title: Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.
abstract: The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.
authors.affiliation: Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Nolin
authors.forename: Dominique
authors.initials: D
authors.lastname: Ploton
authors.forename: Laurence
authors.initials: L
authors.lastname: Wortham
authors.forename: Pavel
authors.initials: P
authors.lastname: Tchelidze
authors.forename: Hélène
authors.initials: H
authors.lastname: Bobichon
authors.forename: Vincent
authors.initials: V
authors.lastname: Banchet
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lalun
authors.forename: Christine
authors.initials: C
authors.lastname: Terryn
authors.forename: Jean
authors.initials: J
authors.lastname: Michel
mesh_terms: D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007477:Ions / Q000032:analysis / Q000378:metabolism; D008027:Light; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000379:methods*; D014867:Water / Q000032:analysis* / Q000378:metabolism
chemical_list: D007477:Ions; D014867:Water