SPECIMEN CHARGING

我们已经开发了快速检测和评估的方法 在生物样本上形成的电荷 通过电子冷冻显微镜检查。充电是通过图像进行评估的 悬浮在薄层葡萄糖或玻璃体中的蛋白质晶体 冰。收费的影响表现为明显增加了 电子束照射区域的质量和有效的 增加物镜的强度。我们评估 影响充电现象的因素是多方面的。 提出了一种收费机制，该机制针对二氧化碳的排放 样品照射时产生的二次电子 在样品处具有可变强度的静电透镜。本研究 提高了我们对标本收费的理解，并演示了 通过将部分 辐照试件区域的相邻碳膜。这 构成了对标本进行高分辨率成像的可行策略 容易被充电的人。为了沉积一层 已经嵌入玻璃体内的生物标本上的碳膜 冰，我们对Gatan涂布机进行了改装，为它配备了一个新的腔室 用碳的热蒸发取代了离子束沉积 棒子。这个腔体的尺寸更大，以减少 标本。它有一个气闸来插入一个低温标本和一个 冷冻保护罩，以保护标本免受污染。一个单独的 连接到定时器和大功率继电器的可变变压器 允许控制蒸发到样品上的碳量 浮出水面。安装在Gatan 626中的冷冻、水合标本 将-168℃的低温保持器插入并涂覆在改进的涂布机中 使用~100碳层并转移到JEOL4000EX电子 工作在400千伏的冷冻显微镜。冰包埋过氧化氢酶晶体 在孔上制备的样品作为测试样品来估计 在低温冷冻机上涂覆一层薄碳层的效果 最大限度地减少充电。对图像进行记录、数字化和分析 对于它们的衍射质量，如IQ图所示。剧情显示 智商4到3的反射决议。获取的图像来自 碳涂层过氧化氢酶晶体倾斜到50-也显示出反射 相同质量，最高可达4决议。这些高分辨率图像 数据表明，使用我们改进的涂布机可以减少装料量。

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.