RAW264.7巨噬细胞的扫描电镜样品制备与观察  

Preparation and observation of scanning electron microscopy specimen of RAW 264.7 macrophages

作　　者：刘博[1] 路菊[2] 江澈[3] 陶忠芬[2] 

机构地区：[1]解放军第210医院检验科,辽宁大连116021 [2]第三军医大学基础医学部中心实验室,重庆400038 [3]重庆三峡中心医院培训中心,重庆万州404000

出　　处：《局解手术学杂志》2011年第1期62-65,共4页Journal of Regional Anatomy and Operative Surgery

摘　　要：目的探索贴壁细胞扫描电镜样本最佳制备方法及仪器工作参数调节与图像质量之间的关系。方法通过对样品制备中清洗、脱水、镀膜步骤的对比试验,调整样品处理的次数、时间、样品台距离、喷涂厚度,获得最佳制备条件;优化电镜加速电压、物镜光阑、工作距离,得到最佳工作参数。结果制备RAW 264.7巨噬细胞扫描电镜样品的最佳方法是用0.1 mol/L PBS清洗2次,每次5 m in;30%、50%、70%、90%、100%乙醇梯度脱水,每次3 m in;镀膜导电时靶与样品之间距离为5 cm,真空度1.33~13.33 Pa,电流10 mA,时间150 s,间断镀膜。观察样品时最佳工作参数设置为加速电压10 kV,物镜活动光阑孔50μm,工作距离12 mm。结论制备样品程序中清洗、脱水、镀膜条件的设定及仪器的加速电压、物镜光阑孔大小、工作距离的优化,有利于获得清晰、细节丰富、反差适中、立体感强的扫描电镜图像。Objective To explore the best method for preparing the scanning electron microscopy（SEM） samples of adherent cells and to investigate the relationship between operating parameters and image quality.Methods The best preparing condition was obtained by adjusting the processing time and frequency,distance to the specimen stage and the coating thickness after comparing the procedures in cleaning,dehydrating and coating.The optimum working parameters were fixed through optimizing the SEM accelerating voltage,objective aperture and working distance.Results The best method for preparing SEM samples of macrophages in RAW264.7 rats was as follows： wash twice with 0.1 mol/L PBS,5 min once,dehydrate with graded ethanol（30%,50%,70%,90%,100%）,3 min once,conduct the coating procedure inconsistently for 150s and fix the distance between the target and the specimen at 5 cm,the vacuum pressure at 1.33～13.33 Pa and the current at 10 mA.The best operating parameters for specimen observation were designed as follows： speeding voltage 10 kV,objective active aperture 50 μm,the working distance 12 mm.Conclusion It is helpful to obtain ideal images with abundant details,proper contrast and strong stereoscopic vision by designing the procedures in cleaning,dehydrating and coating and optimizing the accelerating voltage,objective aperture and working distance

关 键 词：扫描电镜 贴壁细胞 制备样品 工作参数 

分 类 号：Q-336[生物学]