Visualizing macromolecules with fluoronanogold: From photon Microscopy to electron tomography

Fluoronanogold (FNG) contains a fluorochrome molecule (FITC, Alexa, and so on) and a 1.4-nm cluster of gold atoms; these permit visualization both by photon microscopy (direct visualization) and by electron microscopy (EM) (after silver or gold enhancement). Classical applications of FNG concern immunolabeling of any antigen and its visualization with cellular imaging in two dimensions (2D), that is, using wide-field optical microscopy and EM (ultrathin sections). In the present chapter, we show that FNG can be localized in three dimensions (3D), both by confocal light microscopy and electron tomography. These approaches are useful for investigating the volumetric organization of proteins within well-preserved organelles at different levels of resolution (200 nm in confocal microscopy and 10 nm in electron tomography), as shown with two different nucleolar proteins.