Methods in Molecular Biology Volume 950, 2013, pp 397-416

Correlative Fluorescence and EFTEM Imaging of the Organized Components of the Mammalian Nucleus

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Abstract

The cell nucleus contains many distinct subnuclear compartments, domains, and bodies that vary in their composition, structure, and function. While the cellular constituents that occupy the subnuclear regions may be well known, defining the structural details of the molecular assembly of the constituents has been more difficult. A correlative fluorescence and energy-filtering transmission electron microscopy (EFTEM) imaging method has the ability to provide these details. The correlative microscopy method described here allows the tracking of subnuclear structures from specific cells by fluorescence microscopy and then, using electron energy loss imaging in the transmission electron microscope, reveals the ultrastructural features of the nuclear components along with endogenous elemental information that relates directly to the biochemical composition of the structure. The ultrastructural features and composition of well-characterized PML bodies and interchromatin granule clusters are compared to those of ligand-activated glucocorticoid receptor (GR) foci, with GR foci containing fibrogranular nucleic acid-containing features and PML bodies being devoid of nucleic acid.