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.
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