In-situ high-resolution cryo-EM reconstructions from CEMOVIS

Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method to prepare thin samples (lamellae), however, the material removed by the milling process is lost, the imageable area is usually limited to a few square microns, and the surface layers sustain damage from the ion beam. We have examined cryo-electron microscopy of vitreous sections (CEMOVIS), a preparation technique based on cutting thin sections with a knife, as an alternative to FIB-milling. CEMOVIS sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae making CEMOVIS an alternative to FIB-milling with distinct advantages. Using 2-dimensional template matching on images of CEMOVIS sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.