title: Correlated live cell light and electron microscopy using tetracysteine tags and biarsenicals.
abstract: Fundamental to obtaining a more complete understanding of the roles played by macromolecular complexes in cells is the ability to map their location, movement, and transient interactions at high temporal and high spatial resolution. Unfortunately, probes capable of allowing direct correlation of real-time or time-lapse light microscopy (LM) with electron microscopic observations are relatively few. Genetically encoded fluorescent reporters such as green fluorescent protein (GFP) have revolutionized live cell imaging studies but are not directly visible by electron microscopy (EM). Fluorescent nanoparticles or quantum dots are a type of label for LM that can also be visualized directly by EM, but targeting these to cytoplasmic proteins in living cells remains difficult. One method that does allow for highly correlated LM and EM with excellent preservation of cellular ultrastructure is fluorescence photoconversion, in which a fluorescent compound causes the deposition of a reaction product that can be rendered electron-dense and directly visualized by EM. We have used this method in combination with a class of genetically encoded peptide tags that can be labeled in living cells by fluorophores bearing two appropriately spaced arsenic atoms (biarsenicals). The tetracysteine motif is short, easily inserted into or attached to the termini of the host protein, and can be used in combination with other molecular tags such as GFP and its derivatives. This article presents methods to label cells with biarsenicals, conduct live cell imaging recording sessions, and generate specimens that can be evaluated by EM for a correlated LM/EM analysis.
authors.forename: Guido M
authors.initials: GM
authors.lastname: Gaietta
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
mesh_terms: D001152:Arsenicals / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001152:Arsenicals; D003545:Cysteine