An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Conventional electron microscopy (EM) has limitations in visualizing intracellular structures in a native liquid environment.
• Atomic force microscopy (AFM) offers potential for high-resolution imaging but requires improved methods for intracellular access.

Purpose of the Study:

• To develop and validate an improved unroofing method for AFM to visualize intracellular components.
• To compare the resolution and detail of AFM imaging with conventional EM for cytoskeletal elements and membrane structures.

Main Methods:

• An improved unroofing technique was employed to allow AFM cantilever access into cells.
• Intracellular structures including actin filaments, microtubules, clathrin coats, and caveolae were imaged in phosphate-buffered saline (PBS).
• High-resolution AFM imaging was performed and compared to EM standards.

Main Results:

• Actin filaments showed distinct short (5-6 nm) and long helical periodicities, with polarity indicated by striation shape.
• Microtubules were identified by their characteristic thickness.
• Clathrin coats and caveolae were visualized on the cytoplasmic membrane surface, revealing molecular details and surface structures comparable to EM.

Conclusions:

• The improved unroofing method enables high-resolution intracellular AFM imaging in liquid.
• AFM provides imaging quality equivalent or superior to EM for various cellular structures.
• This technique is poised to yield significant advancements in cell biology and histology research.