Electron spectroscopic imaging of DNA.

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Traditional electron microscopy for biological samples often requires heavy metal staining, which can introduce artifacts.
• Accurate quantification of nucleic acid content in nucleoprotein complexes is crucial for understanding gene regulation and DNA packaging.

Purpose of the Study:

• To develop and validate a heavy metal-free electron spectroscopic imaging (ESI) method for analyzing DNA and nucleoprotein complexes.
• To assess the capability of ESI in determining relative nucleic acid levels within these complexes.
• To explore the potential of ESI for characterizing chromatin ultrastructure.

Main Methods:

• Utilized a fixed beam electron microscope with a parallel electron energy filter for electron spectroscopic imaging (ESI).
• Employed inelastically scattered electrons targeting phosphorus LII,III shell ionization to generate phosphorus distribution maps.
• Analyzed purified plasmid DNA and deoxyribonucleoprotein complexes, including chromatin from Saccharomyces cerevisiae.

Main Results:

• Generated micrographs of plasmid DNA without heavy metal stains or shadows.
• Created phosphorus distribution maps demonstrating accurate reflection of known stoichiometric phosphorus levels in single, double, and quadruple DNA strands.
• Identified three distinct ultrastructural classes within the basic chromatin fiber of nucleosomal and transcriptionally active chromatin.

Conclusions:

• Electron spectroscopic imaging (ESI) provides a viable heavy metal-free method for visualizing and quantifying nucleic acid distribution in biological samples.
• ESI is capable of determining relative nucleic acid content in nucleoprotein complexes, offering insights into stoichiometry.
• ESI shows promise for high-resolution ultrastructural characterization of complex biological assemblies like chromatin.