Single-Molecule Studies with AFM and High-Speed AFM: From Nucleosomes to Chromosomes.

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Atomic force microscopy (AFM) is a key single-molecule imaging technique.
• High-speed AFM (HS-AFM) advancements enable visualization of biological molecule dynamics and kinetics.
• Understanding chromatin dynamics is crucial for gene regulation and cellular processes.

Purpose of the Study:

• To describe methods for preparing chromatin for AFM and HS-AFM analysis.
• To enable sub-nanometer topographical measurements of nucleosome and chromatin structures.
• To facilitate quantitative analysis of nucleosome dynamics using HS-AFM data.

Main Methods:

• In vitro reconstitution and in vivo extraction of chromatin.
• Air mode AFM and high-speed AFM (HS-AFM) imaging.
• Data analysis techniques for quantitative dynamic measurements.

Main Results:

• Successful preparation of chromatin for high-resolution AFM imaging.
• Acquisition of static and time-resolved topographical data of nucleosomes and chromatin.
• Development of methods for quantitative analysis of nucleosome dynamics from HS-AFM data.

Conclusions:

• HS-AFM is a powerful tool for studying chromatin structure and dynamics at the single-molecule level.
• The described methods allow for detailed investigation of nucleosome and chromatin conformations.
• Quantitative analysis of HS-AFM data provides insights into the kinetics of biological molecules.