title: Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box.
abstract: Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images. Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of Saccharomyces cerevisiae.
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Jason
authors.initials: J
authors.lastname: Pierson
authors.forename: José Jesús
authors.initials: JJ
authors.lastname: Fernández
authors.forename: Erik
authors.initials: E
authors.lastname: Bos
authors.forename: Shoaib
authors.initials: S
authors.lastname: Amini
authors.forename: Helmut
authors.initials: H
authors.lastname: Gnaegi
authors.forename: Matthijn
authors.initials: M
authors.lastname: Vos
authors.forename: Bennie
authors.initials: B
authors.lastname: Bel
authors.forename: Freek
authors.initials: F
authors.lastname: Adolfsen
authors.forename: José L
authors.initials: JL
authors.lastname: Carrascosa
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Peters
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D016616:Cryoultramicrotomy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods*; D006813:Humidity; D012270:Ribosomes / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae; D055672:Static Electricity