Probing nuclear ultrastructure by electron spectroscopic imaging

Abstract

Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20-30 nm. This complexity hinders the study of structure-function relationships within the mammalian nucleus. Element-specific mapping using the energy-filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of strains relative to unstained material when biochemical-specific staining protocols, such as EDTA-regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogenous. ESI shows substantial promise for the investigation of structure-function relationships in biological specimens.