Correlative light-electron microscopy as a tool to study in vivo dynamics and ultrastructure of intracellular structures

Methods Mol Biol. 2013:931:413-22. doi: 10.1007/978-1-62703-056-4_20.

Abstract

Correlative light-electron microscopy (CLEM) is a very effective technique that combines live-cell imaging and immuno-electron microscopy for ultrastructural morphological characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the user to follow the movements and/or behavior of intracellular structures in a live cell and to fix it at the moment of interest. The subsequent immuno-electron microscopy processing can then reveal the three-dimensional architecture of the same structure, together with precise recognition of the GFP-labeled protein. The process resembles the taking of a high-resolution snapshot of an interesting live scene. Considering that CLEM is a very useful but technically demanding and time-consuming technique, accurate protocols will be helpful to simplify the work of scientists who are willing to apply this method for their own purposes. Here, we present a detailed protocol that describes all of the "tricks" and know-hows involved in carrying out the crucial steps of a CLEM experiment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / biosynthesis
  • Adaptor Proteins, Vesicular Transport / genetics
  • Cell Nucleus / metabolism
  • Cell Nucleus / ultrastructure*
  • Gold / chemistry
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • HeLa Cells
  • Humans
  • Indicators and Reagents / chemistry
  • Metal Nanoparticles / chemistry
  • Microscopy, Confocal
  • Microscopy, Electron, Transmission / methods*
  • Microscopy, Fluorescence
  • Microtomy
  • Plastic Embedding
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Single-Cell Analysis / methods
  • Staining and Labeling
  • Time-Lapse Imaging
  • Transfection

Substances

  • Adaptor Proteins, Vesicular Transport
  • GGA adaptor proteins
  • Indicators and Reagents
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Gold