title: Visualizing live dynamics and ultrastructure of intracellular organelles with preembedding correlative light-electron microscopy.
abstract: One of the very effective methods to perform correlative light-electron microscopy (CLEM) is to combine video imaging of live cells with immuno-electron microscopy. This technique can thus provide detailed, high-resolution characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the movements and/or behavior of intracellular structures in a live cell to be followed, which can then be fixed at the moment of interest. The subsequent immuno-electron microscopy analysis reveals the three-dimensional (3D) architecture of the same structure, together with the precise identification of the GFP-labeled protein pattern. The process resembles taking a high-resolution snapshot of an interesting and/or rare live event. Conceptually, it consists of a switch of wavelengths, from that of photons to that of electrons, with the associated huge gain in resolution. In this respect, CLEM can be considered as the first, and probably one of the most powerful, super-resolution microscopy techniques. This switch, however, requires complex manipulations of the sample. Considering that CLEM is a very valuable but technically challenging and time-consuming method, accurate protocols are needed to simplify the efforts of researchers who are willing to apply this method for their own purposes. Here, we present a detailed description of the preembedding CLEM procedures that explains the know-how and the "tricks of the trade" that are involved in carrying out the crucial steps of CLEM.
authors.affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Polishchuk
authors.forename: Elena V
authors.initials: EV
authors.lastname: Polishchuk
authors.forename: Alberto
authors.initials: A
authors.lastname: Luini
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron / Q000379:methods; D008867:Microtomy; D015388:Organelles / Q000648:ultrastructure*; D016613:Plastic Embedding; D059010:Single-Cell Analysis; D013194:Staining and Labeling; D059008:Time-Lapse Imaging
chemical_list: D049452:Green Fluorescent Proteins