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

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.

Membrane Stiffening in Osmotic Swelling

“…The exact form of the Hertz model depends on the geometry of the cantilever tip. Although this is the most commonly used model to estimate cellular elastic moduli from AFM experiments (Haase & Pelling, 2015; Schillers et al, 2017; Thomas, Burnham, Camesano, & Wen, 2013), the Hertz model was developed based on several assumptions regarding the material being tested, including sample homogeneity, isotropy, and a linear stress-strain response. These assumptions present a limitation to the model, given that cells are structurally heterogeneous and anisotropic.…”

Nanorheology of living cells measured by AFM-based force–distance curves

“…31,32 Living cells show a variety of viscoelastic phenomena which remain insufficiently understood. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] This lack of understanding has fundamental and practical implications. The accuracy of Young's modulus measurements determined without considering viscoelastic effects is highly questionable.…”

Atomic force microscopy methodology and AFMech Suite software for nanomechanics on heterogeneous soft materials

“…The specific steps of the proposed methodology can be summarized as following:Fine mechanical calibration of AFM through rheological measurements on homogeneous gels, applying recursively SNAP method 13 .Systematic analysis of heterogeneous samples (composite gels and biological systems) in physiological conditions using AFM space-resolved indentation.Reconstruction of AFM experiments using FEM parametrized by AFM results 25 . Production of parametric array of simulations for data interpretation.…”

“…Analysis is focused in obtaining (1) elastic constant K (N m −1 ) from thermal noise measurements in air or liquid and (2) calculation of optical lever sensitivity Zsens (nm V −1 ) from FC or FV on rigid, undeformable substrates (with negligible indentation). Interface allows the user to use advanced features such as: photodetector non-linearity correction and SNAP procedure 13 , in order to produce high precision results.Morphology: Visualize and perform operations on the raw height after acquisition. Raw morphology can be flattened removing sample tilt.…”