Three-dimensional analysis of intermediate filament networks using SEM tomography

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Intermediate filaments (IFs) are crucial for cell mechanics.
• Understanding IF network organization is key to cell elasticity.
• Pancreatic cancer cells exhibit altered mechanical properties.

Purpose of the Study:

• To develop a high-contrast 3D imaging method for IF networks in pancreatic cancer cells.
• To analyze the structural organization and morphology of IF networks.
• To link IF network structure to cell elasticity and mechanical response.

Main Methods:

• Scanning electron microscopy (SEM) tilt series acquisition.
• Tomographic reconstruction of secondary electron signals.
• Development of an image analysis algorithm for IF network graph extraction.
• Statistical analysis of extracted IF network graphs.

Main Results:

• Tomographic reconstruction of SEM tilt series provides high-contrast 3D data of IF networks.
• A novel algorithm successfully extracts the graph structure of IF networks from tomograms.
• Analysis reveals network morphology linked to cell mechanical properties.
• Structural organization principles of IF networks were identified.

Conclusions:

• SEM-based tomographic reconstruction is suitable for visualizing IF networks in detergent-extracted pancreatic cancer cells.
• The developed algorithm enables high-resolution analysis of IF network morphology.
• IF network organization significantly impacts cell elasticity and mechanical response.