Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

Abstract

Silicon nitride (SiN) membranes that are merely nanometers-thick have been introduced as new support films for electron microscopy (EM) applications. The use of these thin membranes for high-resolution imaging is widely attractive due to their consistent manufacture, flatness, and durability. Here, we utilized SiN support films to actively monitor key steps in rotavirus RNA synthesis at the nanoscale. Rotaviruses are gastrointestinal pathogens that infect pediatric populations and pose a major health problem in developing countries. We demonstrate, for the first time, that cryo-EM specimens of these viral assemblies may be prepared using SiN membranes, that we have termed "cryo-SiN". Cryo-SiN microchips containing inactive and actively transcribing rotavirus complexes were vitrified using gentleblot conditions to yield reproducibly thin ice-embedded specimens. EM imaging results indicate that cryo-SiN samples exhibited greater visual contrast than specimens prepared on conventional support films under the same conditions. The improved contrast of the cryo-SiN samples permitted us to calculate 3D structures of rotavirus complexes using a fraction of the images required for conventionally prepared specimens. We anticipate the use of cryo-SiN microchip will enable us to better investigate critical viral life-cycle processes at the molecular level under near-native conditions. AbbreviationsDouble-Layered Particles (DLPs); Silicon Nitride (SiN); Electron Microscopy (EM); Transmission Electron Microscope (TEM); Nickel-Nitrilotriacetic Acid (Ni-NTA); Nanometers (nm)

Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame

“…Thin films, such as Kapton and especially Ultralene, offer high signal transmission, purity, and are frequently used as experimental surfaces for X-ray studies. For cryo-X-ray fluorescence microscopy, the frame of the silicon nitride window acts as a thermal conducting surface that can support the low temperatures for a frozen specimen, , with the additional benefit of its nitride membrane being an X-ray transparent, nonmetal support to avoid conflicting with the elemental detection of metals within the sample, , which would be an issue for TEM grids. Prior to testing, the silicon nitride windows were cleaned with oxygen plasma, with a mask placed over the central portion of the window where the supporting silicon was etched away.…”