Experimental validation of atomic force microscopy-based cell elasticity measurements

Area of Science:

• Biophysics
• Cell Mechanics
• Microscopy

Background:

• Atomic force microscopy (AFM) is a common technique for measuring living cell elasticity.
• Reported AFM elasticity values vary widely (100 Pa–100 kPa) and differ from other methods.
• Tip geometry influences AFM elasticity measurements, with pyramidal tips yielding higher values than spherical tips.

Purpose of the Study:

• To investigate the accuracy of contact mechanics models used in AFM elasticity measurements.
• To directly measure the tip-cell contact area and indentation depth during AFM experiments.
• To identify sources of error in AFM-based cell elasticity measurements, particularly concerning tip geometry.

Main Methods:

• Combined Atomic Force Microscopy (AFM) and confocal microscopy were used.
• Epithelial cells expressing a GFP-tagged membrane marker were utilized.
• Experimentally measured indentation geometry and depth were compared with AFM force-indentation curve analyses.

Main Results:

• For spherical tips, the experimentally measured contact area closely matched predicted values.
• For pyramidal tips, contact area was significantly underestimated at forces >0.2 nN.
• This underestimation led to a >2-fold overestimation of cell elasticity when using pyramidal tips.

Conclusions:

• Standard AFM contact mechanics models may overestimate cell elasticity, especially with pyramidal tips.
• A re-examination of previously reported cellular elasticity values obtained via AFM may be required.
• Guidelines are proposed to mitigate measurement artifacts and improve the accuracy of AFM elasticity measurements.