SPECIMEN CHARGING

样品加料

• 批准号: 6611288
• 负责人: MICHAEL B SHERMAN
• 金额: $ 13.47万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611288
• 关键词: biomedical equipment development; biomedical resource; informatics; technology /technique

We have developed methods for the rapid detection and evaluation of the charge which builds up on biological specimens when they are examined by electron cryomicroscopy. Charging is assessed from images of protein crystals suspended in thin layers of glucose or vitreous ice. Effects of charging are manifested as an apparent increase in mass in areas illuminated by the electron beam and an effective increase in the strength of the objective lens. We assess the influence of a variety of factors related to the charging phenomena. A mechanism of charging is proposed in terms of the emission of secondary electrons upon irradiation of the specimen creating an electrostatic lens of variable strength at the specimen. This study has improved our understanding of specimen charging and demonstrated that charging can be substantially reduced by including part of the adjacent carbon film in the irradiated specimen area. This constitutes a viable strategy for high resolution imaging of specimens that are susceptible to charging. In order to deposit a layer of carbon film on the biological specimens already embedded in vitreous ice, we modified the Gatan coater by outfitting it with a new chamber that replaced ion-beam deposition with thermal evaporation of carbon rods. This chamber is larger in size to reduce thermal load on the specimens. It has an airlock to insert a cryo-specimen and a cryo-shield to protect the specimen from contamination. A separate variable transformer connected to a timer and a high-power relay allowed control of the amount of carbon evaporated onto the specimen surface. Frozen, hydrated specimens mounted in the Gatan 626 cryoholder at -168- C were inserted and coated in the modified coater with ~100
layer of carbon and transferred into a JEOL4000EX electron cryomicroscope operating at 400 kV. Ice-embedded catalase crystals prepared over holes were used as test specimens to estimate the effectiveness of coating cryospecimens with a thin carbon layer to minimize charging. The images were recorded, digitized and analyzed for their diffraction quality as shown in the IQ-plot. The plot shows reflections of IQ } 4 up to 3
resolution. Images obtained from carbon-coated catalase crystals tilted to 50- also show reflections of the same quality up to 4
resolution. These high resolution image data demonstrate the reduction of charging using our modified coater.

我们开发了快速检测和评估的方法 在生物标本上建立的费用 通过电子冷冻显微镜检查。 从图像评估充电 悬浮在葡萄糖或玻璃体薄层的蛋白质晶体的 冰。 充电的影响表现为明显增加 由电子束照亮的区域，有效 提高客观镜头的强度。 我们评估 与充电现象有关的多种因素的影响。 提出了一种充电机制 标本照射后，辅助电子产生 样品处可变强度的静电镜片。 这项研究 提高了我们对标本充电的理解和证明 通过包括一部分 辐照样品区域中的相邻碳膜。 这 构成了标本高分辨率成像的可行策略 容易收取费用。 为了存放一层 在已经嵌入玻璃体中的生物标本上的碳膜 冰，我们通过为新室装备了Gatan座椅 用碳的热蒸发代替了离子束沉积 杆。 该腔室的尺寸更大，以减少在 标本。 它有一个气锁可以插入冷冻特性和 冷冻屏蔽以保护样品免受污染。 一个单独的 可变变压器连接到计时器和高功率继电器 允许控制蒸发到样品的碳量 表面。 冷冻的水合标本安装在Gatan 626中 将-168 -C处的冷冻持有人插入并覆盖在修饰的座椅上 用〜100层碳转移到JEOL4000EX电子中 冷冻显微镜以400 kV工作。 冰块的过氧化氢酶晶体 在孔中准备的作为测试标本来估计 涂层的涂层具有薄碳层的有效性 最小化充电。 记录，数字化和分析图像 如智商图所示，它们的衍射质量。 情节显示 IQ} 4最多3分辨率的反射。 从获得的图像 倾斜至50的碳涂层过氧化氢酶晶体也显示了 相同的质量最多4分辨率。 这些高分辨率图像 数据证明了使用我们的修改后的夹具减少充电。