Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae

  • Christophe Normand
  • Maxime Berthaud
  • Olivier Gadal
  • Isabelle Léger-Silvestre
Part of the Methods in Molecular Biology book series (MIMB, volume 1455)


Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller’s spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated “pipeline” for imaging.

Key words

Correlative light and electron microscopy (CLEM) Nucleolus RNA polymerase I (Pol I) Ribosomal RNA genes (rDNA) Pol I transcription Transmission electron microscopy (TEM) Fluorescence microscopy Yeast Saccharomyces cerevisiae 



This work is supported by ATS-Nudgene and Emergence-CLEMgene of the Toulouse-IDEX. O.G. and C.N. are supported by Agence Nationale de la Recherche (ANDY). TEM images were taken with the facilities on the METI platform, part of TRI-Genotoul.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratoire de Biologie Moléculaire du CNRSUniversity of ToulouseToulouseFrance
  2. 2.CRCNA-UMR 892 INSERMNantesFrance
  3. 3.CNRSNantesFrance
  4. 4.University of NantesNantesFrance

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