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

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.
This work was supported by grants to D.N., T.L., N.C., and J.J.U. (Spanish Ministry of Economy and Competitiveness FIS-PI14/00280), M.L. and J.B. (National Science Center NCN DEC-2011/01/M/ST3/00711), H.O. (Deutsche Forschungsgemeinschaft, Koselleck 68/1 and Centre of Excellence, Cells in Motion (CIM), University of Münster, Germany), S.B. and F.L. (ANR-10-EQPX-04-01) and A.D., E.T.H., R.G. (CSD2010-00024, ERC-AdG-340177). A.P. thanks the Dept. of Physics of the University of Milano for financial support under the project “Piano di Sviluppo dell’Ateneo per la Ricerca 2014”, actions: “A. Upgrade of instrumentation”, and “B. Support to young researchers”. We acknowledge the support of the International AFMBioMed Conference Association.