Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Podosomes are key cellular structures involved in matrix adhesion, probing, and remodeling.
• Their precise molecular architecture and function are not fully understood.
• Advanced imaging techniques are needed to study podosome dynamics at the molecular level.

Purpose of the Study:

• To review recent advancements in imaging podosome architecture.
• To introduce a new live-cell super-resolution microscopy technique, HAWK.
• To combine fluorescence localization microscopy with atomic force microscopy for simultaneous visualization of molecular components and mechanical properties.

Main Methods:

• Development and application of HAWK (High-Accuracy Widefield-Kikuchi) microscopy for artifact-free live-cell super-resolution imaging of podosome ring proteins.
• Integration of fluorescence localization microscopy (STORM/PALM) with atomic force microscopy (AFM) on a single platform.
• Utilizing iFluor-647 and the photoswitchable protein mEOS3.2 for two-color live-cell imaging.

Main Results:

• HAWK enables high-resolution, artifact-free live-cell imaging of podosome ring proteins.
• Combined STORM/PALM and AFM reveals the spatial distribution and dynamics of podosome components alongside matrix stiffness variations.
• The chosen fluorescent labels (iFluor-647 and mEOS3.2) facilitate multi-color live-cell imaging without buffer exchange.

Conclusions:

• The developed HAWK technique and integrated AFM-microscopy platform offer unprecedented insights into podosome molecular architecture and mechanics.
• This approach advances the study of cellular mechanosensitive structures and their roles in biological processes.
• Future research can leverage these advanced imaging capabilities to further elucidate podosome function in various cellular contexts.