title: A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography.
abstract: Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.
authors.affiliation: Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Bos
authors.forename: Celso
authors.initials: C
authors.lastname: SantAnna
authors.forename: Helmut
authors.initials: H
authors.lastname: Gnaegi
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Pinto
authors.forename: Raimond B G
authors.initials: RB
authors.lastname: Ravelli
authors.forename: Abraham J
authors.initials: AJ
authors.lastname: Koster
authors.forename: Wanderley
authors.initials: W
authors.lastname: de Souza
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Peters
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D016616:Cryoultramicrotomy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D004847:Epithelial Cells / Q000648:ultrastructure; D006801:Humans; D006813:Humidity; D007150:Immunohistochemistry / Q000379:methods*; D019169:Jurkat Cells; D008747:Methylcellulose / Q000737:chemistry; D058996:Quality Improvement; D058989:Vitrification
chemical_list: D008747:Methylcellulose