Atomic Force Microscopy Using Thermal Fluctuation of Cantilever for Observing Soft Biological Macromolecules

Area of Science:

• Biophysics
• Materials Science
• Nanotechnology

Background:

• Atomic Force Microscopy (AFM) is vital for imaging, but tip-sample interactions can damage soft biological samples.
• Minimizing interaction forces is crucial for preserving the integrity of delicate macromolecules during imaging.

Purpose of the Study:

• To develop a novel AFM scanning mode for imaging soft biological macromolecules with sub-10 pN load force.
• To demonstrate a noninvasive imaging approach that preserves sample structure.

Main Methods:

• A new AFM scanning mode was developed, monitoring cantilever thermal fluctuation reduction at subnanometer tip-sample separations.
• Sample height was controlled to maintain constant cantilever thermal fluctuation magnitude.
• Force interactions were formulated using statistical mechanics, attributing repulsion to entropic effects from reduced cantilever fluctuation.

Main Results:

• Two proteins, GroEL and bacteriorhodopsin, were successfully imaged with marginal load force.
• The fragile GroEL structure remained intact after multiple scans.
• The flexible C-terminal region of bacteriorhodopsin was clearly visualized.

Conclusions:

• The developed AFM scanning mode enables noninvasive imaging of soft and fragile biological macromolecules.
• This technique offers high-resolution topography imaging while preserving sample integrity.