红细胞膜蛋白颗粒冷冻蚀刻图像反差不良的原因分析  

Reason Analysis of Freeze-etching Image About Intramembrane Protein Particles of Erythrocyte in Fuzzy Contrast

作　　者：林曦[1,2] 钟秀容[1] 陈新[2] 

机构地区：[1]福建医科大学基础医学院病理学系电子显微镜室,福建福州350004 [2]福州大学物理与信息工程学院,福建福州350108

出　　处：《中国医学物理学杂志》2012年第6期3818-3821,共4页Chinese Journal of Medical Physics

基　　金：福建省教育厅A类科技项目(No.JA10149)

摘　　要：目的:探讨了红细胞膜蛋白颗粒冷冻蚀刻图像反差不良的相关因素,解决在应用电镜冷冻蚀刻技术制备红细胞膜蛋白颗粒样品的过程中,部分颗粒图像模糊、反差不良,颗粒粘连严重,导致辨识与计数困难的问题。方法:本实验通过采集DMD基因携带者外周静脉血样品,经过抗凝、离心、固定和防冰晶等处理步骤后,应用HUS-5型真空喷镀仪及配套装置,在-100℃条件下制备复型膜。在以往工作的基础上,分析蛋白颗粒图像成因,采取在喷镀过程中控制喷镀电流与真空度,以及在喷镀前精选喷镀源,并在仪器准备阶段注重真空系统良好的清洁和维护等多种手段,制备出了质量良好的冷冻蚀刻复型膜。结果:制备出的复型膜在电镜下显示的蛋白颗粒图像清晰可辨,反差良好。部分改善了图像质量,收到良好的效果。结论:采用精心烧制并优选喷镀源,在冷冻蚀刻实验过程中注重喷镀电流与真空度的配合,结合改进实验器材的准备工作等方法,可提高膜蛋白颗粒冷冻蚀刻图像的质量,制备出边界清晰,背景杂质较少,易于识别的蛋白颗粒复型膜。Objective： The relevant factors of freeze-etching image about intramembrane protein particles of erythrocyte in fuzzy contrast were discussed. We would solved the problem that the phenomena of fuzzy contrast and severe adhesion between particles had led to identifying and counting the protein particles difficulty in some of particles image, during the process of applying freeze-etching electron microscopy in fabricating the intramembrane protein particles samples of erythrocyte. Methods： The peripheral venous blood samples of the Ducherme muscular dystrophy （DMD） gene carrier had been gathered at first. After the processing steps of anticoagulation, centrifugal process, fixing and preventing ice process, the blood samples of DMD cartier could been used to form protein particles replica by HUS-5 vacuum evaporation apparatus with its accessory devices at a condition of-100℃. According to the analysis based on the previous work of protein particles image formation, we had adopted some methods and obtained the freeze-etching replica of good quality. During the spraying process, we had made good coordination between spraying currents and vacuum. Before spraying the specimen, we chose the spraying source carefully. In equipment preparation stage, we focused on cleaning and maintenance vacuum system seriously. Results： The protein particles image on the freeze-etching replica could be distinguished clearly and was good contrast under the electron microscope. The measures we had taken improved the image quality partly and received the better effects.Conclusions： We had improved the image quality by burning and choosing the spraying source carefully, focusing on spraying current interface with vacuum in freeze-etching experimental process and making the preparatory work of experimental equipment better. The boundaries of the protein particles replica were clear and distinguishable. And there were fewer impurities on the protein particles image background. A satisfactory result about protein particles image h

关 键 词：冷冻蚀刻 膜蛋白颗粒 反差不良 喷镀源 

分 类 号：R318.52[医药卫生—生物医学工程]