Correlative LightElectron Microscopy as a Tool to Study In Vivo Dynamics and Ultrastructure of Intracellular Structures

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.