加纳居住的地方，将用植物吸引剂（如等值酮）诱饵的田地陷阱放在漏斗陷阱上。吸引木虫成人进入陷阱。在 pH 7.2 下，将成人氯氟烃卡拉加纳的清洁身体保持 0.1 摩尔/升 PBS，以体积谷胱甘肽保持 2.5% 的重量，以体积 Tween-20 保持 0.06% 体积。将样品在四摄氏度下固定三天。从保鲜液中去除尸体，并在 PBS 中冲洗。在立体显微镜下，使用钳子去除附属物，并在PBST中以40千赫的超声波清洁。清洁 100 秒后，将样品转移到显微镜上，检查样品是否清洁。清洁时间不要超过400秒，以免损坏。然后将样品以一系列浓度浸入乙醇中，每次20分钟进行脱水。脱水后，在立体显微镜下，使用碳双面胶带将三个观察表面（后侧、腹体和侧侧）分别固定到存根上。确保所有观察表面保持清洁且无污染。将样品阶段放在含有硅胶干燥剂的培养皿中 48 小时。接下来，在离子溅射器上，将主阀旋转到打开位置，拆下样品室盖，将样品放入腔室。打开电源开关，确保就绪指示灯已打开。将溅射时间设置为 45 秒，将涂层厚度设置为 70.875 安热罗姆。一旦机械泵真空表盘指数降至 7 以下，按压放电并开始喷洒铂金。在步骤结束时，关闭电源，将样品从腔室中带走。计算喷雾膜厚度 D 等于 K 倍的 T.K 是由溅射金属和气体决定的常数。I 是等离子流量（以毫安为单位），而 V 是施加的千伏电压，T 是时间（以秒为单位）。将包含样品的存根插入 SEM 的阶段。确保样品存根的样本阶段有足够的高度，以便获得良好的图像。打开 SEM 软件并选择从 20 千伏开始的所需工作电压。从成人氯氟氏菌类获得附属物后，在超声波浴中清洗PBST中的样品三小时，然后在25摄氏度下用PBS中的三氧化二氮体积将样品以1%的重量固定一小时。在室温下连续处理20分钟，在室温下用体积乙醇脱水样品，50%60%70%80%85%90%90%100%和100%体积。使用钳子，将加热树脂涂抹在扁平嵌入模具中，并将样品嵌入树脂中。确保样品位于板的底部，并尽可能靠近凹槽的边缘。在60摄氏度下标记并孵育含有样品的板，72小时。孵育后，从培养箱中取出板，并验证树脂是否已聚合。样品凝固后，将每个树脂块置于荧光显微镜下，移动显微镜的荧光光源，从上方用蓝光照射样品并拍摄。确保树脂块中的感官清晰可见。测量树脂块边缘与目标传感器之间的距离，以定位感官。参考小巴的SEM图像，用靠近目标受体的剃须刀切割树脂块。接下来，在蓝光荧光显微镜下，从上面调整光源，以便清晰观察感官。将目标千分尺添加到荧光显微镜阶段。拍摄大致切割的树脂块，然后使用 ImageJ 测量目标与树脂边缘之间的距离。将树脂样品放在超微体上，切割 50 至 60 纳米厚的部分，直到达到目标位置。在 Petri 盘中安装涂有外衣涂层的 100 个网状铜网格上的部分。在室温下用饱和的醋酸化溶液染色网格。在染色过程中盖住各部分，以阻挡由光线引起的沉淀物。污渍10分钟。在烟气罩中，用50%甲醇冲洗两次，然后在过滤后气水中冲洗两次。将准备好的铅柠檬酸盐污渍滴在塑料培养皿的方块上，让它们坐8分钟。用脱气过滤水冲洗并干燥。将网格安装到 TEM 机器内的样品级。将电压设置为 80 千伏，并检查各部分。在此协议中，使用 Tween-20 的清洁和固定解决方案，观察到比没有 Tween-20 的清晰的 SEM 图像。Tween-20 固定溶液允许谷胱甘肽固定溶液穿透组织，在 TEM 图像中清楚地看到微管结构，而未制备的 Tween-20 样品的内部结构模糊不清。第一型尖牙树枝在最大唇上的钉钉的连续横截面视图显示，树突状护套包围着外树突状段，并延伸到尖端孔隙。七个未分的外突状片段存在于被外腔包围的内受体淋巴腔内。管状体由树突护套与每个森硅插座基座的其他外树突状段隔开。在硅体区域，确定了8个不同直径的树突，表明存在8个双极神经元。该技术可用于进一步研究神经的功能，如单细胞记录或原位杂交。

Research

JoVE Journal

Environment

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

一种优化富集技术的隔离<em&gt;节杆菌</em&gt;噬菌体种从土壤样品隔离

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

协议测量过冷的合成砂水 - 气 - 甲烷水合物样品的热性能

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

热扫描电导 (TSC) 作为研究和控制导电物理凝胶相行为的一般方法

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

用薄膜旋转圆盘电极法制备和测试燃料电池催化剂的研究

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

利用低成本、小型、易于建造的Quercivorus测量豚草甲虫、鸭嘴兽的飞行能力 (村山富市)

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

木本植物的赛莱默水分布,用低温扫描电子显微镜可视化

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...