A hybrid high-speed atomic force-optical microscope for visualizing single membrane proteins on eukaryotic cells.

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• High-speed atomic force microscopy (HS-AFM) is crucial for protein structure and dynamics research.
• Previous HS-AFM applications were limited to purified proteins in controlled environments.
• Imaging cellular structures and dynamics in vivo remained a significant challenge.

Purpose of the Study:

• To integrate optical microscopy with HS-AFM to overcome limitations of current techniques.
• To enable high-resolution imaging of cellular components, particularly membrane proteins, in their native environment.
• To advance HS-AFM capabilities from molecular systems to live cell biology.

Main Methods:

• Integration of an optical microscopy path into a HS-AFM system.
• Development of a hybrid HS-AFM/optical microscopy setup for precise tip positioning.
• Acquisition of high-speed imaging data (960 ms/frame) of cellular membrane dynamics.

Main Results:

• The hybrid system maintains HS-AFM performance while enabling bright-field and fluorescence microscopy.
• Demonstrated visualization of aquaporin-0 arrays and single molecule dynamics in intact eye lens cells.
• Achieved imaging speeds approximately 1,000 times faster than conventional AFM/optical microscopy setups.
• Enabled first-time visualization of unlabeled membrane proteins on eukaryotic cells under physiological conditions.

Conclusions:

• The hybrid HS-AFM/optical microscopy system significantly expands the application scope of HS-AFM.
• This technology allows for the study of cellular membrane processes like signaling, transport, and diffusion in real-time.
• Represents a major advancement for analyzing cellular dynamics at the membrane, bridging molecular and cell biology.