Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples

TLDR

Measurements of soft hydrogel samples with defined elastic moduli using different AFMs revealed that uncertainties in deflection sensitivity and cantilever spring constant determination were the main sources of error. The authors present SNAP, a standardized AFM procedure that reliably determines the elastic (Young’s) modulus of soft samples, including living cells, by precisely calibrating the optical lever system and computing deflection sensitivity from vibrometer‑derived spring constants. SNAP eliminates errors by calculating the correct deflection sensitivity based on spring constants measured with a vibrometer, thereby standardizing AFM measurements across instruments, laboratories, and operators. Validation across European laboratories demonstrated that SNAP reduces hydrogel modulus variability to 1% and doubles the consistency of living cell elasticity measurements, improving the use of cell mechanics as a quantitative marker for distinguishing cell types and conditions.

Abstract

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.