title: The future is cold: cryo-preparation methods for transmission electron microscopy of cells.
abstract: Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.
authors.affiliation: Institut Curie, Centre de Recherche, Cell and Tissue Imaging Facility - IBiSA, Paris F-75248, France. ilse.hurbain@curie.fr
authors.forename: Ilse
authors.initials: I
authors.lastname: Hurbain
authors.forename: Martin
authors.initials: M
authors.lastname: Sachse
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D055032:Electron Microscope Tomography; D006801:Humans; D008544:Melanocytes / Q000648:ultrastructure; D008867:Microtomy; D016613:Plastic Embedding; D012763:Shigella flexneri / Q000648:ultrastructure; D016707:Tissue Fixation; D058989:Vitrification