title: Correlative light-electron microscopy as a tool to study in vivo dynamics and ultrastructure of intracellular structures.
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.
authors.affiliation: Institute of Protein Biochemistry, Naples, Italy.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Polishchuk
authors.forename: Roman S
authors.initials: RS
authors.lastname: Polishchuk
authors.forename: Alberto
authors.initials: A
authors.lastname: Luini
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000096:biosynthesis / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008856:Microscopy, Fluorescence; D008867:Microtomy; D016613:Plastic Embedding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods; D013194:Staining and Labeling; D059008:Time-Lapse Imaging; D014162:Transfection
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006046:Gold