AFM Force Volume for Extracellular Vesicle Detection and Membrane Blebbing Analysis Through Mechanical Signature...

Area of Science:

• Cell Biology
• Biophysics
• Nanotechnology

Background:

• Membrane blebbing is crucial for cell functions like migration and apoptosis, but difficult to study in live cells.
• Traditional imaging methods require fixed samples, limiting understanding of dynamic membrane events.
• Quantifying blebs and extracellular vesicles (EVs) in real-time is essential for biological insights.

Purpose of the Study:

• To develop and validate a novel Atomic Force Microscopy (AFM) approach for detecting bleb- and EV-like structures on live cells.
• To enable quantitative mapping and temporal monitoring of membrane dynamics using AFM force spectroscopy.
• To explore the mechanical signatures of blebs and EVs through force-distance (FD) curves.

Main Methods:

• Utilized AFM force spectroscopy to capture force-distance (FD) curves from live cells.
• Developed a custom MATLAB algorithm to identify EV-like mechanical signatures (breakthrough points) in FD curves.
• Validated the method on isolated EVs and live MDA-MB-231 breast cancer cells, comparing with AFM imaging of fixed cells.

Main Results:

• Successfully detected and mapped bleb- and EV-like structures on live cells using AFM force spectroscopy.
• Observed a dynamic increase in EV-like events up to Day 10 in cell cultures, followed by a decline.
• Demonstrated preferential localization of these structures at the cell periphery, with results consistent with traditional AFM imaging.

Conclusions:

• AFM force spectroscopy provides a non-invasive, high-content tool for detecting nanoscale membrane phenomena in live cells.
• The novel data analysis pipeline enables quantitative insights into the temporal evolution and distribution of blebs and EVs.
• This advancement significantly enhances the capability of AFM for live-cell analysis beyond standard elasticity measurements.