Fiche publication
Date publication
janvier 2015
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MICHEL Jean
,
Pr PLOTON Dominique
,
Dr TERRYN Christine
Tous les auteurs :
Nolin F, Ploton D, Wortham L, Tchelidze P, Bobichon H, Banchet V, Lalun N, Terryn C, Michel J
Lien Pubmed
Résumé
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.
Mots clés
Cell Culture Techniques, Cell Nucleus, metabolism, Cryoelectron Microscopy, methods, HeLa Cells, Humans, Ions, analysis, Light, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Nanotechnology, methods, Water, analysis
Référence
Methods Mol. Biol.. 2015 ;1228:145-58
