Correlative Atomic Force and Single-Molecule Fluorescence Microscopy of Nucleoprotein Complexes

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy Techniques

Background:

• Structurally heterogeneous biomolecular assemblies require advanced imaging methods.
• Orthogonal information extraction is crucial for understanding complex biological systems.
• Limitations exist in current techniques for high-resolution analysis of biomolecular structures.

Purpose of the Study:

• To describe an integrated setup for correlative fluorescence and atomic force microscopy (AFM).
• To present protocols for sample preparation, data acquisition, and processing for correlative imaging.
• To demonstrate the capabilities of the integrated approach for characterizing biomolecular assemblies.

Main Methods:

• Integrated setup for correlative fluorescence and atomic force microscopy.
• Detailed protocols for sample preparation, data acquisition, and data processing.
• Application to simultaneous characterization of mesoscale geometry and composition.

Main Results:

• Achieved nanoscale topographic resolution.
• Demonstrated high image registration accuracy.
• Attained single-fluorophore sensitivity in correlative imaging.
• Successfully characterized a multipart nucleoprotein complex.

Conclusions:

• Correlative fluorescence and AFM microscopy is a versatile tool for analyzing heterogeneous biomolecular assemblies.
• The described integrated setup and protocols enable high-quality nanoscale imaging.
• This approach facilitates simultaneous characterization of structure and composition in complex biological systems.