木钻甲虫附着物扫描和透射电子显微镜的样品制备方法
1The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry,Beijing Forestry University, 2School of Forestry,Inner Mongolia Agricultural University, 3Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College, 4School of Forestry,Fujian Agriculture and Forestry University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 样品固定和清洁 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…
Representative Results
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。 图3：SEM下…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Anatomical lens | Chongqing Auto Optical limited liability company |
1425277 | |
| Carbon adhesive tape | SPI Supplies, Division of Structure Probes, Inc. | 7311 | |
| Carbon tetrachloride | Sigma | 56-23-5 | |
| Copper grids | GilderGrids | G300 | |
| Disodium hydrogen phosphate | Sinopharm group chemical reagent co., LTD | 10039-32-4 | |
| Ethanol | J.T. Baker | 64-17-5 | |
| Flat embedding molds | Hyde Venture (Beijing) Biotechnology Co., Ltd. | 70900 | |
| Fluorescence microscope | LEICA | DM2500 | |
| Glutaraldehyde | Sigma-Aldrich | 111-30-8 | Anhydrous EM Grade |
| Isophorone | Sigma | 78-59-1 | |
| Lead citrate | Sigma | 512-26-5 | |
| Methanol | Sigma | 67-56-1 | |
| Monobasic sodium phosphate | Its group chemical reagent co., LTD | 7558-80-7 | |
| Objective micrometer | Olympus | 0-001-034 | |
| Osmium tetroxide | Sigma | 541-09-3 | |
| Petri dish | Aldrich | 1998 | |
| Razor blade | Gillette | ||
| Resin | Spurr | ERL4221 | |
| Scalpel | Lianhui | GB/T19001-2008 | |
| SEM | Hitachi | S-3400 | |
| Silica gel desiccant | Suzhou Longhui Desiccant Co., Ltd. | 112926-00-8 | |
| Small brush | Martol | G1220 | |
| Sodium hydroxide | Sigma | 1310-73-2 | |
| Sputter ion instrument | Hitachi Koki Co. Ltd., Tokyo, Japan | E-1010 | |
| Stereo microscope | Leica | EZ4 HD | |
| TEM | Hitachi | H-7500 | |
| Tween 20 | Tianjin Damao Chemical Reagent | 9005-64-5 | |
| Ultramicrotome | Leica | UC6 | |
| Ultrasonic cleaner | GT Sonic | GT-X1 | |
| Uranyl acetate | Sigma | 6159-44-0 |
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Citar este artigo
Zhang, Y., Qiao, H., Ren, L., Wang, R., Lu, P. Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle. J. Vis. Exp. (156), e59251, doi:10.3791/59251 (2020).