SPECIMEN CHARGING

我们已经开发了快速检测和评估的方法 当生物样本被收集时， 通过电子低温显微镜检查。 根据图像评估充电 蛋白质晶体悬浮在葡萄糖或玻璃体的薄层中 冰 收费的影响表现为 电子束照射区域的质量和有效的 增加物镜透镜的强度。 我们评估 与充电现象相关的各种因素的影响。 提出了一种收费机制， 二次电子在照射样品时产生 在样品上的可变强度的静电透镜。 本研究 提高了我们对样品充电的理解，并证明了 充电可以大大减少，包括部分的 在受照射的样品区域中的相邻碳膜。 这 构成了一个可行的战略，高分辨率成像的标本 容易被充电 为了存款一层 在已经包埋在玻璃体中的生物标本上的碳膜 冰，我们修改了加坦涂布机配备了一个新的腔室 用碳的热蒸发代替离子束沉积 棒。 该腔室的尺寸较大，以减少热负荷 标本 它有一个气闸，可以插入冷冻标本， 冷冻防护罩，以保护标本免受污染。 单独的 可变Transformer，连接到定时器和大功率继电器 允许控制蒸发到样品上的碳的量 面 冷冻、水合标本安装在Gatan 626 在-168 ℃的低温保持器中插入并在改进的涂布器中涂布 约100
碳层并转移到JEOL 4000 EX电子 低温显微镜在400 kV下工作。 冰包埋过氧化氢酶晶体 制备的过孔被用作测试样本，以估计 用薄碳层涂覆低温试样的有效性， 最大限度地减少收费。 对图像进行记录、数字化和分析 其衍射质量如IQ图所示。 该图显示 IQ 4至3的反射
分辨率 图像来自 碳包被的过氧化氢酶晶体倾斜到50 °，也显示出 相同质量高达4
分辨率 这些高分辨率图像 数据表明使用我们改进的涂布机减少了充电。

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.